Anti-adipogenic 18,19-seco-ursane stereoisomers and oleane-type saponins from Ilex cornuta leaves

Article abstract of DOI:10.1016/j.phytochem.2020.112363

Three undescribed seco-ursane stereoisomers, ilexcornutosides A–C, two undescribed triterpenoid saponins, ilexcornutosides D–E, and 11 known triterpenoids were isolated from the leaves of Ilex cornuta Lindl. & Paxton. Ilexcornutosides A–C and F with the same planar structures are unique 13(18)-ene-18,19-seco-ursane skeleton triterpenoids, identified as (3S,12R)-3-O-[β-D-glucopyranosyl-(1 → 2)-α-L-arabinopyranosyl]-12-hydroxyl-19-oxo-18,19-secours-13(18)-en-28,21-lactone. Among them, ilexcornutosides A and F (or ilexcornutosides B and C) are a pair of diastereomers at the C-20 position; ilexcornutosides A and C (or ilexcornutosides B and F) are a pair of diastereomers with epimerization at the C-21. Their structures were established by extensive spectroscopic (IR, 1D and 2D NMR, and HR-ESI-MS) and chemical analyses. The absolute configurations of ilexcornutosides A, B, D and F were determined by a single crystal X-ray diffraction analysis with a Cu Kα radiation. The inhibitory effect of ilexcornutosides A–F on the PPARγ expression was assessed in the 3T3-L1-Lenti-PPARγ-Luc cells using a single luciferase reporter assay. Ilexcornutosides A and C showed a comparable activity in decrease of the PPARγ expression to the positive control (T0070907) at 5 μM.

Full text of DOI:10.1016/j.phytochem.2020.112363

Phytochemistry175(2020)112363
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Anti-adipogenic 18,19-seco-ursane stereoisomers and oleane-type saponins
from Ilex cornuta leaves
Haiyan Fenga,1, Jihe Tanga,1, Peiliang Zhanga, Yu Miaoa, Tao Wua,∗∗, Zhihong Chengb,∗
a Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
b Department of Natural Medicine, School of Pharmacy, Fudan University, Shanghai 201203, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Three undescribed seco-ursane stereoisomers, ilexcornutosides A–C, two undescribed triterpenoid saponins,
ilexcornutosides D–E, and 11 known triterpenoids were isolated from the leaves of Ilex cornuta Lindl. & Paxton.
Ilexcornutosides A–C and F with the same planar structures are unique 13(18)-ene-18,19-seco-ursane skeleton
triterpenoids, identified as (3S,12R)-3-O-[β-D-glucopyranosyl-(1 → 2)-α-L-arabinopyranosyl]-12-hydroxyl-19-
oxo-18,19-secours-13(18)-en-28,21-lactone. Among them, ilexcornutosides A and F (or ilexcornutosides B and C)
are a pair of diastereomers at the C-20 position; ilexcornutosides A and C (or ilexcornutosides B and F) are a pair
of diastereomers with epimerization at the C-21. Their structures were established by extensive spectroscopic
(IR, 1D and 2D NMR, and HR-ESI-MS) and chemical analyses. The absolute configurations of ilexcornutosides A,
B, D and F were determined by a single crystal X-ray diffraction analysis with a Cu Kα radiation. The inhibitory
effect of ilexcornutosides A–F on the PPARγ expression was assessed in the 3T3-L1-Lenti-PPARγ-Luc cells using a
single luciferase reporter assay. Ilexcornutosides A and C showed a comparable activity in decrease of the PPARγ
expression to the positive control (T0070907) at 5 μM.
Ilex cornuta Lindl. & Paxton
Aquifoliaceae
Triterpenoids
Seco-ursanes
PPARγ
Ilexcornutoside
1. Introduction
ursanes (1–3), two undescribed triterpenoid saponins (4 and 5), and 11
known triterpenoids (6–16). In addition, the PPAR-γ inhibitory activ-
Ilex cornuta Lindl. & Paxton (Aquifoliaceae) is an evergreen shrub
ities of these components were estimated using the 3T3-L1-Lenti-
widely distributed in China. Its leaves have been used in traditional
PPARγ-Luc cells.
Chinese medicines as
a heat-clearing and Yin-nourishing agent
(Editorial Committee of Chinese Pharmacopoeia, 2015). Leaves of the
herb are also widely used to prepare a functional tea product, named
kudingcha in China, with the benefit of potential weight reduction.
