Stigmastane-type steroids with unique conjugated Δ7,9(11) diene and highly oxygenated side chains from the twigs of Vernonia amygdalina

Article abstract of DOI:10.1016/j.phytochem.2018.10.036

Veramyosides A-J, eleven undescribed stigmastane-type steroids, including one aglycone and ten glycosides, along with three known homologues were isolated from the twigs of Vernonia amygdalina Delile (compositae). All compounds featured a stigmastane-type steroid skeleton with a unique conjugated Δ^7,9(11) diene segment and highly oxygenated side chains with a γ-lactone or an α, β-unsaturated five-membered lactone ring. The structures of veramyosides A-J and their absolute configurations were unambiguously elucidated by HR-ESI-MS, extensive NMR spectroscopy, in situ dimolybdenum CD methods, modified Mosher's method, quantum chemical calculation of their ECD curves, and CD comparison methods on basis of their biogenetic pathway. In addition, all isolates were investigated for their effects on RXRα transcription, and their effects on the NF-κB signaling pathway were also evaluated.

Full text of DOI:10.1016/j.phytochem.2018.10.036

Phytochemistry158(2019)67–76
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Stigmastane-type steroids with unique conjugated Δ7,9(11) diene and highly
oxygenated side chains from the twigs of Vernonia amygdalina
Xiangzhong Liu1, Wenjing Tian1, Guanghui Wang, Qiannan Xu, Mi Zhou, Shuo Gao, Daren Qiu,
Xin Jiang, Cuiling Sun, Rong Ding, Ting Lin∗∗, Haifeng Chen∗
Fujian Provincial Key Laboratory of Innovative Drug Target, School of Pharmaceutical Sciences, Xiamen University, Xiamen, 361005, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Veramyosides A-J, eleven undescribed stigmastane-type steroids, including one aglycone and ten glycosides,
along with three known homologues were isolated from the twigs of Vernonia amygdalina Delile (compositae).
All compounds featured a stigmastane-type steroid skeleton with a unique conjugated Δ7,9(11) diene segment and
highly oxygenated side chains with a γ-lactone or an α, β-unsaturated five-membered lactone ring. The structures
of veramyosides A-J and their absolute configurations were unambiguously elucidated by HR-ESI-MS, extensive
NMR spectroscopy, in situ dimolybdenum CD methods, modified Mosher's method, quantum chemical calcu-
lation of their ECD curves, and CD comparison methods on basis of their biogenetic pathway. In addition, all
isolates were investigated for their effects on RXRα transcription, and their effects on the NF-κB signaling
pathway were also evaluated.
Vernonia amygdalina Delile (compositae)
Stigmastane-type steroids
Absolute configuration
RXRα
NF-κB
1. Introduction
dysentery, gastrointestinal disorders and so on. Two major categories of
constituents, steroids and sesquiterpene lactones, were found in V.
Stigmastane-type steroids, a family of phytosterols, are structurally
characterized by the presence of 1,2-cyclopentanoperhydrophenan-
threne ring system composed of 17 carbon atoms arranged in four rings
and 10 carbon atoms in side chains. The core structures of stigmastane-
type steroids can be classified into two categories that differ from each
amygdalina and were reported to have activities including antimalarial
(Nerdy, 2015), anti-breast cancer (Luo et al., 2011; Yedjou and
Tchounwou, 2017), and anti-nasopharyngeal carcinoma activities
(Kupchan et al., 1969). However, the Δ7,9(11) stigmastane-type steroids
were the most common steroids in V. amygdalina, with only 20 distinct
compounds discovered (Ohigashi et al., 1991; Jisaka et al., 1992, 1993;
Kamperdick et al., 1992; Igile et al., 1995; Schmittmann et al., 1994;
Quasie et al., 2016; Wang et al., 2018), which shows great potential in
medicinal chemistry study of this plant. In our efforts to find more
active Δ7,9(11) stigmastane-type steroids with undescribed structures,
eleven undescribed Δ7,9(11) stigmastane-type steroids along with three
known steroids (Fig. 1) were isolated from the twigs of V. amygdalina.
Compounds 1–11 featured a Δ7,9(11) stigmastane-type steroid skeleton
with highly oxygenated side chains characterized by the existence of a
21, 23-epoxy bridge leading to the formation of a γ-lactone or an α, β-
unsaturated five-membered lactone ring. Herein, we report the isola-
tion, structural elucidation, and biological evaluation of the isolated
compounds.
other by the position of the conjugated diene, at Δ7,9(11) or Δ8,9(14)
.
Nevertheless, only a few Δ7,9(11) stigmastane-type steroids have been
reported, and they were primarily found in the genus Vernonia. The
reported Δ7,9(11) stigmastane-type steroids were characterized by highly
oxidized side chains with 21, 23-epoxy, 22, 24-epoxy or other cyclized
moieties as well as polyhydric substituted acyclic fragments, and these
compounds showed a vast array of biological activities, such as anti-
malarial, antimicrobial, anthelmintic and antithrombotic activities
(Audu et al., 2012). Modifications of side chains with different cycli-
zation patterns, attached groups and configurations led to the structural
diversity of Δ7,9(11) stigmastane-type steroids. Thus, the Δ7,9(11) stig-
mastane-type steroids can be considered as a rich source of undescribed
structures and active lead compounds.
Vernonia amygdalina Delile (Compositae), a member of the genus
Vernonia, shows great efficacy in the treatment of gout, amoebic
∗ Corresponding author.
∗∗ Corresponding author.
E-mail addresses: linting@xmu.edu.cn (T. Lin), haifeng@xmu.edu.cn (H. Chen).
1 These two authors contributed equally to this research.
https://doi.org/10.1016/j.phytochem.2018.10.036
Received 12 August 2018; Received in revised form 9 October 2018; Accepted 29 October 2018
0031-9422/©2018ElsevierLtd.Allrightsreserved.

X. Liu et al.
Phytochemistry158(2019)67–76
Fig. 3. Key NOESY correlations of 1–11.
Δ7,9(11) stigmastane-type steroid core accounts for 6 degrees of un-
saturation, and the remaining 3 degrees of unsaturation and the re-
maining C10H16O4 in the side chains indicated the presence of one
olefin, one carbonyl and an additional ring. The 1H−1H COSY corre-
lations of H-22/H-23 and the HMBC correlations from H-22 to C-21 and
from H-23 to C-20/C-21 suggested that C-21 and C-23 were linked via
an oxygen bridge to form an α, β-unsaturation five-membered lactone
ring (Fig. 2). The 1H−1H COSY correlations of H-25/H3-26, H-25/H3-
27 and H-28/H3-29 as well as the HMBC correlations from H-23 to C-
24/C-25/C-28, from H3-26 to C-24, and from H-28 to C-24, together
with the chemical shifts of C-24 (δC 79.7) and C-28 (δC 71.3) indicated
the presence of a 1,2-OH-1-isopropyl-propyl group, which was con-
nected to the lactone ring at C-23 and C-24 (Fig. 2). In addition, the
HMBC correlations from H-17 to C-20/C-21/C-22 constructed the
linkage of the side chain to C-17 of the steroid core (Fig. 2). Thus, the
planar structure of 1 was deduced.
Fig. 1. Chemical structures of veramyosides A-J (1-14).
