Lipid-Lowering Activities of Cucurbitacins Isolated from Trichosanthes cucumeroides and Their Synthetic Derivatives

Article abstract of DOI:10.1021/acs.jnatprod.0c00364

In the ongoing efforts to discover natural cholesterol-lowering compounds, dihydrocucurbitacin B, isolated from Trichosanthes cucumeroides roots, was found to promote LDL uptake by upregulating LDLR protein in a PCSK9-dependent process. In this study, an in-depth investigation of T. cucumeroides roots afforded 27 cucurbitacins (1-27), including seven new cucurbitacins (1-7), and their structures were elucidated by spectroscopic data analyses. In order to gain insight into their structure-activity relationship, cucurbitacin derivatives (B1-11 and DB1-11) were synthesized. Evaluation of lipid-lowering activities of these cucurbitacins by an LDL uptake assay in HepG2 cells revealed that most of the compounds improved the LDL uptake rate, among which hexanorisocucurbitacin D (6) and isocucurbitacin D (21) exhibited the highest activities (rates of 2.53 and 2.47, respectively), which were comparable to that of the positive control, nagilactone B (rate of 2.07). According to a mechanistic study by Western blot analysis, compounds 6 and 21 dose-dependently increased LDLR protein levels and reduced PCSK9 protein levels, representing promising new lipid-lowering drug candidates.

Full text of DOI:10.1021/acs.jnatprod.0c00364

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Article
Lipid-Lowering Activities of Cucurbitacins Isolated from
Trichosanthes cucumeroides and Their Synthetic Derivatives
Xianjing Zhang, Huihui Li, Wenqiong Wang, Tong Chen, and Lijiang Xuan*
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ABSTRACT: In the ongoing efforts to discover natural cholesterol-
lowering compounds, dihydrocucurbitacin B, isolated from Trichosanthes
cucumeroides roots, was found to promote LDL uptake by upregulating
LDLR protein in a PCSK9-dependent process. In this study, an in-depth
investigation of T. cucumeroides roots afforded 27 cucurbitacins (1−27),
including seven new cucurbitacins (1−7), and their structures were
elucidated by spectroscopic data analyses. In order to gain insight into their structure−activity relationship, cucurbitacin derivatives
(B1−11 and DB1−11) were synthesized. Evaluation of lipid-lowering activities of these cucurbitacins by an LDL uptake assay in
HepG2 cells revealed that most of the compounds improved the LDL uptake rate, among which hexanorisocucurbitacin D (6) and
isocucurbitacin D (21) exhibited the highest activities (rates of 2.53 and 2.47, respectively), which were comparable to that of the
positive control, nagilactone B (rate of 2.07). According to a mechanistic study by Western blot analysis, compounds 6 and 21 dose-
dependently increased LDLR protein levels and reduced PCSK9 protein levels, representing promising new lipid-lowering drug
candidates.
Therefore, a follow-up study of cucurbitacins from T.
cucumeroides roots was conducted. Cucurbitacins (1−27),
including seven new compounds (1−7), were identified, and
cucurbitacin derivatives (B1−11 and DB1−11) were synthe-
sized. Most of these compounds were evaluated for lipid-
lowering activity by the LDL uptake assay, and the effects of
two active compounds on LDLR and PCSK9 protein levels
were further tested by Western blot analysis.
ardiovascular disease (CVD) is the leading cause of
1
Cmortality worldwide.
Statins are the first-line drug
therapy in the treatment of hyperlipidemia or CVD and act
by lowering plasma low-density lipoprotein cholesterol (LDL-
C). Despite the efficacy of statin therapy, some patients still
face substantial residual risks associated with high levels of
LDL-C.
One reason for the continued high LDL-C levels is that
statins increase PCSK9 expression at higher drug doses, an
effect believed to attenuate a further reduction in LDL-C
levels.2 Hence, inhibition of PCSK9 to enhance the effects of
statins provides an alternative therapeutic option for patients
who need additional lowering of LDL-C. For instance, PCSK9
antibodies have been shown to be clinically beneficial.3
However, their drawbacks, such as high cost and inconvenient
subcutaneous administration, lead to the need for small, orally
available, and low-cost chemicals that regulate PCSK9.3
Recently, several compounds such as berberine and
curcumin have been reported to inhibit PCSK9 expression,
initiating a growing interest in discovering small molecules
with PCSK9-modulating activity derived from natural prod-
ucts.4,5
Dihydrocucurbitacin B, isolated from the roots of
Trichosanthes cucumeroides, was first reported by our group to
elevate LDL uptake in HepG2 cells.6 A mechanistic study
indicated that dihydrocucurbitacin B upregulated LDLR
expression by increasing SREBP2 protein levels and down-
regulated PCSK9 expression by decreasing HNF1α protein
levels.6 These findings initiated our interest in the structure−
activity relationships of cucurbitacins.
RESULTS AND DISCUSSION
■
Isolation and Structure Elucidation. Seven new
cucurbitacins (1−7) and 20 known cucurbitacins (8−27)
were obtained from the 95% EtOH extract of T. cucumeroides
roots using various chromatographic techniques.
Compound 1 was obtained as a white amorphous powder.