Phytochemical studies showed that triterpenoids and their corre-
sponding glycosides were the main components in this herb (Li et al.,
2010; Nakanishi et al., 1982; Qin et al., 1986; Zhou et al., 2012), and
have been reported for several biological activities, such as lipid low-
ering (Wang et al., 2015; Wang and Xie, 2015), antibiotic, antiviral
(Jesus et al., 2015), cardioprotective (Singh and Chaudhuri, 2018),
antioxidant (Zeng et al., 2011), anti-myocardial ischemia (Zuo et al.,
2011), and myocardial cell protective effects (Li et al., 2014). As part of
our continuous investigation of I. cornuta (Li et al., 2010), this research
investigated the chemical components in the 75% ethanol extract of I.
cornuta leaves. This study included the isolation and structure identi-
fication of three undescribed diastereomers of 13(18)-ene-18,19-seco-
2. Results and discussion
Repeated column chromatography (CC) and preparative TLC of 75%
ethanol extract of I. cornuta leaves led to the isolation of 16 triterpe-
noids including five undescribed ones (Fig. 1). Purification of the
CH2Cl2-MeOH (6:1) fraction on a silica gel column provided a sub-
fraction enriched with four isomers (1–3 and 6). TLC analysis of this
fraction showed only a purple dense spot after spraying with 10%
H2SO4 in ethanol and heating. Multiple attempts to separate the isomers
using repeated silica gel CC with different solvent systems were un-
successful. Fortunately, the four isomers could be well separated using a
semi-preparative HPLC with MeCN-H2O (33:67) as the eluting solvent
(Fig. 2).
Compound 1 was obtained as colorless needles with a molecular
formula C41H64O14 according to the positive-ion HR-ESI-MS (m/z
∗ Corresponding author. Department of Natural Medicine, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China.
∗∗ Corresponding author. Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China.
E-mail addresses: laurawu2000@163.com (T. Wu), chengzhh@fudan.edu.cn (Z. Cheng).
1 Haiyan Feng and Jihe Tang contributed equally to this work.
https://doi.org/10.1016/j.phytochem.2020.112363
Received 1 November 2019; Received in revised form 18 March 2020; Accepted 19 March 2020
0031-9422/©2020ElsevierLtd.Allrightsreserved.

H. Feng, et al.
Phytochemistry175(2020)112363
Fig. 1. Chemical structures of compounds 1–16.
798.4630 [M+NH4]+) and the 13C NMR data, indicating 10 degrees of
unsaturation. The 1H NMR spectrum showed seven methyl signals at δH
0.81 (3H, s), 0.97 (3H, d, J = 7.2 Hz), 1.01 (3H, s), 1.23 (6H, s), 1.26
(3H, s) and 2.16 (3H, s), a typical oxygenized proton at δH 3.24 (1H, dd,
J = 11.8, 4.5 Hz, H-3), an olefinic proton at δH 6.16 (1H, s) and two
anomeric protons at δH 4.97 (1H, d, J = 5.8 Hz) and δH 5.18 (1H, d,
J = 7.7 Hz). A combination of 13C NMR, DEPT and HSQC spectra re-
vealed 41 carbon signals, and the 30 signals of that were attributed to a
triterpenoid aglycone and the other 11 to a disaccharide part. Some
diagnostic carbon signals for the triterpenoid aglycone included a ke-
tone carbon at δc 209.7 (C-19), an ester carbonyl carbon at δc 179.0 (C-
28), a quaternary olefinic carbon at δC 151.4 (C-13), a tertiary olefinic
carbon at δC 119.9 (C-18), seven methyls at δc 12.2, 17.0, 17.1, 19.2,
22.5, 28.7 and 29.9, and three oxygenized methines at δc 69.6, 77.8
and 89.1. The 11 sugar carbon signals (Table 1) and their corresponding
anomeric proton signals were indicative of a terminal β-glucose and 2′-
O-substituted α-arabinose (Matsuo et al., 2009). These NMR data were
characteristics for a triterpenoid diglycoside with a 13(18)-ene-18,19-
seco-ursane skeleton (Potterat et al., 1992; Zhang et al., 2011) and were
almost identical to those of compound 6 [(3β,12β)-3-(β-D-glucopyr-
anosyl-(1
→
2)-α-L-arabinopyranosyl)-12-hydroxyl-19-oxo-18,19-se-
cours-13(18)-en-28,21-lactone isolated previously from I. cornuta] (Lee
et al., 2015). But, a slight difference (Δ1.4 ppm) was clearly observed
for the chemical shift of C-30 (Table 1). This suggested that compound
1 might have the same planar structure with 6. The assignment of the
13(18)-ene-18,19-seco-ursane skeleton in 1 was verified by HMBC
2