2. Results and discussion
2.1. Isolation and structural elucidation
The powdered twigs of V. amygdalina were extracted with 60%
EtOH(aq.) and partitioned consecutively with petroleum ether, di-
chloromethane, ethyl acetate and n-butyl alcohol. The dichloromethane
extract was repeatedly separated by a variety of chromatographic
methods to afford eleven undescribed (1–11) and three known (12–14)
stigmastane-type steroids (Fig. 1).
Veramyoside A (1), [ ]2D5.7-40.00 (c 1.30, CH3OH), was obtained as
an amorphous white powder. Its molecular formula was assigned as
The relative configuration of 1 was determined by a NOESY ex-
periment (Fig. 3). The NOESY correlations of H-1α/H-3/H-5, H-2β/H3-
19/H-4β, H3-19/H3-18/H-15β and H-17/H-14 suggested the trans fu-
sion of the A/B and C/D rings and the β-orientation of OH-3, CH3-19,
CH3-18 and the C-17 side chain. Additionally, the NOESY correlations
of H-22/H-12β, H-22/H3-18 and H-23/H3-18 indicated the relative
position of lactone ring E and the β-orientation of H-23. The relative
configurations of C-24 and C-28 were deduced from the NOESY cor-
relations of H-23/H-25, H-23/H-26, H-22/H-28, H-22/H-29, H-23/H-
28 and H-23/H-29.
C
29H42O5 based on its HR-ESI-MS data with a pseudo molecular ion at
m/z 493.2921 [M+Na]+ in the positive mode, corresponding to 9 in-
dices of hydrogen deficiency. The absorption bands at 3411, 1738, and
1641 cm−1 in its IR spectrum suggested the presence of hydroxy
groups, an ester carbonyl and olefinic functionalities. The 1H NMR
spectrum shows three olefinic protons [δH 7.52 (1H, t, J = 1.4 Hz), 5.53
(1H, d, J = 6.5 Hz) and 5.46 (1H, br. s)], an isopropyl group [1.03 (3H,
d, J = 7.1) and 0.99 (3H, d, J = 7.0)], two angular methyls [0.91 (3H,
s) and 0.47 (3H, s)] and another methyl [1.30 (3H, d, J = 6.6 Hz)]. The
13C NMR and DEPT spectra exhibited 29 carbon resonances, including
the signals characteristic of a carbonyl [δC 176.5], six olefinic carbons
[(δC151.9, 145.5, 137.3, 134.8, 121.9, and 119.6)] and five methyl
carbons [δC 19.8, 18.7, 18.7, 18.6, and 13.0]. The above analysis in-
dicated that 1 was a derivative of a stigmastane-type steroid. In addi-
tion, the characteristic UV absorptions at 234, 243, and 250 nm sug-
gested the existence of a unique conjugated Δ7,9(11) moiety in the
steroid core, which are present in Δ7,9(11) stigmastane-type steroids,
which was further confirmed by the HMBC correlations (Fig. 2). The
The absolute configuration of C-3 was determined by a modified
Mosher's method (Tian et al., 2016; Ohtani et al., 1991). The Δδ values
between the (S) and (R)-MTPA esters indicated the S configuration for
C-3 (Fig. 4). In addition, the absolute configurations of the vic-diols at
C-24 and C-28 were determined by the in situ dimolybdenum CD
method (Bari et al., 2001; Marcin et al., 2007; Liu et al., 2010). Ac-
cording to the empirical rule proposed by Snatzke, the negative Cotton
effect at approximately 400 nm (Fig. 5) observed in the induced CD
spectrum permitted the assignment of the absolute configuration of the
vic-diol as 24S, 28R. Consequently, the absolute configuration of 1 was
established as 3S, 5R, 10S, 13S, 14R,17S, 23R, 24S, 28R.
Veramyoside B (2), an amorphous white powder, gave the mole-
cular formula C35H52O10, corresponding to 10 degrees of unsaturation,
as revealed by its HR-ESI-MS signal at m/z 655.3441 [M + Na]+
(calcd. 655.3453). Both the 1D and 2D NMR data of 2 were similar to
those of 1, except for the presence of a glucosyl group [δC 102.6,79.1,
78.9, 75.8, 72.1, and 63.3], which accounted for the additional one
degree of unsaturation. (Fig. 2). The β orientation of the anomeric
proton was assigned based on the J1H,2H
coupling constant
value
(J = 7.7 Hz) (Ma et al., 2016). The absolute configuration of the sugar
moiety was determined as β-D-glucose by HPLC analysis of its deriva-
tive after acid hydrolysis (Ye et al., 2017; Tanaka et al., 2007; Niu et al.,
2017). The HMBC correlation from H-1′ to C-3 indicated that the β-D
glucosyl group was linked to C-3 of the core structure, which was
Fig. 2. Key HMBC correlations of 1–11.
68

X. Liu et al.
Phytochemistry158(2019)67–76
The ECD curve of 2 is a highly consistent with that of biogenetically
related compound 1, which indicates that these two compounds have
the same absolute configuration (Fig. 7). Therefore, the absolute con-
figuration of 2 was defined as 3S, 5R, 10S, 13S, 14R,17S, 23R, 24S, 28R.
Veramyoside C (3) was isolated as a white solid. Its molecular for-
mula was determined as C37H54O12 according to its molecular ion [M
+Na]+ at m/z 713.3415 in the HR-ESI-MS. Extensive analysis of its
NMR data revealed that 3 was closely related to 2 except for an addi-
tional acetoxy substituent [δC 171.1 and 21.3] at C-16, which was
further confirmed by the obvious downfield shift of C-16 (δC 77.9) and
the HMBC correlations from H-16 (δH 6.03) to C-1′ (δC 171.1) and from
H3-2′′ (δH 1.98) to C-1′ (δC 171.1). According to the NOESY spectrum,
the relative configurations of C-3, C-5, C-10, C-13, C-14, and C-17 in 3
were identical to those of 2. In addition, the NOESY correlations of H-
22/H3-18 and H-22/H-16β suggested the relative position of the D/E
rings and the α-orientation of the acetoxy unit. The NOESY cross-peaks
between H-23 and H3-18 indicated that H-23 was β-oriented. In addi-
tion, the relative configurations of C-24 and C-28 were deduced by the
NOESY correlations (Fig. 3) of H-23/H-28, H-22/H3-29, H-23/H3-26
and H-25/H3-29. The experimental ECD curves of 3 and 2 showed the
same Cotton effects (Fig. 7), which allowed the assignment of the ab-
solute configuration of 3 as 3S, 5R, 10S, 13S, 16R, 17R, 23S, 24R, 28S.
Veramyoside D-F (4–6) were obtained as amorphous white powders
with the same molecular formula, C35H50O10, identified by their HR-
ESI-MS data. The IR, UV, and NMR data of 4–6 were identical to those
of known compound 12, which has the same planar structure, and this
assignment was further confirmed by their 1H−1H COSY and HMBC
spectra (Fig. 2). A thorough comparison of the NMR data of these four
compounds suggested that they were stereoisomers with variations in
the configurations at C-23 and C-24, as proved by the differences in the
NOESY correlations of their side chains. In the NOESY spectrum of 4,
the correlations of H3-27/H-23/H3-18/H-22/H-25 and H3-29/H-17 al-
lowed the determination of the relative configurations of C-23 and C-24
as (23R*, 24R*), as shown in Fig. 3. The relative configurations of C-23
and C-24 in 5 were established as (23S*, 24S*) by the NOESY corre-
lations of H3-18/H-22/H-16β, H3-18/H-23/H3-26 and H-25/H-22/H3-
29. Similarly, the NOESY correlations of H3-18/H-23, H3-18/H-22/H-
25 and H3-27/H-23/H3-29 in 6 indicated the relative configurations of
C-23 and C-24 as 23R*, 24S* (Fig. 3). The relative configuration of
these two chiral carbons in known compound 12 was reported by Wang
J as 23S*, 24R* (Wang et al., 2018). Thus, these four compounds ac-
counted for all the possibilities of the configurations of C-23 and C-24:
(23R*, 24R*)-4; (23R*, 24S*)-5; (23S*, 24S*)-6; and (23S*, 24R*)-12.