The molecular formula was determined as C31H46O8 based on
the 13C NMR data and HRESIMS ion at m/z 569.3080 [M +
Na]+ (calcd for C31H46O8Na, 569.3085). The 1H NMR
spectrum exhibited signals for nine methyl groups, a
hydroxymethine (δH 4.32), and an olefinic proton (δH 5.75).
The 13C NMR spectrum displayed 31 carbon signals including
nine methyls, six methylenes, four methines (one oxygenated),
three quaternary carbons, three oxygenated tertiary carbons,
Received: April 7, 2020
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Chart 1
two olefinic carbons (δC 120.7, 137.0), two ester carbonyl
groups (δC 170.5, 172.1), and two ketocarbonyl groups (δC
211.8, 214.0). These data showed a signal pattern similar to
that of 23,24-dihydrocucurbitacin B7 except for the A-ring.
The partial structure of the A-ring was established by 2D
NMR data. In addition to the 1H−1H COSY correlation
between H2-1 (δH 2.16, 2.50) and H-10 (δH 2.76), the HMBC
correlations from H2-1 to C-2 (δC 172.1) and C-5 (δC 136.9),
as well as from H3-28 and H3-29 (δH 1.51 and 1.53,
respectively) to oxygenated C-4 (δC 84.0), suggested a
lactone-type structure in the A-ring. The NOESY correlations
from H-10 to H3-30, H-17, and H3-21 suggested α-orientations
of these protons and methyl groups, while those from H3-19 to
H-8, H3-18, and H-16 revealed β-orientations.
The ECD spectrum (Figure S1.9, Supporting Information)
of compound 1 showed Cotton effects at 300 nm (Δε +2.8)
for the carbonyl n−π* transition and 200 nm (Δε +5.6) for
the olefinic π−π* transition, confirming the (8S, 9R, 10R, 13R,
and 14S) absolute configuration of compound 1.8 The NOESY
cross-peaks of H3-18/H-16 and H3-30/H-17 indicated the
(16R) and (17R) configuration. Because of the restricted
rotation of the single bond between C-17 and C-20,9 the NOE
correlation of H3-21/H-17 supported the (20R) configuration.
Thus, the structure of neocucurbitacin E (1) was defined as
shown. It represents the fifth example of a lactone-type
norcucurbitacin.10−12
Compound 2 was assigned a molecular formula of C31H48O9
deduced from the HRESIMS ion at m/z 587.3192 [M + Na]+
(calcd for C31H48O9Na, 587.3191). The 1H and 13C NMR data
of 2 revealed a similar structure to that of 1, with major
differences in the A-ring. Compared to 1, H2-1a (δH 2.51) still
showed a correlation with carboxylic carbon C-2 (δC 180.2) in
the HMBC spectrum, which was consistent with its eight
indices of hydrogen deficiency and the IR absorption band at
1694 cm−1. Moreover, H3-28 and H3-29 (δH 1.35 and 1.62,
respectively) were correlated to C-4 (δC 89.9). The ECD
Cotton effects and NOE correlations of 2 were similar to those
of 1,9 indicating that these two compounds had the same
configuration (8S, 9R, 10R, 13R, 14S, 16R, 17R, and 20R). As
the fifth example of a ring-A seco-cucurbitacin,13 2 was named
colocynthenin E.
Compounds 3 and 4 had the same molecular formula of
C31H48O8, as indicated by their HRESIMS ions at m/z
571.3244 and 571.3245 [M + Na]+ (calcd for C31H48O8Na,
571.3241), respectively. The NMR spectra of 3 suggested that
it was closely related to cucurbitacins H (8) and G (9).
However, the hydroxy group at C-24 was replaced by a
methoxy group, which was supported by the HMBC
B
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1
Figure 1. H−1H COSY (red) and select HMBC correlations (H → C, blue) of 1−5 and 7.
Figure 2. Key NOESY correlations of 1−5 and 7.
correlation from the methoxy protons (δH 3.43) to C-24 (δC
84.7). The NMR data of 4 resembled those of compound 3 but
differed in the coupling constants of H2-23 and H-24, implying
that 4 was a C-24 epimer of 3. Comparison of the ECD and
NMR spectra of 3 and 4 with those of 2 confirmed that these
cucurbitacins had the same configuration (8S, 9R, 10R, 13R,
14S, 16R, 17R, and 20R). According to the rule summarized by
Gan Maolong,9 the J23a,24 and J23b,24 values (H2-23a is defined
to be deshielded to a greater extent than H2-23b) may be used
to define the C-24 absolute configuration in cucurbitacins. The
value of J23a,24 (6.9 Hz) was larger than that of J23b,24 (3.6 Hz)
in 3, confirming the (24R) configuration, while the value of
J23a,24 (2.6 Hz) was smaller than that of J23b,24 (8.5 Hz) in 4,
establishing the (24S) configuration.
(calcd for C32H46O9Na, 597.3034). Comparison of its NMR
data with those of 3-epi-isocucurbitacin B14 revealed their
structural similarity, except for an oxymethine (δH 4.13; δC
66.5) instead of a C-7 methylene (δC 24.8) in 3-epi-
isocucurbitacin B and the deshielded resonance of C-8 (δC
53.8) in 5. These data indicated a hydroxy group at C-7 in 5,
which was confirmed by the molecular formula as well as the
1H−1H COSY cross-peak between H-7 (δH 4.13) and H-6 (δH
1
6.04). The H NMR signal for H-8 (δH 2.10) appeared as a
broad singlet, suggesting a small coupling constant between H-
7 and H-8, which corresponded to a dihedral angle of
approximately 90° and indicated a β-OH group at C-7.15
Therefore, the structure of compound 5 was defined as 7β-
hydroxy-3-epi-isocucurbitacin B.