H. Feng, et al.
Phytochemistry175(2020)112363
NOESY spectrum. The correlations between H-3 (δH 3.24)/H-5 (δH
0.73), between H-9 (δH 1.54)/H-12 (δH 4.60) and H-5, and between H-
5/H-23(δH 1.23) indicated that they were cofacial and designated as α-
oriented (Fig. S1). The NOESY correlations from H-16a (δH 1.60) to H-
22a (δH 2.11) and Me-27 (δH 1.23) as well as correlations between H-
16b (δH 1.98) and H-21 (δH 4.82) revealed that the plane of the spiro γ-
lactone is vertical to that of the plane defined by the A/B/C/D rings.
The relative configuration of the other stereocenters except for C-20 in
1 was also consistent with that of 6. Together with their different NMR
chemical shifts at Me-30, this finding indicated that 1 is a stereoisomer
of the known compound 6 at C-20. However, the C-20 configuration of
6, as well as the absolute configuration of the γ-lactone, was not as-
signed in the original report (Lee et al., 2015). Thus, a single-crystal X-
ray diffraction study of 1 and 6 (Cu Kα radiation) was carried out
(Fig. 4), from which their planar structures were fully confirmed and
the absolute configuration was fully determined as 17R, 20S, 21S for 1
and 17R, 20R, 21S for 6, respectively. Finally, the structure of 1 was
identified as (3S, 12R, 17R, 20S, 21S)-3-O-[β-D-glucopyranosyl- (1 →
2)-α-L-arabinopyranosyl]-12-hydroxy-19-oxo-18,19-secours-13(18)-en-
28,21- lactone, named ilexcornutoside A. For the first time, the absolute
configuration of compound 6 was established, and was named ilex-
cornutoside F.
Compounds 2 and 3 showed the same molecular formula as those of 1
and 6. Their similar NMR data closely resembled those of 1 and 6. The
only small difference between 2 and 3 in NMR was the chemical shift of
Me-30 (δH 0.94 and δC 12.2 for 2; and δH 1.18 and δC 13.5 for 3), as
similarly observed between 1 and 6 (δH 0.97 and δC 12.2 for 1; and δH
1.19 and δC 13.6 for 6) (Table 1). This indicated that compounds 2 and 3
might be a pair of C-20 stereoisomers. The absolute configuration of 2
was unambiguously established as 17R, 20R, 21R by the single-crystal X-
ray diffraction (Fig. 4). Thus, the absolute configuration of 3 can be
deduced as 17R, 20S, 21R, based on the NMR data of Me-30 of 3. Finally,
the structures of 2 and 3 were identified as (3S,12R,17R,20R, 21R)-3-O-
[β-D-glucopyranosyl-(1 → 2)-α-L-arabinopyranosyl]-12-hydroxyl-19-oxo-
18,19-secours-13(18)-en-28,21-lactone and (3S,12R,17R,20S, 21R)-3-O-
[β-D-glucopyranosyl-(1 → 2)-α-L-arabinopyranosyl]-12-hydroxy-19-oxo-
18,19-secours-13(18)-en-28,21-lactone, respectively, and they were
named ilexcornutosides B and C.
Compound 4 was obtained as colorless needles with a molecular
formula C41H66O14 determined by the negative-ion HR-ESI-MS peak at
m/z 827.4453 [M-H+HCOOH]- (calcd for C42H67O16, 827.4429) and
the 13C NMR data, indicating 9 degrees of unsaturation. A comparison
of the NMR data of 4 and 1–3 revealed that 4 shared the similar A, B, C,
D and E ring systems as that of 1–3 except that a C-19 hydroxyl group
was observed in 4 instead of a keto group at the position in 1–3. The
HMBC correlations from Me-30 to C-21, C-22 and C-19; and from Me-29
and H-20 to C-19 confirmed the assigned positions (Fig. 3). Further
inspection of the NMR data of 4 found that the structure of its aglycone
moiety was the same as that of (3β,12β)-3-O-α-L-arabinopyranosyl-
12,19-dihydroxyl-18,19-secours-13(18)-ene-28,21-lactone (Lee et al.,
2015), with C-3 α-L-arabinopyranosyl group replaced by a β-D-gluco-
pyranosyl-(1 → 2)-α-L-arabinopyranosyl group in 4. However, the
stereo-configuration of C-19 and C-20 could not be defined (Lee et al.,
2015).