The absolute configuration of 4 was determined by calculating its
ECD curve. Based on the deduced relative configurations, the ECD
spectra of (3S, 5R, 10S, 13S, 17S, 23R, 24R)-4 was calculated. As a
result, the overall pattern of its experimental ECD curve was highly
consistent with the calculated curve. Thus, the absolute configuration of
4 was assigned as 3S, 5R, 10S, 13S, 17S, 23R, 24R. Additionally,
HO
HO
H
O
-0.0038
-0.0157H
H
O
H
-0.0292
H
-0.0798
H
-0.0651
H
H
H
0.0078
RO
H0.0449
H
H
0.0280 H
H
0.0393
0.0013
0.0713
values (in ppm) =
R (A-B)
S
Fig. 4. Δδ values (in ppm) = δS – δR for the (S)- and (R)-MTPA esters (A and B)
of 1.
Fig. 5. Mo2(OAc)4-induced ECD spectrum of 1 and a Newman projection of the
diol moiety of 1, with the helicity rule applied.
further confirmed by the glycosidation shifts in the resonances of C-2
(δC 30.5) and C-4 (δC 34.9) (Fig. 2). The relative configurations of the
chiral centers at C-3, C-5, C-10, C-13, C-14, C-17, C-23, C-24, and C-28
in 2 were consistent with those of 1 on the basis of their NOESY data.
Expt. ECD of
4
Expt. ECD of 9
Expt. ECD of 10
Calcd. ECD of 10
Calcd. ECD of
4
Calcd. ECD of 9
30
25
20
15
10
5
15
10
5
16
14
12
10
8
0
6
0
4
-5
-10
-15
2
-5
-10
-15
0
-2
200
250
300
350
400
200
250
300
350
400
200
250
300
350
400
wavelength nm
wavelength nm
wavelength nm
Fig. 6. Experimental and calculated ECD spectra of 4, 9 and 10.
69

X. Liu et al.
Phytochemistry158(2019)67–76
1
9
2
3
4
5
6
7
8
10
11
13
14
30
20
15
10
5
10
12
0
0
-5
-10
-15
-10
-20
200
250
300
350
400
200
250
300
350
400
wavelength nm
wavelength nm
Fig. 7. Comparison of the experimental ECD spectra of 1–14.
considering the biogenetic relationship among these compounds, the
absolute configurations of 5, 6 and 12 were determined as (3S, 5R, 10S,
13S, 17S, 23S, 24S), (3S, 5R, 10S, 13S, 17S, 23R, 24S) and (3S, 5R, 10S,
13S, 17S, 23S, 24R), respectively, based on a comparison of their
Cotton effects with the ECD spectrum of 4 (Fig. 7).
indicated that 10 was structurally similar to 9. The obvious upfield shift
of C-16 (δC 75.0) in 10 indicated that the carbonyl at C-16 in 9 had been
converted to a hydroxy group. This conjecture was supported by the
1H-1H COSY correlations of H-15/H-16 and H-16/H-17 (Fig. 2). The
relative configurations of C-20, C-23 and C-24 in the side chain of 10
were identical to those of 9 based on the similarities in their NOESY
spectra (Fig. 3). The α-orientation of 16-OH was deduced by the NOESY
correlation between H-20 and H-16. According to the deduced relative
configurations, the ECD curve of (3S, 5R, 10S, 13S, 14R, 17R, 20R, 23S,
24S)-10 was calculated for. As a result, the overall pattern of the cal-
culated and experimental ECD spectra of 10 show the same Cotton ef-
fect. (Fig. 6). Thus, the absolute configuration of 10 was determined to
be 3S, 5R, 10S, 13S, 14R, 17R, 20R, 23S, 24S.
Veramyoside H (7) and veramyoside I (8), amorphous white pow-
ders, gave the same molecular formula, C35H50O11, based on their HR-
ESI-MS data, with an additional oxygen compared with the molecular
formula (C35H50O10) of 4–6. Careful comparison of their NMR data
revealed that 7 and 8 were structural analogues of 4–6 except for the
hydrogen at C-16 in 4–6 was substituted by a hydroxyl group in 7 and 8
based on the obvious downfield shift of C-16 (δC 76.0) in 7 and C-16 (δC
74.3) in 8. Their planar structures were further confirmed by their
1H-1H COSY and HMBC spectra (Fig. 2). The NOESY spectra (Fig. 3) of 7
and 8 both showed correlations of H3-18/H-22/H-16 suggesting that H-
16 in each of these two compounds was β-oriented. The relative con-
figurations of C-23 and C-24 in 7 and 8 were deduced as (23S*, 24R*)-7
and (23R*, 24S*)-8 from their NOESY spectra (Fig. 3), and these con-
figurations are identical to 6 and 12, respectively. The CD spectra of 7
and 8 showed the same Cotton effects as those of biogenetically related
compound 4 (Fig. 7). Thus, the absolute configurations of 7 and 8 were
unambiguously determined as 3S, 5R, 10S, 13S, 14R, 16R, 17S, 23S,
24R and 3S, 5R, 10S, 13S, 14R, 16R, 17S, 23R, 24S, respectively.
Veramyoside J (9) was isolated as an amorphous white powder,
with the same molecular formula (C35H50O11) as 7 and 8 as deduced
from its HR-ESI-MS data (molecular ion [M + Na]+ detected at m/z
653.3204). The NMR data of 9 were identical to those of 7 and 8 except
for the obvious upfield shift of C-20 (δC 38.2) and C-22 (δC 27.4) as well
as a significant downfield shift of C-16 (δC 214.6), which suggested the
hydrogenation of the C]C bond between C20 and C22 as well as the
oxidization of 16-OH to a carbonyl. These assignments were further
confirmed by the 1H-1H COSY correlations of H-20/H-22 and the HMBC
correlations from H-17/H-20 to C-16 (Fig. 2). The relative configuration
of 9 was determined from its NOESY spectrum (Fig. 3). The β-orienta-
tions of H-20 and H-23 were deduced from the correlations of H3-18/H-
20 and H-20/H-23. The relative configuration of C-24 was assigned
based on the NOESY correlations of H-23/H3-27, H-23/H-25, H3-18/H-
22β, H-22α/H3-29 and H-22α/24-OH. Based on the deduced relative
configurations, the ECD curve of (3S, 5R, 10S, 13S, 14R, 20R, 23S,
24S)-9 was calculated, which matched well with the experimental ECD
curve of 9 (Fig. 6). Consequently, the absolute configuration of the 9
was defined as 3S, 5R, 10S, 13S, 14R, 20R, 23S, 24S.