Compound 5 had a molecular formula of C32H46O9 on the
Compound 6 was assigned a molecular formula of
C24H34O5, as determined by its HRESIMS ion at m/z
basis of its sodium adduct ion at m/z 597.3040 [M + Na]+
C
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Chart 2
425.2296 [M + Na]+ (calcd for C24H34O5Na, 425.2298). Its
spectroscopic data were comparable to those of hexanorcu-
curbitacin D,15 with the difference being a sharp singlet (δH
3.91) assigned to an α-OH group at C-3.16 Therefore, the
structure of compound 6 was defined as hexanorisocucurbita-
cin D.
isocucurbitacin D (20),20 isocucurbitacin D (21),19 23,24-
dihydroisocucurbitacin D (22),21 23,24-dihydro-3-epi-isocu-
curbitacin D (23),21 24α-hydroxyisocucurbitacin D (24),13
24β-hydroxyisocucurbitacin D (25),13 cucurbitacin F (26),22
and hexanorcucurbitacin D (27).15
Synthesis of Cucurbitacin Derivatives. Since the
separation of the T. cucumeroides extract led to the isolation
of cucurbitacin B (14, B, 3 g) and dihydrocucurbitacin B (15,
DB, 2 g) in large amounts, semisynthetic derivatives were
prepared to examine the structure−activity relationships
(SARs) of cucurbitacins.
In order to study the influence of the hydroxy groups at C-2
and C-16 on the biological activity, we introduced
modifications at C-2 and C-16. The reaction of B or DB
with Dess-Martin periodinane yielded B1,2 or DB1,2.23
Compounds B or DB were treated with acetic, propionic,
succinic, or benzoic anhydrides to yield esters B3−7 or DB3−
7.24 To analyze the importance of HO-3 in cucurbitacin,
compounds with a 2-oxo-3-hydroxy A-ring were explored. 3-
epi-Isocucurbitacin B (obtained from the interconversion of B
with silica gel in CH2Cl2) was treated with Ac2O to yield B8−
10, and 3-epi-iso-dihydrocucurbitacin B (obtained from the
interconversion of DB) was treated with Ac2O to yield DB8−
10.24,25 To explore the role of the C-7 allylic position, B11 and
DB11 were synthesized from B5 and DB3, respectively, by
oxidation with CrO3 and pyridine in CH2Cl2.24
Compound 7 had a molecular formula of C32H48O10 based
on its HRESIMS ion at m/z 615.3137 [M + Na]+ (calcd for
C32H48O10Na, 615.3140). Its NMR data showed similarity to
those of dihydrocucurbitacin B with major differences in the A-
ring. The HMBC correlations from H2-1 (δH 2.46, 2.75) to the
carboxylic C-2 (δC 180.2), as well as from H3-28 and H3-29
(δH 1.32 and 1.44, respectively) to the carboxylic C-3 (δC
184.2), suggested a ring-A seco-cucurbitacin, which was
consistent with its nine indices of hydrogen deficiency. The
NOESY correlations of H-10/H3-30/H-17/H3-21 and of H3-
19/H-8/H3-18/H-16, as well as the ECD Cotton effects at 300
nm (Δε +3.4) and 202 nm (Δε +17.4), indicated its (8S, 9R,
10R, 13R, 14S, 16R, 17R, 20R) absolute configuration, which
was different from the compound reported in a patent17 [same
2D structure with an (8R, 9R, 10S, 13R, 14S, 16R, 17R, 20R)
absolute configuration]. As the sixth example of a ring-A seco-
cucurbitacin,12 7 was named colocynthenin F.
By comparison of the spectroscopic data with reported data,
known compounds 8−27 were identified as cucurbitacin H
(8),13 cucurbitacin G (9),13 cucurbitacin I (10),18 cucurbitacin
L (11),18 cucurbitacin D (12),18 dihydrocucurbitacin D
(13),18 cucurbitacin B (14),18 23,24-dihydrocucurbitacin B
(15),7 arvenin I (16),19 arvenin II (17),19 7β-hydroxycucurbi-
tacin B (18),15 7β-hydroxydihydrocucurbitacin B (19),15 3-epi-
LDL Uptake Activity. An LDL uptake assay was first
conducted to evaluate the effects of 47 cucurbitacins (5 μM)
on LDL uptake in HepG2 cells. The results in Figure 3 show
that most of the compounds improved the LDL uptake rates,
D
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carbonyl group may promote activity. A 24-methoxy moiety
could enhance activity, as shown by comparison of the three
groups 3/4, 8/9, and 12/13.
Influence on LDLR and PCSK9 Protein Levels. Since
LDLR protein is a key cell surface receptor for cholesterol
internalization, Western blot analysis was carried out to
examine whether these compounds impact LDLR protein
levels in HepG2 cells. Compounds 6 and 21, which strongly
promoted LDL uptake, were chosen for this study. It is clear
that LDLR protein levels increased dose-dependently in
response to these two cucurbitacins, which may explain why
these compounds could improve LDL uptake.