Fig. 2. HPLC separation of compounds 1–3 and 6 (A, a mixture of 1–3 and 6; B,
compound 1; C, compound 6; D, compound 2; and E, compound 3). HPLC
conditions: an Agilent 1100 series system with a Capcell Pak MGII C18 column
(4.6 mm × 250 mm, 5 μm); column temperature, 25 °C, the mobile phase:
MeCN-H2O (33:67); flow rate: 1.0 mL/min; detection wavelength, 210 nm.
correlations from H-18 to C-12, C-16 and C-17; from H-22 to C-28; from
Me-30 to C-19, C-21 and C-22; and from Me-29 and H-20 to C-19
(Fig. 3).
The sugar units were identified as D-glucose and L-arabinose by GC-
MS analysis of the acid hydrolysate of 1. The β-anomeric configuration
for the D-glucose was determined by the relative large coupling constant
observed for H-1'' (δH 5.18, d, J = 7.7 Hz). The α-anomeric config-
uration for L-arabinose was determined by the anomeric proton (δH
4.97, d, J = 5.8 Hz) (Che et al., 2011), along with NOESY correlations
from H-1' (δH 4.97) to H-3' (δH 4.35), H-4' (δH 4.37) and H-5′a (δH 3.79)
(Fig. S1) (Li et al., 2015). In addition, the linkage site of the α-L-ara-
binose at the aglycone and the 1 → 2 interglycosidic linkage of the
disaccharide were established through HMBC correlations between H-1'
(δH 4.97) and C-3 (δC 89.1), and between H-1'' (δH 5.18) and C-2' (δC
81.5), respectively (Fig. 3).
The NOESY spectrum of 4 did not provide stereo-configuration in-
formation about the γ-lactone ring and the C-19(20) side chain, though
clear correlations between H-21 (δH 5.06) and H-22a (δH 2.14)/H-16
(δH 2.01); between H-20 (δH 1.77) and H-22b (δH 2.25); and between H-
18 (δH 6.20) and H-22b were observed (Fig. S1). In this study, the
stereochemistry of 4 was established unambiguously from the single-
crystal X-ray analysis (Fig. 4). Thus, the structure of 4 was established
as (3S,12R,17R,19S,20S,21S)-3-O-[β-D-glucopyranosyl-(1 → 2)-α-L-ara-
binopyranosyl]-12,19-dihydroxy-18,19-secours-13(18)-en-28,21-lac-
tone, named ilexcornutoside D.
Compound 5, obtained as white amorphous powders, possessed the
molecular formula of C41H66O13 determined by the positive-ion HR-ESI-
The relative configurations of H-3 and H-12 were determined by the
3

H. Feng, et al.
Phytochemistry175(2020)112363
Table 1
1H NMR (600 MHz) spectra data of 1–6 (δ in ppm, J in Hz, C5D5N).
Pos.