Veramyoside L (11) was obtained as an amorphous white powder
with the molecular formula C37H54O12 as determined from its HR-ESI-
MS data. Analyses of the NMR data of 10 and 11 indicated they had
similar planar structures, except for the presence of an additional acetyl
group in 11. The 1H−1H COSY correlations of H-15/H-16 and H-16/H-
17, in conjunction with the HMBC cross-peaks from H-16 to δC 171.2
suggested that the additional acetyl group was linked to C-16 of the
core structure (Fig. 2). The relative configuration of 11 was identical to
10 based on their NOESY spectra (Fig. 3). Their consistent CD data
combined with the biogenetic relationship between 11 and 10 sug-
gested they shared the same absolute configuration (Fig. 7). Conse-
quently, the absolute configuration of 11 was assigned as 3S, 5R, 10S,
13S, 14R, 17R, 20R, 23S, 24S.
The CD spectra of the isolated compounds could generally be se-
parated into two groups (Fig. 7), which could be attributed to the dis-
tinguishing lactone unit as well as the different configurations and
conformations in the side chains. According to the octant-sector rule
(Zhang et al., 2018), Compounds 1–8 and 12 showed the first CD
pattern with Cotton effects at 220 nm (positive) and 240 nm (negative),
which were presumed to arise from the π → π* electronic transition of
the α, β-unsaturated lactone. Another ECD pattern, with a positive
Cotton effect at 240 (compounds 9–11 and 13–14) may be primarily
attributed to the lactone moiety in the side chain. Compared with the
CD spectra of 10–11 and 13–14, the synergistic Cotton effects at
300 nm (negative) in the CD spectrum of 9 would be due to the n → π*
electronic transition of the additional carbonyl at C-16. (Fig. 8).
2.2. RXRα and NF-κB transcriptional activities
Veramyoside K (10), an amorphous white powder, exhibited a
molecular at ion m/z 647.3459 [M-H]- in its HR-ESI-MS data, which
suggested a molecular formula of C35H52O11. Its 1H and 13C NMR data
Retinoid X receptor-α (RXRα), a unique intracellular target for
pharmacologic interventions, regulates diverse biologic processes and
can be considered an intriguing target for cancer therapies (Bloch,
70

X. Liu et al.
Phytochemistry158(2019)67–76
9
associated with Bcl-2 and execute proapoptotic effects (Cao et al., 2004;
Zhou et al., 2014). Nuclear factor κB (NF-κB) is a family of transcription
factors, that are also involved in the development and progression of
cancer (Dolcet et al., 2005). TNFα can stimulate the NF-κB and Akt
pathways to enhance cell survival and provoke cancer cell apoptosis by
activating the caspase-8–mediated death pathway under certain cir-
cumstances (Croft et al., 2013). Recently, studies have shown that some
ligands of RXRα can inhibit TNFα activation of NF-kB and promote
cancer cell apoptosis. Thus, RXRα and NF-κB could be considered
therapeutic targets for the treatment of cancer (Zeng et al., 2015). All
isolated compounds were investigated for their effects on the tran-
scription of RXRα and NF-κB. Compounds 2, 3, 4, 5 and 6 could inhibit
the 9-cis induced transcription of RXRα significantly compared with
what was seen in the 9-cis group (*P < 0.05) (Fig. 9). In addition,
compared with the TNF-α group, compounds 5 and 6 inhibited the NF-
κB pathway according to the dual luciferase reporter assay, and their
(-)
10
(+)
(-)
15
10
5
O
O
Me
R
R=
OH
O
H
O
(+)
0
-5
-10
*
n
-15
200
250
300
350
400
wavelength nm
Fig. 8. Comparison of the experimental ECD spectra of 9 and 10, and applying
the octant rule to 9 accounts for the negative Cotton effect at approximately
300 nm.
effects were significant different (*P
< 0.01) (Fig. 10). The pre-
liminary structure-activity relationship analysis suggested that the
Δ7,9(11) stigmastane-type steroids with α, β-unsaturated lactone moieties
in their side chains, except for compounds 7–8 with a hydroxyl group
substituent at C-16, were more effective than compounds bearing a
lactone moiety.
293T cell
40
3. Conclusion
*
30
In conclusion, eleven undescribed stigmastane-type steroids, ver-
amyosides A-J (1–11), along with three known homologues (12–14)
were isolated from the twigs of V. amygdalina. Compounds 1–11 fea-
tured a Δ7,9(11) stigmastane-type steroid skeleton with a unique con-
jugated Δ7,9(11) diene segment and highly oxygenated side chains with a
γ-lactone or an α, β-unsaturated five-membered lactone ring. Due to the
flexible groups and multiple chiral centers in the side chains, de-
termining the relative and absolute configurations of this type of
compound is still a great challenge. In this paper, we have used the in
situ dimolybdenum CD method, modified Mosher's method, quantum
chemical calculation of their ECD and CD comparison methods com-
bined with their biogenetic pathways to address these challenges. In
addition, the effects of the compounds on the transcription of RXRα and
NF-κB were evaluated. Compound 6 showed intriguing transcriptional
inhibitory activities on both RXRα and NF-κB, indicating this com-
pound may have anti-cancer properties.
20
*
*
***
*
***
10
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
con.
sample
sample and 9-cis-retinoic acid
Fig. 9. Inhibition and activation effects of the isolates on RXRα transcription.
Data are presented as the mean
pared with the 9-cis group.
SD, with *P < 0.05, ***P < 0.01 com-
293T cell
4. Experimental
20
15
10
5
4.1. General experimental procedures
Optical rotations were measured with a Rudolph Autopol IV/IV-T
automatic polarimeter (NJ, USA). UV spectra were recorded on a
Shimadzu UV-2600 UV–visible spectrophotometer (Shimadzu, Japan).
IR spectra were obtained on a Bruker ALPHA FT-IR spectrometer
(Bruker, Germany) with KBr pellets. NMR spectra were acquired on
Bruker Avance 600Ⅲ spectrometers (Bruker, Germany) with tetra-
methylsilane as the internal standard, using methanol-d4 or pyridine-d5
as solvents. HRESIMS experiments were conducted on a Thermo
***
***
Scientific
Q Exactive Quadrupole- Orbitrap mass spectrometer
(Thermo, USA). ECD spectra were acquired on a Jasco J-810 circular
dichroism spectrometer (Jasco, Japan) in methanol. Column chroma-
tography separations were performed on silica gel (200–300 mesh,
Qingdao Marine Chemical Inc., Qingdao, People's Republic of China),
Sephadex LH-20 (Amersham Pharmacia Biotech, Sweden), and ODS
(YMC, Japan). Analytical HPLC separations were performed with a
Shimadzu LC-20AT pump system (Shimadzu, Japan) with a DAD, and
semipreparative separations were performed with a Shimadzu LC-8A
pump system (Shimadzu, Japan) with a DAD. Fractions were monitored
by TLC (Qingdao Marine Chemical Inc., Qingdao, People's Republic of
China) and visualized under a UV lamp at 254 nm or 365 nm after
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14
con.
TNF-
Fig. 10. Inhibitory effect of the isolates against the NF-κB pathway. Data are
presented as the mean
group.
SD, with ***P < 0.01 compared with the TNF-α
1965). Some antagonists of RXRα can induce RXRα and Nur77 trans-
location from the nucleus to the cytoplasm, where they become
71

X. Liu et al.
Phytochemistry158(2019)67–76
spraying with 5% H2SO4 in ethanol, followed by heating.