We further assessed whether these two cucurbitacins
regulate cholesterol metabolism in a PCSK9-dependent
process. The results show that they dose-dependently reduced
PCSK9 protein levels, suggesting that PCSK9 is involved in the
LDLR uptake elevation induced by compounds 6 and 21.
To study the lipid-lowering activities of cucurbitacins, a
small library of this class of compounds was prepared. Among
compounds 1−27 isolated from T. cucumeroides roots, 1 was a
novel lactone-type norcucurbitacin, while 2 and 7 were rare
ring-A seco-cucurbitacins. Modifications of the groups
contributing to activity of cucurbitacins yielded 22 derivatives.
Most of natural cucurbitacins and their derivatives were first
screened for lipid-lowering activities. The results revealed that
most of the compounds could improve the LDL uptake rate,
among which hexanorisocucurbitacin D (6) and isocucurbita-
cin D (21) displayed the strongest effects compared to the
positive control, nagilactone B. In addition, some general
trends were observed that may be useful for the discovery of
more active compounds: a 2-oxo-3α-hydroxy A-ring con-
stitution is essential for activity, while modifications of the
hydroxy groups at C-2, C-3, or C-16 make no difference;
additionally, 7-carbonyl and 24-methoxy groups may be
favorable in certain types of compounds.
Figure 3. Effects of cucurbitacins on LDL uptake rates in HepG2 cells
(n = 3). (a) Effects of cucurbitacins isolated from T. cucumeroides
roots on LDL uptake rates. (b) Effects of cucurbitacin derivatives on
LDL uptake rates. Nagilactone B (ZBM30, 5 μM) was used as a
positive control. The red dotted line indicates an uptake rate of 1.0.
The results are presented as the means SEM, n = 3.
especially those of hexanorisocucurbitacin D (6, rate of 2.53)
and isocucurbitacin D (21, rate of 2.47), which were
comparable to that of the positive control, nagilactone B
(designated ZBM30, rate of 2.07).26
According to the observed lipid uptake rates, the preliminary
SARs were summarized as shown in Figure 3. The 2-oxo-3α-
hydroxy A-ring was found to be essential for activity: the
compounds with a 2-oxo-3α-hydroxy A-ring, such as 6 and 21,
had a distinct advantage over those with a 2β-hydroxy-3-oxo A-
ring, such as 12 and 13. Similar activities were recorded for B
with B1−10 and DB with DB1−10, indicating that either
substitution or oxidation at C-2, C-3, or C-16 was not
significant for activity. Comparing the LDL uptake rate of B
with B11, and of DB with DB11, suggested that the C-7
Further assays indicated that compounds 6 and 21 dose-
dependently increased LDLR protein levels and reduced
PCSK9 protein levels, suggesting that 6 and 21 are potent
PCSK9 modulators for the treatment of hyperlipidemia.
Nevertheless, additional studies to determine such information
as the intact mechanism, effects in animals, in vivo
pharmacokinetics, and drug safety should be carried out on
cucurbitacins in the future.
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured on a Rudolph Autopol VI automatic polarimeter. IR
■
Figure 4. Effects of compounds 6 and 21 on LDLR and PCSK9 protein levels in HepG2 cells. (a) Compounds 6 and 21 dose-dependently
upregulated LDLR protein levels. (b) Compounds 6 and 21 dose-dependently downregulated PCSK9 protein levels. The results are presented as
the means SEM, n ≥ 5. *P < 0.05, **P < 0.01, ***P < 0.001 vs vehicle control by one-way ANOVA (Dunnett’s multiple comparisons test).