1 (20S, 21S)
2 (20R, 21R)
3 (20S, 21R)
4 (19S, 20S, 21S)
5
6 (20R, 21S)
δH
δH
δH
δH
δH
δH
3
3.24, dd (11.8, 4.5)
0.73, d (11.9)
1.54, m
3.23, dd (11.7, 4.6)
0.73, d (11.7)
1.55, m
3.23, dd (11.8, 4.5)
0.72, d (11.8)
1.54, m
3.23, dd (11.8, 4.4)
0.72, d (11.8)
1.53, m
3.21, dd (11.7, 4.5)
0.76, d (11.7)
1.80, m
3.24, dd (4.6, 11.8)
0.74, d (11.7)
1.53, m
5
9
11
1.69, m
2.16, m
2.16, m
2.14, overlapped
1.67, m
1.98, m
2.14, m
2.16, overlapped
4.60, overlapped
2.28, td (13.6, 3.2)
1.16, dt (13.5, 3.9)
1.98, overlapped
1.60, m
1.64, overlapped
4.64, m
1.65, m
1.66, m
12
15
4.63, m
4.59, dt (7.7, 4.6)
2.32, td (13.8, 3.9)
1.15, dq (12.1, 3.9)
2.01, dt (13.7, 4.2)
1.62, m
5.57, s
4.60, m
2.88, td (13.7, 3.9)
1.18, m
2.87, td (13.7, 3.8)
1.18, overlapped
1.93, overlapped
1.58, m
1.27, overlapped
2.17, overlapped
2.84, m
2.32, td (13.6, 3.5)
1.16, dt (13.8, 4.0)
2.01, m
16
1.94, m
1.64, overlapped
6.39, s
1.24, overlapped
3.67, s
1.60, m
18
19
20
21
6.16, s
6.43, s
6.20, s
6.16, s
4.01, m
3.64, m
2.74, dq (9.1, 7.1)
2.75, dq (9.3, 7.1)
2.79, m
1.77, td (7.0, 4.6)
5.06, dt (10.5, 5.2)
2.75, m
4.82, ddd (10.4, 9.2, 5.5)
4.85, ddd (10.5, 9.1, 5.5)
4.82, ddd (10.2, 7.3, 5.6)
1.27, overlapped
2.17, m
4.80, ddd, (10.1, 7.4, 5.7)
22
2.11, dd (12.5, 5.6)
1.98, overlapped
1.23, s
1.83, m
2.21, dd (12.6, 5.6)
1.93, overlapped
1.22, s
2.25, dd (13.0, 10.8)
2.14, overlapped
1.22, s
1.33, m
2.07, dd (12.8, 10.1)
2.22, m
2.07, dd (12.5, 5.5)
1.23, s
1.48, m
23
1.24, s
1.24, s
24
1.01, s
1.00, s
1.00, s
1.00, s
1.06, s
1.02, s
25
0.81, s
0.82, s
0.81, s
0.81, s
0.88, s
0.82, s
26
1.26, s
1.11, s
1.12, s
1.28, s
1.06, s
1.27, s
27
1.23, s
1.24, s
1.22, s
1.22, s
1.66, s
1.22, s
29
2.16, s
2.13, s
2.13, s
1.32, d (6.2)
1.01, d (7.9)
4.97, d (5.7)
4.59, dt (7.7, 4.6)
4.34, m
1.14, s
2.15, s
30
0.97, d (7.2)
4.97, d (5.8)
4.60, overlapped
4.35, m
0.94, d (7.2)
4.97, d (5.8)
4.60, m
1.18, d (7.1)
4.97, d (5.8)
4.59, t (6.5)
4.37, overlapped
4.37, overlapped
4.30, overlapped
3.80, m
1.21, s
1.19, d (7.1)
4.98, d (5.8)
4.62, m
Ara -1′
Ara -2′
Ara -3′
Ara -4′
Ara -5′
4.97, d (5.8)
4.61, t (6.5)
4.35, m
4.37, m
4.38, overlapped
4.38, overlapped
4.30, dd (11.8, 4.3)
3.79, m
4.37, m
4.38, m
4.36, m
4.38, m
4.30, dd (11.9, 4.3)
3.79, m
4.30, dd (11.8, 4.3)
3.78, m
3.78, m
4.33, m
4.30, m
3.82, m
Glc-1″
Glc-2″
Glc-3″
Glc-4″
Glc-5″
Glc-6″
5.18, d (7.7)
4.10, t (7.7)
4.20, t (9.0)
4.33, d (9.3)
3.82, m
5.18, d (7.7)
4.09, t (8.5).
4.19, t (9.0)
4.33, d (9.2)
3.82, dt (9.5, 3.5)
4.44, d (3.5)
5.17, d (7.8)
4.09, t (8.4)
4.19, t (9.1)
4.30, overlapped
3.79, m
5.17, d (7.7)
4.08, m
5.19, d (7.7).
4.10, t (8.4)
4.20, t (9.0)
5.20, d (7.7)
4.11, t (8.4)
4.21, t (9.0)
4.33, d (9.2)
3.83, dt (9.6, 3.5)
4.45, d (3.5)
4.18, td (9.1, 3.1)
4.32, m
3.81, m
3.82, m
4.44, d (3.5)
4.43, d (3.2)
4.43, dd (5.8, 3.4)
4.44, d (3.5)
MS peak at m/z 789.4415 [M+Na]+ and the 13C NMR data. The 1H
NMR spectrum showed seven methyl signals at δH 0.88 (3H, s), 1.06
(6H, s), 1.14 (3H, s), 1.21 (3H, s), 1.24 (3H, s) and 1.66 (3H, s), a
typical 3α-H signal at δH 3.21 (1H, dd, J = 11.7, 4.5 Hz), and an ole-
finic proton at δH 5.57 (1H, s), together with two anomeric protons at
oic acid, named ilexcornutoside E.