1639, 1374, 1076, 1022 cm−1; ECD (MeOH) λmax (Δε) 219 (+3.50)
nm, λmax (Δε) 242 (−8.80) nm; 1H and 13C NMR data, see Table 1;
positive-ion HRESIMS [M +Na]+ m/z 655.3442 (calcd. for
4.2. Plant material
C35H52O10Na, 655.3453).
Tender stems of Vernonia amygdalina Delile (compositae)were col-
lected from Taiwan, China, in April 2016, and authenticated by
Professor Zhenji Li, College of the Environment Ecology, Xiamen
University. A voucher specimen (VA-308201604) has been deposited in
the School of Pharmaceutical Sciences, Xiamen University.
4.3.4. Veramyoside D (4)
Amorphous white powder; [ ]2D3.5-31.02 (c 1.87, CH3OH); UV
(MeOH) λmax (log ε) 235 (3.12) nm, λmax (log ε) 242 (3.09) nm, λmax
(log ε) 250 (2.90) nm; IR (KBr) νmax 3439, 2961, 2926, 2876, 1751,
1712, 1358, 1073, 1028 cm−1; ECD (MeOH) λmax (Δε) 217 (+3.92)
nm, λmax (Δε) 242 (−5.03) nm; 1H and 13C NMR data, see Table 1;
positive-ion HRESIMS [M +Na]+ m/z 653.3215 (calcd. for
4.3. Extraction and isolation
The dried, powdered tender stems of Vernonia amygdalina Del.
(5.5 kg) were soaked overnight in 40 L of 60% EtOH, then refluxed
three times in 60% EtOH for 2.5 hours each time. Concentrating the
filtrate under reduced pressure yielded crude extract (1073 g), and then
the crude extract was suspended in water and partitioned successively
C35H50O10Na, 653.3296).
4.3.5. Veramyoside E (5)
Amorphous white powder; [ ]2D3.4-31.14 (c 1.67, CH3OH); UV
(MeOH) λmax (log ε) 235 (2.83) nm, λmax (log ε) 242 (2.86) nm, λmax
(log ε) 250 (2.67) nm; IR (KBr) νmax 3439, 2961, 2926, 2878, 1753,
1711, 1364, 1076, 1029 cm−1; ECD (MeOH) λmax (Δε) 222 (+4.98)
nm, λmax (Δε) 241 (−4.90) nm; 1H and 13C NMR data, see Table 2;
positive-ion HRESIMS [M +Na]+ m/z 653.3204 (calcd. for
C35H50O10Na, 653.3296).
with petroleum ether (3
×
4 L), CH2Cl2 (3
×
4 L), EtOAc (3
×
4 L) and n-
BuOH (3
×
4 L). After removing the solvent, the CH2Cl2 extract (97 g)
was separated by silica gel column chromatography eluted with a
gradient of CH2Cl2-CH3OH (100:0 → 80:20, v/v) to yield eleven frac-
tions (Fr. 1-11). Fr. 4 (3.5 g) was subjected to a Sephadex LH-20 column
(MeOH) and then separated on a silica gel column eluted with CH2Cl2-
(CH3)2CO (100:1 → 5:1, v/v) to yield ten fractions (Fr. 4.1–4.10).
Among them, Fr. 4.5 (300.9 mg) was successively separated by a
Sephadex LH-20 column (MeOH) and an ODS column [CH3OH-H2O
(15:85 → 100:0, v/v)], and finally purified by prep-HPLC (CH3OH-H2O,
80:20, v/v) to afford compound 1 (2 mg). Fr. 8(16.2 g) was further
separated by silica gel column chromatography [CH2Cl2-CH3OH
(99:1 → 80:20, v/v)] to yield nine fractions (Fr. 8.1–8.9). Then Fr. 8.8
(12.7 g) was separated into twelve fractions (Fr. 8.8.1–8.8.12) by ODS
column chromatography [CH3OH-H2O (50:50, v/v)] and Fr. 8.8.3
(588.8 mg) was purified by repeated prep-HPLC [CH3OH-H2O (60:40,
v/v)] separations to afford compounds 3 (6.0 mg), 7 (10.7 mg) and 8
(1.9 mg). Fr. 8.8.5 (664.5 mg) separated by prep-HPLC [CH3OH-H2O
(60:40, v/v); CH3CN-H2O (35:65, v/v)] to yield 9 (26.9 mg), 10
(5.2 mg) and 13 (32.8 mg). Furthermore, Fr. 8.8.7 (195.0 mg) was
purified by repeated prep-HPLC [CH3OH-H2O (60:40, v/v)] separations
to afford 4 (2.8 mg) and 5 (2.5 mg). Fr. 8.8.8 (195.0 mg) was purified by
prep-HPLC with [CH3OH-H2O (60:40, v/v) to afford 2 (30.3 mg) and by
prep-HPLC with [CH3CN-H2O (35:65, v/v)] to obtain 11 (5.0 mg) and
14 (30.0 mg). Fr. 8.8.12 (271.9 mg) was purified by prep-HPLC
[CH3OH-H2O (70:30, v/v)] to afford 6 (2.0 mg) and 12 (2.4 mg).
4.3.6. Veramyoside F (6)
Amorphous white powder; [ ]2D3.99.02 (c 1.33, CH3OH); UV (MeOH)
λ
max (log ε) 235 (3.11) nm, λmax (log ε) 242 (3.10) nm, λmax (log ε) 250
(2.90) nm; IR (KBr) νmax 3439, 2926, 2878, 1753, 1711, 1363, 1077,
1029 cm−1; ECD (MeOH) λmax (Δε) 223 (+4.58) nm, λmax (Δε) 245
(−2.14) nm; 1H and 13C NMR data, see Table 2; positive-ion HRESIMS
[M+Na]+ m/z 653.3299 (calcd. for C35H50O10Na, 653.3296).
4.3.7. Veramyoside G (7)
Amorphous white powder; [ ]2D3.7-17.98 (c 1.78, CH3OH); UV
(MeOH) λmax (log ε) 235 (3.18) nm, λmax (log ε) 242 (3.17) nm, λmax
(log ε) 250 (2.96) nm; IR (KBr) νmax 3440, 2931, 2882, 1753, 1713,
1362, 1071, 1045 cm−1; ECD (MeOH) λmax (Δε) 223 (+9.62) nm, λmax
(Δε) 244 (−4.92) nm; 1H and 13C NMR data, see Table 2; positive-ion
HRESIMS [M+Na]+ m/z 669.3250 (calcd. for C35H50O10Na,
669.3245).
4.3.8. Veramyoside H (8)
Amorphous white powder; [ ]2D3.8-36.22 (c 1.27, CH3OH); UV
(MeOH) λmax (log ε) 235 (3.40) nm, λmax (log ε) 242 (3.39) nm, λmax
(log ε) 250 (3.19) nm; IR (KBr) νmax 3440, 2964, 2935, 2879, 1753,
1709, 1358, 1077 cm−1; ECD (MeOH) λmax (Δε) 223 (+6.95) nm, λmax
(Δε) 245 (−5.08) nm; 1H and 13C NMR data, see Table 2; positive-ion
HRESIMS [M+Na]+ m/z 669.3249 (calcd. for C35H50O10Na,
669.3245).