E
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Table 1. 1H NMR Data of Compounds 1−4 (δ in ppm, J in
Hz)
Table 2. 1H NMR Data of Compounds 5−7 (δ in ppm, J in
Hz)
a
b
c
c
a
b
c
1
2
3
4
5
6
7
1
2.50, m
2.16, dd
(16.2, 13.5)
2.51, m
2.46, m
2.10, ddd
(12.3, 5.8, 3.6) (12.3, 5.8, 3.6)
1.20, m
4.58, m
2.11, ddd
1
2.21, m
2.03, m
4.12, s
6.04, dd (5.0, 2.4)
4.13, dd (5.0, 2.1)
2.25, m
2.13, m
3.91, s
5.94, d (5.6)
2.47, ddd
(19.1, 5.1, 2.6)
2.04, dd (19.1, 6.6)
2.01, d (7.6)
2.83, br d (13.1)
3.31, d (14.8)
2.33, d (14.8)
1.91, dd (13.0, 9.0)
1.45, d (13.0)
4.79, t (7.8)
3.15, d (6.7)
0.66, s
2.75, dd (20.0, 3.0)
2.46, dd (20.0, 5.4)
1.41, m
4.58, dd (13.0,
5.9)
5.83, dd (6.1,
2.3)
3
6
7
2
6
7
5.84, dt (5.3, 2.5)
2.35, ddd
(19.2, 8.4, 3.1)
2.05, m
2.05, d (7.9)
3.70, dd (5.4, 3.0)
3.25, d (15.1)
2.60, d (15.1)
1.83, dd (13.3, 8.8)
1.43, m
4.30, t (7.8)
2.52, d (7.0)
0.99, s
5.75, dd (5.2, 6.06, dd (5.1, 5.82, dt (6.0,
2.7)
2.40, ddd
2.8)
2.43, m
2.2)
2.42, dd (19.7, 2.43, ddd
8.2)
2.03, dd (19.7, (19.3, 8.4, 2.8)
5.6)
8
10
12
2.10, br s
3.12, m
3.21, d (14.6)
2.52, d (14.6)
2.08, m
(20.2, 8.2, 3.9) 2.03, m
2.04, m
2.02, d (8.3)
2.04, m
2.00, d (8.1)
3.01, br d
(12.4)
8
10
2.00, d (8.0)
1.98, d (8.1)
2.96, m
15
2.76, d (13.5) 3.26, m
1.54, m
12
15
3.12, d (14.8) 3.35, d (14.7) 3.43, d (15.4) 3.47, d (14.7)
2.68, d (14.8) 2.56, d (14.7) 2.61, d (15.4) 2.63, d (14.7)
1.84, dd (13.4, 1.81, dd (13.3, 1.89, dd (13.2, 1.87, dd (13.4,
16
17
18
19
21
22
23
4.61, t (7.5)
2.51, d (7.3)
0.89, s
1.16, s
1.40, s
8.7)
9.0)
1.40, m
4.45, t (7.9)
2.53, d (7.2)
0.90, s
1.13, s
1.37, s
2.83, ddd
9.0)
8.8)
0.82, s
2.12, s
1.24, s
1.25, s
1.06, s
1.40, s
1.40, m
4.32, t (8.3)
2.49, d (6.6)
0.96, s
1.05, s
1.40, s
1.40, m
4.57, t (8.2)
2.60, d (6.5)
0.93, s
1.05, s
1.39, s
3.05, dd (18.5, 3.08, dd (17.7,
6.9) 2.6)
2.97, dd (18.5, 2.84, dd (17.7,
3.6) 8.5)
1.40, m
4.46, t (7.7)
2.59, d (7.3)
0.96, s
1.06, s
1.42, s
16
17
18
19
21
23
6.84, d (15.8)
2.86, ddd
(17.5, 10.0, 5.6)
2.58, ddd
(17.5, 11.0, 6.5)
2.05, m
1.45, s
1.47, s
1.32, s
1.44, s
2.81, m
24
26
27
28
29
30
32
6.97, d (15.8)
1.54, s
1.57, s
1.27, s
0.98, s
1.11, s
2.52, m
(18.0, 10.3,
5.5)
2.70, ddd
(18.0, 10.3,
5.5)
1.29, s
2.00, s
1.37, s
1.96, s
24
2.06, dd (9.9, 1.98, m
5.3)
3.57, dd (6.9, 3.63, dd (8.5,
3.6)
1.19, s
1.14, s
1.29, s
1.30, s
1.40, s
2.6)
1.17, s
1.20, s
1.30, s
1.31, s
1.40, s
a
b
Recorded at 600 MHz, in methanol-d4. Recorded at 400 MHz, in
methanol-d4. Recorded at 600 MHz, in CDCl3.
26
27
28
29
30
32
OCH3
1.43, s
1.46, s
1.51, s
1.53, s
1.33, s
1.96, s
1.44, s
1.44, s
1.35, s
1.62, s
1.32, s
1.95, s
c
(500 g). The extract was suspended in water (2 L) and extracted with
EtOAc to afford the EtOAc-soluble fraction (120 g). The EtOAc layer
was divided into fractions A−H by silica gel CC using gradients of
increasing polarity from petroleum ether/acetone (10:1) to acetone.
Fraction C (5 g) was applied to an ODS column (CH3OH/H2O,
50:50 to 100:0) to give subfractions Fr. C1−C7. Fr. C2 was purified
first by silica gel CC (CH2Cl2/EtOAc, 6:1 to 1:1) and with an RP-C18
column (CH3OH/H2O, 65:35) to obtain 1 (3 mg) and 7 (6 mg). Fr.
C5 was subjected to silica gel CC (petroleum ether/EtOAc, 4:1 to
2:1) followed by semipreparative HPLC (CH3CN/H2O, 68:32) to
yield 2 (7 mg).
Fraction D (60 g) was separated by silica gel CC (CH2Cl2/
CH3OH, 50:1 to 5:1) to give Fr. D1−D5. Fr. D3 was subjected to
silica gel CC (petroleum ether/acetone, 5:1 to 3:1, with 0.1% formic
acid) to produce Fr. D31−D35. Fr. D31 and Fr. D33 were purified by
an ODS column (CH3CN/H2O, 25:65 to 45:55) and HPLC
(CH3CN/H2O, 43:57); Fr. D31 produced 8 (8 mg) and 9 (8 mg),
while Fr. D33 produced 20 (10 mg) and 27 (7 mg).