In addition, the other 10 known triterpenoids were identified as
ursolic acid (7) (Zhang et al., 2008), lupeol (8) (Fotie et al., 2006),
uvaol (9) (Wu et al., 2005), pomolic acid 28-O-β-D-glucopyranoside
(10) (Cheng and Cao, 1992), ziyuglycosides
I (12) (Reher and
δH 4.97 (1H, d, J = 5.8 Hz) and 5.19 (1H, d, J = 7.7 Hz) (Table 1). The
Budesinsky, 1992) and II (11) (Cheng and Cao, 1992), ilexside I (13)
(Nakanishi et al., 1982), 3β-O-[α-L-arabinopyranosyl-(1 → 2)-β-D-glu-
curonopyranosyl] −19α-hydroxyurs-12-en-28-oic acid 28-O-β-D-gluco-
pyranosyl ester (14) (Wang et al., 2014), ilexoside II (15) (Nakanishi
et al., 1982), and ilexpublesnin Q (16) (Zhou et al., 2014), by com-
parison of their spectral data with those in the literatures. Among these,
compound 16 with a HSO3 group on the sugar moiety was obtained for
the first time from I. cornuta.
13C NMR spectrum combined with the DEPT-135 and HSQC spectra
showed the presence of 41 carbon signals, which included a carbonyl
carbon signal at δC 181.9, two diagnostic olefinic carbon signals at δC
123.3 and 145.5, two anomeric carbon signals at δC 105.3 and 106.4,
two oxygenated methines at δc 89.3 and 81.5, and seven methyl carbon
signals at δc 15.8, 17.1, 17.9, 25.2, 25.3, 28.6 and 29.3. All these NMR
data indicated that 5 had a 3,19-dihydroxy-Δ12(13) oleane-type skeleton
(Su et al., 2004) with the same sugar moiety as 1–4. Also noted was that
the presence of D-glucose and L-arabinose in 5 was confirmed by acidic
hydrolysis and GC analysis. A direct comparison between 13C NMR data
of 5 and 3β-O-[(α-L-arabinopyranosyl-19α-hydroxyolean-12-en-28-oic
acid (Liu et al., 2005) revealed that their carbon signals of their agly-
cone and arabinose moieties were almost identical, except for the
downfield shift of C-2′ arabinose and the additional glucose residue in
5. All the above assignments were verified by the HMBC spectrum. As
shown in Fig. 3, HMBC correlations from Me-29 (δH 1.14) to C-19 (δC
81.7), from H-19 (δH 3.65) to C-17 (δC 46.5), and from H-1'' (δH 5.19) to
C-2' (δC 81.5) suggested the locations of C-19 hydroxyl and C-2″
terminal glucose. The relative configurations of the hydroxyls at C-3
and C-19 were determined as β- and α-oriented, respectively, by NOESY
correlations from H-3 and H-5 to Me-23; and from H-19 to Me-29 (Fig.
S1). Therefore, the structure of 5 was established as 3β-O-[β-D-gluco-
pyranosyl-(1 → 2)-α-L-arabinopyranosyl]-19α- hydroxyolean-12-en-28-
To date, only eight compounds possessing the same skeleton (18,19-
seco-ursane) as 1–4 and 6 have been reported. Among these, three 19-
hydroxy substituted analoges (alatosides A–C) were reported from the
aerial parts of Sesamum alatum and the absolute configuration of their
aglycones were determined as 17R, 19R, 20S, and 21R by a X-ray dif-
fraction analysis (Potterat et al., 1992). Another three 19-hydroxy
substituted analogues (dunnianaolactones A–C) of 4 were reported in
Ilex dunniana leaves (Zhang et al., 2011), among which the configura-
tion of dunnianaolactone A (with a mono-O-glucosyl group at C-19
hydroxyl) was fully determined as 19R, 20S, and 21R by a X-ray crystal
analysis. However, they all possess a different configuration at C-19
hydroxyl with 4 in this study. In 2015, two 18,19-seco-ursane glycosides
(including a 19-hydroxy substituted and a 19-keto substituted analoge)
were also isolated from I. cornuta; however, their configurations re-
mained unassigned (Lee et al., 2015). These stereoisomers (1–4 and 6)
might be derived from E-secoursanes such as cornutaosides A and B
4

H. Feng, et al.
Phytochemistry175(2020)112363
Fig. 3. Key HMBC correlations of 1–6.