4.3.1. Veramyoside A (1)
Amorphous white powder; [ ]2D5.7-40.00 (c 1.30, CH3OH); UV
(MeOH) λmax (log ε) 235 (3.41) nm, λmax (log ε) 242 (4.04) nm, λmax
(log ε) 250 (4.04) nm; IR (KBr) νmax 3411, 2962, 2930, 2878, 1738,
1378, 1096, 1053, 1029 cm−1; ECD (MeOH) λmax (Δε) 218 (+0.20)
nm, λmax (Δε) 241 (−4.64) nm; 1H and 13C NMR data, see Table 1;
positive-ion HRESIMS [M +Na]+ m/z 493.2921 (calcd. for
C29H42O5Na, 493.2924).
4.3.9. Veramyoside H (9)
Amorphous white powder; [ ]2D3.752.63 (c 1.33, CH3OH); UV
(MeOH) λmax (log ε) 235 (2.69) nm, λmax (log ε) 242 (2.74) nm, λmax
(log ε) 250 (2.55) nm; IR (KBr) νmax 3505, 3413, 2951, 2930, 2906,
1758, 1709, 1353, 1086, 1042 cm−1; ECD (MeOH) λmax (Δε) 241
(+1.00) nm, λmax (Δε) 298 (−0.89) nm; 1H and 13C NMR data, see
Table 3; negative-ion HRESIMS [M-H]+ m/z 645.3304 (calcd. for
4.3.2. Veramyoside B (2)
Amorphous white powder; [ ]2D5.7-23.40 (c 4.10, CH3OH); UV
(MeOH) λmax (log ε) 235 (3.21) nm, λmax (log ε) 242 (3.20) nm, λmax
(log ε) 250 (2.98) nm; IR (KBr) νmax 3422, 2937, 2877, 2834, 1725,
1639, 1374, 1076, 1022 cm−1; ECD (MeOH) λmax (Δε) 219 (+3.50)
nm, λmax (Δε) 242 (−8.80) nm; 1H and 13C NMR data, see Table 1;
positive-ion HRESIMS [M +Na]+ m/z 655.3442 (calcd. for
C35H52O10Na, 655.3453).
C35H49O11, 645.3269).
4.3.10. Veramyoside I (10)
Amorphous white powder; [ ]2D5.012.77 (c 1.41, CH3OH); UV
(MeOH) λmax (log ε) 235 (2.66) nm, λmax (log ε) 242 (2.69) nm, λmax
(log ε) 250 (2.52) nm; IR (KBr) νmax 3501, 3413, 2950, 2906, 2882,
1756, 1709, 1359, 1088, 1025 cm−1; ECD (MeOH) λmax (Δε) 239
(+2.86) nm; 1H and 13C NMR data, see Table 3; positive-ion HRESIMS
[M+Na]+ m/z 671.3315 (calcd. for C35H52O11 Na, 671.3402).
4.3.3. Veramyoside C (3)
Amorphous white powder; [ ]2D5.0-21.67 (c 1.20, CH3OH); UV
(MeOH) λmax (log ε) 235 (2.98) nm, λmax (log ε) 242 (2.88) nm, λmax
(log ε) 250 (2.69) nm; IR (KBr) νmax 3422, 2937, 2877, 2834, 1725,
72

X. Liu et al.
Phytochemistry158(2019)67–76
Table 1
1H (600 MHz) and 13C NMR (150 MHz) Data for Compounds 1–4 (δ in ppm, J in Hz).
No.
1a
2b
3b
4b
δC
δH
δC
δH
δC
δH
δC
δH
1α
1β
36.0
32.3
1.34, dd (13.0, 3.5)
1.97, t (3.5)
1.85, br. d (13.0)
1.46, br. d (13.0)
3.51, m
35.4
30.5
1.23, dd, (13.6, 2.9)
1.82, overlap
2.13, m
35.3
30.5
1.21, overlap
1.80, m
2.14, m
1.69, m
3.97, m
2.02, m
1.39, m
1.31, m
1.80, m
35.3
30.5
1.21, m
1.80, m
2.13, m
1.70, m
3.97, m
2.04, m
1.41, m
1.31, m
1.81, m
2α
2β
1.71, m
3
71.4
38.5
77.3
34.9
3.98, m
77.3
34.8
77.3
34.9
4α
4β
1.69, m
2.04, m
1.29, overlap
1.42, m
1.41, q (12.4)
1.32, m
5
40.6
31.0
39.5
30.6
39.5
30.4
39.5
30.5
6α
6β
1.31, m
1.82, overlap
1.92, m
7
121.9
137.3
145.5
37.0
5.46, br. s
121.5
136.6
144.7
36.6
5.38, br. s
122.0
135.3
144.7
36.6
5.33, br. s
121.6
136.4
144.7
36.5
5.37, m
8
9
10
11
12α
12β
13
14
15α
15β
16α
16β
17
18
19
20
21
22
23
24
25
26
27
28
29
16-AcO
119.6
41.4
5.53, d (6.5)
2.26, d, (17.3)
2.02, m
119.2
41.1
5.49, d (6.0)
2.48, m
118.6
40.8
5.44, br. s
2.54, d (17.2)
2.23, m
119.0
40.9
5.44, m
2.44, m
2.19, m
2.29, dd (17.4, 6.0)
45.0
52.6
24.5
44.5
51.9
24.1
44.7
49.8
33.1
44.6
51.9
24.1
2.37, m
1.93, m
1.63, m
2.03, m
2.34, m
2.68, m
2.33, overlap
1.85, m
1.82, m
2.25, m
1.56, dd (11.6, 5.9)
2.01, m
1.93, m
1.55, m
27.7
27.1
77.9
6.03, t (8.0)
27.1
1.87, m
1.91, m
47.9
13.0
19.9
134.8
176.5
151.9
84.4
79.7
33.3
18.7
18.7
71.3
18.6
2.65, t, (9.9)
0.47, s
47.7
13.1
19.8
136.6
175.0
151.2
83.8
79.5
33.2
19.2
19.3
70.9
19.5
2.79, t (11.3)
0.57, s
55.0
14.1
19.8
131.4
174.8
152.8
83.9
79.7
33.0
19.0
19.1
70.6
19.4
171.1
21.3
102.7
75.7
79.1
72.1
78.9
63.3
3.15, d (8.0)
0.62, s
47.6
13.1
19.8
136.9
173.9
147.5
83.9
85.0
34.2
17.6
17.5
211.3
28.1
2.74, t (9.5)
0.54, s
0.91, s
0.81, s
0.76, s
0.80, s
7.52, t (1.4)
7.86, s
5.78, s
8.05, s
5.77, s
7.57, overlap
5.65, s
5.33, dd, (1.4, 0.9)
2.11, quint (7.1)
0.99, d (7.1)
1.03, d (7.1)
3.92, q (6.6)
1.30, d (6.6)
2.49, m
2.47, m
2.49, m
1.26, d (7.0)
1.26, d (7.0)
4.44, m
1.21, d (6.4)
1.21, d (6.4)
4.41, m
1.02, d (7.0)
1.21, d (7.0)
1.61, d (6.5)
1.58, d (6.4)
2.33, s
1.98, s
1′
2′
3′
4′
5′
6′
102.6
75.8
79.1
72.1
78.9
63.3
5.04, d (7.6)
4.07, t (7.6)
4.32, m
5.04, d (8.0)
4.07, t (8.0)
4.32, m
102.6
75.7
79.0
72.1
78.9
63.3
5.03, overlap
4.07, m
4.32, m
4.29, m
4.29, m
4.28, m
4.01, m
4.02, m
4.01, m
4.60, br. d (11.3)
4.44, m
4.61, br. d (11.6)
4.44, m
4.60, d (11.7)
4.43, dd (11.7, 5.3)
a
b
Recorded in methanol-d4.