Fraction E (20 g) was subjected to silica gel CC (CH2Cl2/CH3OH,
100:1 to 1:1) to give Fr. E1−E6. Fr. E2 was separated by silica gel CC
(petroleum ether/acetone, 5:1 to 3:1, with 0.1% formic acid) to
produce six fractions. Fr. E23 was purified by HPLC (CH3CN/H2O,
55:45) to yield 6 (2 mg), 14 (3 g), and 15 (2 g). Fr. E24 was
separated by silica gel CC (petroleum ether/EtOAc, 6:1 to 1:1, with
0.1% formic acid) and HPLC (CH3OH/H2O, 65:35) to obtain 3 (1
mg), 4 (5 mg), 10 (6 mg), 11 (5 mg), 12 (600 mg), 21 (12 mg), 22
(4 mg), and 23 (7 mg).
3.43, s
3.43, s
a
b
Recorded at 500 MHz, in CDCl3. Recorded at 400 MHz, in
methanol-d4. Recorded at 600 MHz, in methanol-d4.
c
spectra were recorded on a Thermo Scientific Thermo IS5
spectrometer. HRESIMS data were collected using an Agilent 1290
Infinity HPLC and an Agilent 6224 TOF mass spectrometer. NMR
spectra were recorded on Bruker AM-400, AVANCE III 500, and
AVANCE III 600 spectrometers. Semipreparative HPLC was
performed on an Agilent 1100 series HPLC system equipped with a
YMC-Pack ODS-A column (250 × 10 mm, 5 μm). Silica gel (200−
300 mesh, Qingdao Haiyang Chemical Co., Ltd., People’s Republic of
China) and ODS (20−45 μm, Fuji Silysia Chemical Ltd., Japan) were
used for column chromatography (CC). Silica gel GF254 and RP-18
F254S TLC plates were obtained from Qingdao Haiyang Chemical
Co. Ltd. and Merck (Darmstadt, Germany), respectively. All solvents
were purchased from Sinopharm Chemical Reagent Co., Ltd.
Plant Material. Roots of Trichosanthes cucumeroides were collected
in Yunnan Province, China, in July 2014 and identified by Prof.
Heming Yang. A voucher specimen (no. SIMM239) was deposited at
the Herbarium of the Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai, People’s Republic of China.
Extraction and Isolation. Dried roots of T. cucumeroides
(10.0 kg) were crushed into a coarse powder and cold-soaked in
95% EtOH. The solvent was evaporated to produce a crude extract
Fr. E3 was separated into Fr. E31−E34 by an ODS column
(CH3OH/H2O, 60:40 to 70:30). Fr. E33 and E34 were subjected to
F
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Table 3. 13C NMR (125 MHz) Data of Compounds 1−7 (δ in ppm)
a
b
b
b
b
b
a
1
2
3
4
5
6
7
1
2
3
4
30.4, CH2
172.1, C
31.2, CH2
180.2, C
37.1, CH2
72.8, CH
214.0, C
49.0, C
37.1, CH2
72.8, CH
214.0, C
49.2, C
37.8, CH2
212.4, C
80.7, CH
49.5, C
40.2, CH2
211.8, C
81.4, CH
49.6, C
32.1, CH2
180.2, C
184.2, C
47.2, C
84.0, C
89.9, C
5
6
7
8
136.9, C
120.7, CH
23.9, CH2
42.2, CH
47.8, C
138.4, C
128.2, CH
24.6, CH2
44.1, CH
49.4, C
142.7, C
121.2, CH
24.8, CH2
44.1, CH
49.9, C
140.0, C
121.2, CH
24.8, CH2
44.1, CH
49.8, C
145.3, C
122.7, CH
66.5, CH
53.8, CH
48.1, C
140.1, C
122.5, CH
24.9, CH2
44.6, CH
49.7, C
140.6, C
122.9, CH
24.7, CH2
42.7, CH
48.7, C
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
OCH3
31
32
33.4, CH
211.8, C
48.9, CH2
50.5, C
33.6, CH
215.8, C
49.9, CH2
51.9, C
34.9, CH
215.9, C
49.9, CH2
51.87, C
51.82, C
46.6, CH2
71.6, CH
59.9, CH
20.5, CH3
20.2, CH3
81.1, C
25.0, CH3
216.4, C
40.0, CH2
84.7, CH
73.8, C
34.8, CH
215.7, C
49.9, CH2
51.9, C
34.4, CH
215.7, C
49.8, CH2
50.8, C
37.3, CH
213.6, C
48.1, CH2
51.2, C
34.9, CH
214.4, C
49.9, CH2
50.6, C
48.4, C
52.8, C
51.8, C
48.4, C
50.1, C
48.7, C
45.8, CH2
71.1, CH
57.9, CH
20.0, CH3
18.6, CH3
79.0, C
24.6, CH3
214.0, C
30.8, CH2
34.9, CH2
81.4, C
26.0, CH3
26.3, CH3
31.5, CH3
30.7, CH3
18.3, CH3
46.4, CH2
71.4, CH
59.5, CH
20.5, CH3
20.2, CH3
80.8, C
25.8, CH3
216.6, C
32.9, CH2
35.8, CH2
83.1, C
26.2, CH3
26.3, CH3
25.5, CH3
25.6, CH3
18.8, CH3
46.6, CH2
71.5, CH
58.9, CH
20.6, CH3
20.2, CH3
80.8, C
26.4, CH3
215.2, C
40.2, CH2
85.3, CH
73.8, C
25.2, CH3
25.3, CH3
29.8, CH3
21.9, CH3
19.5, CH3
60.8, CH3
46.5, CH2
71.8, CH
60.1, CH
20.8, CH3
20.4, CH3
80.2, C
25.4, CH3
205.3, C
124.8, CH
151.6, CH
81.1, C
26.4, CH3
26.9, CH3
27.8, CH3
21.9, CH3
19.5, CH3
46.1, CH2
72.5, CH
67.9, CH
20.2, CH3
20.2, CH3
210.6, C
31.8, CH3
24.6, CH3
21.7, CH3
19.5, CH3
45.9, CH2
71.1, CH
58.6, CH
20.8, CH3
20.4, CH3
79.2, C
24.6, CH3
214.6, C
30.9, CH2
35.1, CH2
81.5, C
26.0, CH3
26.3, CH3
28.1, CH3
27.8, CH3
18.8, CH3
25.7, CH3
26.5, CH3
29.8, CH3
21.9, CH3
19.5, CH3
59.7, CH3
170.5, C
22.6, CH3
172.4, C
22.4, CH3
171.9, C
24.2, CH3
170.6, C
22.6, CH3
a
b
Recorded in CDCl3. Recorded in methanol-d4.