Recorded in pyridine-d6.
4.3.11. Veramyoside J (11)
mobile phase (25:75, v/v); detection wavelength of 254 nm; 0.8 mL/
min). The sugar in compound 2 was identified as D-glucose in (tR
18.370 min) [authentic samples, D-glucose (tR, 18.325 min) and L-glu-
cose (tR, 16.820 min)].
Amorphous white powder; [ ]2D5.74.24 (c 3.30, CH3OH); UV (MeOH)
max (log ε) 235 (3.02) nm, λmax (log ε) 242 (3.06) nm, λmax (log ε) 250
λ
(2.88) nm; IR (KBr) νmax 3424, 2964, 2934, 2881, 1769, 1729, 1639,
1379, 1250, 1077, 1029 cm−1; ECD (MeOH) λmax (Δε) 243 (+2.53)
nm; 1H and 13C NMR data, see Table 3; positive-ion HRESIMS [M
+Na]+ m/z 713.3510 (calcd. for C37H54O12 Na, 713.3507).
4.5. Snatzke's method to identify the absolute configuration of the vic-diols
in 1
4.4. General procedure for acid hydrolysis determine of the absolute
configuration of the sugars
Based on the reported procedure (Bari et al., 2001), the sample and
Mo2(OAc)4 were mixed in dry DMSO at a ratio of 1:1.25, and the CD
spectrum was immediately measured. CD curves were recorded every
10 min until a stationary ICD curve was obtained (after approximately 1
hour). Then, the inherent CD was subtracted, and the Cotton effects at
approximately 310 nm and 400 nm were analyzed as the diagnostic
bands, and this bands are closely correlated with the absolute config-
uration of the vic-diol moiety.
Compound 2 (10.0 mg) was hydrolyzed in 1 M HCl (14.0 mL) by
heating at 80 °C for 4 hours. The mixture was extracted with water
saturated dichloromethane to remove the nonpolar components. The
remaining solution was concentrated and dissolved in pyridine and
reacted with L-cysteine methyl ester hydrochloride (5.0 mg) at 60 °C for
1 h. Then, O-tolylisothiocyanate (500 μL) was added, and the mixture
was reacted at 60 °C for an additional 1 h. The reaction product was
analyzed by HPLC (SilGreen C18 5 μm, 4.6 × 250 mm; CH3CN−H2O
73

X. Liu et al.
Phytochemistry158(2019)67–76
Table 2
1H (600 MHz) and 13C NMR (150 MHz) Data for Compounds 5–8 (δ in ppm, J in Hz).
No.
5a
6a
7a
8a
1α
1β
2α
2β
3
35.4
30.5
1.21, m
1.79, m
2.13, m
1.70, m
3.96, m
2.03, m
1.40, m
1.31, m
1.82, m
5.40, overlap
35.4
30.5
1.24, br. d (13.0)
1.82, overlap
2.14, m
35.2
30.5
1.20, overlap
1.80, m
35.3
30.5
1.22, m
1.82, overlap
2.13, br. d (11.4)
1.69, m
2.12, br. d (11.5)
1.68, m
1.70, m
77.3
34.9
77.3
34.9
3.97, m
77.3
34.8
3.95, m
77.3
34.9
3.96, m
4α
4β
5
2.03, br. d (12.0)
1.40, q (12.0)
1.31, m
2.01, br. d (12.0)
1.39, m
2.02, br. d (12.4)
1.39, q (12.4)
1.30, m
39.5
39.5
39.4
1.27, m
39.5
30.6
6
30.6
30.6
1.81, m
30.4
1.78, m
1.80, m
7
121.5
135.6
144.7
36.6
121.6
136.5
144.8
36.6
5.38, br. s
121.7
135.8
144.6
36.5
5.39, overlap
121.7
136.0
144.8
36.6
5.41, br. s
8
9
10
11
12α
12β
13
14
15α
15β
16α
16β
17
18
19
20
21
22
23
24
25
26
27
28
29
1′
119.1
40.7
5.41, overlap
2.44, overlap
2.14, m
119.1
40.9
5.49, br. d (5.5)
2.44, overlap
118.7
40.9
5.40, overlap
118.8
41.0
5.48, br. d (6.1)
2.50, overlap
2.34, m
2.54, d (17.0)
2.30, dd (17.0,5.5)
2.07, dd (17.0, 6.6)
44.7
52.0
24.2
44.7
51.9
24.2
44.9
50.2
35.9
45.3
49.9
36.2
2.37, m
1.87, m
1.62, m
1.95, m
2.33, m
2.91, t (8.6)
2.24, m
2.91, t (8.6)
2.29, m
1.82, overlap
1.58, m
2.25, m
27.7
26.9
2.12, m
76.0
5.01, overlap
74.3
5.35, t (7.2)
1.94, m
47.3
12.8
19.8
136.6
174.7
147.3
85.2
85.6
36.0
17.6
18.1
213.3
29.5
102.7
75.7
79.0
72.1
78.9
63.3
2.81, t (9.7)
0.76, s
47.8
13.1
19.8
136.3
174.4
147.9
85.1
85.9
35.8
17.6
18.1
213.2
29.4
102.7
75.8
79.1
72.1
78.9
63.3
2.77, t, (9.7)
0.58, s
58.5
14.2
19.7
136.0
174.4
147.5
84.0
84.8
33.9
17.3
17.4
211.3
28.2
102.6
75.7
79.0
72.1
78.9
63.2
3.19, d (7.5)
0.65, s
59.5
14.4
19.8
134.7
174.7
148.8
85.2
85.8
35.7
18.1
17.6
213.0
29.3
102.7
75.8
79.1
72.1
78.9
63.3
3.15, d (7.1)
0.71, s
0.80, s
0.80, s
0.78, s
0.79, s
7.31, s
5.60, s
7.42, s
5.71, s
7.73, s
7.57, overlap
5.66, s
5.38, m
2.46, overlap
1.16, d (6.5)
1.15, d (6.5)
2.51, m
2.46, m
2.47, overlap
1.13, d (7.0)
1.17, d (7.0)
1.19, d (6.8)
1.16, d (6.8)
1.17, d (6.8)
0.95, d (6.8)
2.44, s
2.47, s
2.32, s
2.42, s
5.03, overlap
4.06, m
5.04, d (7.7)
4.07, m
5.02, overlap
4.05, t (7.5)
4.29, m
5.03, d (8.0)
4.06, t (8.0)
4.31, m
2′
3′
4.32, m
4.32, m
4′
4.28, m
4.29, m
4.28, m
4.28, m
5′
4.00, m
4.01, m
3.99, m
4.01, m
6′
4.59, d (11.2)
4.43, m
4.60, br. d (10.4)
4.43, m
4.58, d (11.6)
4.41, m
4.60, d (11.2)
4.43, dd (11.2, 4.6)
a
Recorded in pyridine-d6.
4.6. Preparation of the (S)-MTPA (1a) and (R)-MTPA (1b) esters
4.8. RXRα transcriptional activities
Under the protection of nitrogen in NMR tubes, portions of 1
(0.5 mg) were dissolved in pyridine-d5 and separately reacted with (S)-
MTPA chloride (10 μL) and (R)-MTPA chloride (10 μL). The mixtures
were completely reacted at room temperature for 3–4 h to prepare the
(R)-MTPA ester (1b) and the (S)-MTPA ester (1a), respectively.
The RXRα transcriptional activities of the isolates were evaluated by
a dual-luciferase reporter assay. 293T cells were seeded at a density of
1 × 104 cells per well in 96-well plates. Then, the cells were transfected
with pGL5 luciferase reporter vector (30 ng/mL) and pGAL4-RXRα-LBD
vector (15 μg/mL) by Liposome 2000. After 12 hours, the cells were
incubated with 9-cis and the test compounds (50 μM) for 12 hours. The
luciferase activities were measured using the Dual-Luciferase Assay
System Kit (Promega) (Kotani et al., 2010).
4.7. Quantum chemical ECD calculation method
Relative luciferase activity (%) = FL/RL×100%
Monte Carlo conformational searches were carried out by means of
the Spartan's 9 software using the merck molecular force field (MMFF).
The conformers with a Boltzmann-population of over 5% were chosen
for ECD calculations, and the conformers were initially optimized at the
B3LYP/6-31 + g (d, p) level in MeOH using the conductor-like polar-
izable continuum model (CPCM). The theoretical calculations of the
ECD curves were carried out MeOH using time-dependent density
functional theory (TD-DFT) at the B3LYP/6-311 + g (d, p) level for all
conformers of compounds 4, 9 and 10. Rotatory strengths for a total of
50 excited states were calculated. ECD spectra were generated using the
program SpecDis 1.6 (University of Würzburg, Würzburg, Germany)
and GraphPad Prism 5 (University of California San Diego, USA) from
dipole-length rotational strengths by applying Gaussian band shapes
with sigma = 0.3 eV.
4.9. NF-κB transcriptional-inhibitory activities
The 293T cells were plated into 96-well plates at a concentration of
1 × 104 cells per well, and then the pGL4.32[luc2P/NF-ΚB-RE/Hygro]
vector (30 μg/mL) and pC-DNA-Rellina vector (15 ng/mL) were co-
transfected into the 293T cells. Twelve hours later, the test compounds
(50 μM) and TNF-α were added, and the cells were incubated for an
additional 6 hours. Then, the activities of Firefly luciferase (FL) and
Rellina luciferase (RL) were determined using the Dual-Luciferase
Reporter Assay System Kit (Promega).
Relative luciferase activity(%) = FL/RL×100%.
74

X. Liu et al.
Phytochemistry158(2019)67–76
Table 3
1H (600 MHz) and 13C NMR (150 MHz) Data for Compounds 9–12 (δ in ppm, J in Hz).
No.
9a
10a
11a
δC
12a
δC
δH
δC
δH
δH
δC
δH
1α
1β
2α
2β
3
35.2
30.4
1.24, m
35.3
30.6
1.20, m
1.81, m
2.12, m
1.70, m
3.95, m
2.01, m
1.40, m
1.28, m
1.80, m
5.38, br. s
35.4
30.6
1.19, t (13.2)
1.80, m
2.13, m
1.68, m
3.94, m
1.99, m
1.37, m
1.28, m
1.76, m
5.28, s
35.4
30.6
1.21, overlap
1.82, overlap
2.13, overlap
1.70, m
1.85, br. d (13.3)
2.16, m
1.72, m
77.3
34.8
3.98, m
77.3
34.9
77.4
34.9
77.3
34.9
3.97, m
4α
4β
5
2.04, br. d (12.0)
1.40, q (12.0)
1.28, m
2.03, br. d (11.0)
1.40, q (11.0)
1.32, m
39.5
39.5
39.6
39.5
6
30.4
1.77, m
30.5
30.5
30.5
1.81, overlap
5.37, br. s
7
123.0
133.8
145.1
36.7
5.25, br. s
121.3
135.6
144.5
36.5
122.0
135.4
144.7
36.6
121.6
136.5
144.7
36.6
8
9
10
11
12α
12β
13
14
15α
15β
16α
16β
17
18
19
20
21
22
118.1
40.3
5.52, br. d (5.7)
2.40, m
119.1
41.7
5.48, br. d (6.0)
2.73, dd (17.5, 6.0)
2.27, d (17.5)
118.7
41.2
5.44, d (5.5)
2.54, m
119.1
40.8
5.42, br. d (5.6)
2.44, overlap
2.11, overlap
2.28, m
2.24, m
41.3
46.5
38.0
43.6
49.6
36.4
43.4
49.3
33.5
44.5
52.0
24.1
2.60, m
2.40, m
2.12, m
2.78, m
2.12, m
2.52, m
2.36, overlap
1.86, m
2.11, m
1.84, m
1.58, m
214.6
75.0
4.59, br. d (9.8)
77.4
5.53, t (7.8)
27.7
1.96, m
1.90, m
63.1
14.4
19.8
38.2
178.4
27.4
3.00, br. s
0.66, s
60.6
14.2
19.8
40.7
178.9
28.5
2.43, overlap
0.72, s
56.6
14.1
19.9
40.0
178.5
27.9
2.50, m
0.63, s
0.76, s
3.08, m
47.4
2.77, t (9.3)
0.57, s
13.0
0.80, s
0.83, s
19.8
0.80, s
3.09, m
3.10, m
136.7
173.9
147.3
2.87, q (10.8)
2.31, m
3.11, m
2.70, m
2.42, overlap
5.15, m
2.24, m
7.51, s
23
81.6
84.6
35.7
18.0
17.9
213.8
30.0
5.12, q (5.6)
81.4
84.8
35.6
18.0
18.0
214.2
30.2
81.4
84.8
35.6
18.0
17.9
214.3
30.3
5.05, overlap
83.9
84.9
33.9
17.5
17.5
211.3
28.3
5.50, br. s
24
25
2.49, m
2.43, overlap
1.16, d (7.0)
1.15, d (7.0)
2.37, m
2.47, m
26
1.19, d (7.0)
1.14, d (7.0)
1.10, d (6.6)
1.10, d (6.6)
0.99, d (6.8)
1.20, d (6.8)
27
28
29
2.45, s
2.44, s
2.43, s
2.33, s
16-AcO
171.2
21.5
102.8
75.8
79.1
72.2
79.0
63.4
2.00, s
1′
2′
3′
4′
5′
6′
102.7
75.8
79.1
72.1
78.9
63.3
5.05, d (7.8)
4.08, t (7.8)
4.32, m
102.7
75.7
79.0
72.1
78.9
63.3
5.05, overlap
4.05, m
5.00, overlap
4.04, t (7.6)
4.28, m
102.7
75.8
79.1
72.1
78.9
63.3
5.04, d (7.7)
4.06, m
4.30, m
4.31, m
4.29, m
4.28, m
4.26, m
4.28, m
4.01, m
4.00, m
3.99, m
4.01, m
4.61, d (11.5)
4.43, m
4.59, br. d (10.0)
4.42, dd (10.0, 5.1)
4.57, d (11.8)
4.40, dd (11.8, 4.0)
4.60, br. d (11.0)
4.44, m
a
Recorded in pyridine-d6.
Conflicts of interest
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76