silica gel CC (petroleum ether/EtOAc, 3:1 to 1:2, with 0.1% formic
acid) to afford Fr. E331−334 and Fr. E340−343. Fr. E334 was
purified by an ODS column (CH3CN/H2O, 55:45) to yield 13 (700
mg). Compounds 5 (2 mg), 18 (3 mg), and 19 (2 mg) were obtained
from Fr. E340 by HPLC (CH3OH/H2O, 53:47); 24 (3 mg) and 25
(3 mg) were obtained from Fr. E341 by HPLC (CH3CN/H2O,
37:63). Fr. E4 was purified by silica gel CC (petroleum ether/acetone,
4:1 to 1:1, with 0.1% formic acid) followed by an ODS column
(CH3OH/H2O, 45:55 to 65:35) to give 26 (3 mg). Fraction F was
separated by silica gel CC (CH2Cl2/CH3OH, 20:1 to 5:1) and HPLC
(CH3CN/H2O, 50:50) to yield 16 (700 mg) and 17 (800 mg).
Neocucurbitacin E (1): white amorphous powder; [α]2D0 −9 (c 0.1,
CHCl3); ECD (CH3OH) 300 nm (Δε +2.8), 200 nm (Δε +5.6); IR
24α-Methoxydihydrocucurbitacin D (4): white amorphous
powder; [α]2D0 +63 (c 0.1, CH3OH); ECD (CH3OH) 304 nm (Δε
+3.6), 202 nm (Δε +15.8); IR (KBr) νmax 3437, 2976, 2930, 1688,
1
1654, 1384, 1091, 1047 cm−1; H and 13C NMR data, see Tables 1
and 3; HRESIMS m/z 571.3245 [M + Na]+ (calcd for C31H48O8Na,
571.3241).
7β-Hydroxy-3-epi-isocucurbitacin B (5): white amorphous pow-
der; [α]2D0 +6 (c 0.1, CHCl3); ECD (CH3OH) 300 nm (Δε +1.1),
201 nm (Δε +5.0); IR (KBr) νmax 3437, 2956, 2923, 2852, 1718,
1689, 1626, 1459, 1377, 1259, 1125 cm−1; 1H and 13C NMR data, see
Tables 2 and 3; HRESIMS m/z 597.3040 [M + Na]+ (calcd for
C32H46O9Na, 597.3034).
Hexanorisocucurbitacin D (6): white amorphous powder; [α]D20
+109 (c 0.1, CHCl3); ECD (CH3OH) 295 nm (Δε +4.0), 200 nm
(Δε +9.6); IR (KBr) νmax 3446, 2958, 2923, 2853, 1697, 1655, 1458,
1380, 1101 cm−1; 1H and 13C NMR data, see Tables 2 and 3;
HRESIMS m/z 425.2296 [M + Na]+ (calcd for C24H34O5Na,
425.2298).
1
(KBr) νmax 3439, 2924, 1734, 1697, 1654, 1369, 1219, 772 cm−1; H
and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 569.3080 [M
+ Na]+ (calcd for C31H46O8Na, 569.3085).
Colocynthenin E (2): white amorphous powder; [α]2D0 +32 (c 0.1,
CHCl3); ECD (CH3OH) 300 nm (Δε +4.6), 202 nm (Δε +16.6); IR
(KBr) νmax 3443, 2921, 1770, 1695, 1369, 1255, 1128, 1023 cm−1; 1H
and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 587.3192 [M
+ Na]+ (calcd for C31H48O9Na, 587.3191).
Colocynthenin F (7): white amorphous powder; [α]2D0 +18 (c 0.1,
CH3OH); ECD (CH3OH) 300 nm (Δε +3.4), 202 nm (Δε +3.6); IR
(KBr) νmax 3444, 2974, 2925, 1705, 1385, 1370, 1270, 1209, 1129,
1023 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/
z 615.3137 [M + Na]+ (calcd for C31H48O9Na, 615.3140).
Cell Culture. HepG2 cells (ATCC HB-8065) were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine
serum (FBS, v/v) and incubated under a humidified atmosphere of
5% CO2 and 95% O2 at 37 °C. Cells from passages 4 to 11 were used;
subcultures were performed once every 2 days.
24β-Methoxydihydrocucurbitacin D (3): white amorphous
powder; [α]2D0 +60 (c 0.1, CH3OH); ECD (CH3OH) 301 nm (Δε
+3.2), 203 nm (Δε +13.0); IR (KBr) νmax 3446, 2971, 2394, 2923,
1
1504, 1032, 1013 cm−1; H and 13C NMR data, see Tables 1 and 3;
HRESIMS m/z 571.3244 [M + Na]+ (calcd for C31H48O8Na,
571.3241).
G
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Lipoprotein Isolation and DiI-LDL Preparation. Human
plasma was obtained from Shanghai Xuhui Central Hospital, China,
after informed consent and approval from the Ethics Committee. The
procedures were performed under the principles of the Declaration of
Helsinki.27 LDL and lipoprotein-deficient serum (LPDS) were
isolated from the pooled plasma of healthy volunteers by ultra-
centrifugation and dialysis against dialysis buffer and PBS. The
obtained LDL was labeled with the fluorescent probe 1,1′-
dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI,
Biotium, CA, USA) as previously described, with minor modifica-
tions.28
AUTHOR INFORMATION
■
Corresponding Author
Lijiang Xuan − State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, People’s Republic of China;
University of Chinese Academy of Sciences, Beijing 100049,
People’s Republic of China; orcid.org/0000-0002-0593-
3921; Phone: 86-21-20231968; Email: ljxuan@simm.ac.cn
Authors
In short, DiI dissolved in DMSO (15 mg/mL) was added to the
LDL/LPDS mixture (v/v, 1:2) to a final concentration of 300 mg
DiI/mg LDL protein. After incubation at 37 °C for 18 h, the DiI-
labeled LDL (DiI-LDL) was separated by ultracentrifugation and
dialysis against buffer and PBS. After sterilization using 0.45 μm filters
(Millipore, MA, USA), DiI-LDL was stored at 4 °C.
DiI-LDL Uptake Assay. DiI-LDL uptake assays were conducted as
described previously, with slight modifications.28 HepG2 cells seeded
in 24-well plates, after treatment with DMSO or compounds, were
removed and placed in DiI-LDL DMEM (20 μg/mL) at 37 °C for 3 h
in the dark. The cells were rinsed twice with ice-cold PBS containing
0.4% albumin (Sigma-Aldrich, St. Louis, MO, USA) and washed twice
with PBS. After that, 500 μL of isopropanol was added to each well
following a 20 min incubation at room temperature under constant
shaking in the dark. Finally, 200 μL aliquots were used for
fluorescence detection with a SpectraMax M2e microplate reader
(520−578 nm, Molecular Devices, San Jose, CA, USA). Nagilactone
B (ZBM30, 5 μM), a natural product isolated from Podocarpus nagi,
significantly improved LDL uptake in a PCSK9-dependent process26
and thus was used as a positive control in this research.
Xianjing Zhang − State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, People’s Republic of China;
University of Chinese Academy of Sciences, Beijing 100049,
People’s Republic of China
Huihui Li − State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, People’s Republic of China; University of
Chinese Academy of Sciences, Beijing 100049, People’s
Republic of China
Wenqiong Wang − State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, People’s Republic of China
Tong Chen − State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, People’s Republic of China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00364
Western Blot Analysis. HepG2 cells were cultured in six-well
plates for 12 h. The medium was incubated for an additional 24 h,
after the addition of 2% LPDS. After treatment with various
concentrations of compounds for the indicated times, cells were
washed three times with PBS. Total cellular proteins were extracted
with 100 μL of lysis buffer containing a protease and phosphatase
inhibitor cocktail (catalogue number: 539134, Calbiochem, Merck
Millipore, Germany) and centrifuged at 12000g and 4 °C for 10 min.
Cell nuclear and cytoplasmic proteins were extracted using a nuclear
and cytoplasmic protein extraction kit (catalogue number: P0013B,
Beyotime Biotechnology) according to the manufacturer’s instruc-
tions. Protein concentrations were determined with a BCA protein
assay kit (catalogue number: P0010, Beyotime Biotechnology).
Protein (30 μg) was loaded in each well for 8% SDS PAGE and
transferred to PVDF membranes (catalogue number: 1620177, Bio
Rad, Hercules, CA, USA). The membranes were blocked with 5%
skim milk for 2 h at room temperature and incubated with primary
antibodies overnight at 4 °C. After three washes with TBST solution,
the membranes were incubated with secondary antibodies (catalog
numbers: 1706515 and 1706516, Bio-Rad) for 2 h. Finally, the bands
were visualized by Clarity Western ECL blotting substrates (catalogue
number: 1705061, Bio Rad), and the values were normalized to that
of a housekeeping protein, either lamin B1 or β-actin.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge grants from the “Person-
alized Medicines Molecular Signature based Drug Discovery
and Development”, Strategic Priority Research Program of the
Chinese Academy of Sciences (No. XDA12040335), the
National Natural Science Foundation of China (No.
81773863), the Youth Innovation Promotion Association,
and the National Science and Technology Major Project (Nos.
2019ZX09201004-003-041 and 2019ZX09201004-003-042).
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* Supporting Information
The Supporting Information is available free of charge at
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