(Wu et al., 2008), which were isolated from the same herb. Cyclization
of the C-17 side chains of cornutaosides A and B took place via in-
tramolecular dehydration, yielding compounds 1–4 and 6.
concentration of 5 μM (P < 0.001) with the decrease of PPARγ ex-
pression to 19.4% and 20.9% relative to that of the vehicle control,
which was comparable to the T0070907 positive control (with the de-
crease of the PPARγ expression by 29.8% relative to the vehicle con-
trol). Compounds 2 and 6 showed relatively weaker inhibitory activity
at 5 μM with the PPARγ expression decrease to 15.4% and 15.9% re-
lative to that of the vehicle control. Compound 4 did not downregulate
the PPARγ expression at the tested concentration. This strongly in-
dicated that the keto group at the C-19 is required for the inhibitory
effect and the C-20S configuration enhances the activity. In addition,
compared to compounds 9 and 10, the ursane-type triterpene glycoside
compounds 13–16 showed marginal or no inhibition, suggesting that
further increase in glycosylation of the ursane aglycone decreases the
activity. This is the first report on the inhibition of PPARγ expression of
the triterpenoids in I. cornuta.
Peroxisome proliferator-activated receptor γ (PPARγ) was an im-
portant transcription factor of cell differentiation (Nguyen et al., 2017).
Inhibition of PPARγ expression in the 3T3-L1 preadipocytes can inhibit
adipocyte differentiation, exerting anti-adipogenesis activity. In this
study, the cytotoxicity of all the isolates (1–16) was evaluated in the
3T3-L1-Lenti-PPARγ-Luc cells using the MTT assay. As a result, all the
compounds did not have any influence on cell viability at 5 μM. The
inhibitory effect of 1–16 on the PPARγ expression was assessed in the
3T3-L1-Lenti-PPARγ-Luc cells using a single luciferase reporter assay.
Among these compounds, seven triterpenoids (1–3, 6, 9, 10 and 12)
displayed significant inhibitory activity of PPARγ expression (Fig. 5).
The four stereoisomers (1–3 and 6) had different levels of inhibitory
activity. Compounds 1 and 3 showed significant inhibitory activity at a
5

H. Feng, et al.
Phytochemistry175(2020)112363
Fig. 4. ORTEP drawing of compounds 1, 2, 4, and 6 (A different numbering system might be used for the structure in the text).
3. Conclusions
4. Experimental section
In this study, four stereoisomers of 12-hydroxy-19-oxo-18,19-se-
cours-13(18)-en-28,21-lactone (1–3 and 6) and their C-19 hydroxyl
derivative (4) were isolated from the 70% ethanol extract of I. cornuta
leaves using a routine RP-HPLC. Their absolute configurations at C-20
and C-21 were unambiguously determined by a single X-ray crystal-
lographic analysis using a Cu Kα radiation. In addition, an undescribed
oleane-type glycoside (5) was isolated and identified, along with 10
known triterpenoids. Compounds 1 and 6 (or 2 and 3) are a pair of
stereoisomers with epimerization at C-20. All the 16 triterpenoids were
tested for their anti-adipogenic activities. The results showed that four
18,19-seco-ursane stereoisomers (1–3 and 6) with a C-19 keto group
and three ursane-type triterpenoid glycosides (9, 10 and 12) could
down-regulate PPARγ expression significantly at 5 μM. These data
would advance the knowledge on how this herb and its products may
reduce body weight.
4.1. General experimental procedures
The optical rotation was recorded on an Autopol VI polarimeter
(Rudolph Research Analytical, Hackettstown, NJ, USA). IR spectra were
obtained with a PerkinElmer FT-IR spectrophotometer (Waltham, MA,
USA). The melting point was determined on a SGW X-4A digital melting
point apparatus (Shanghai INESA Physico-Optical Instrument Co., Ltd.,
Shanghai, China) and was uncorrected. The 1D and 2D NMR spectra
were recorded on
a Bruker Avance III 600 MHz spectrometer
(Fällanden, Switzerland). HR-ESI-MS spectra were obtained with an
Agilent 6500 HPLC ESI-QTOF mass spectrometer (Santa Clara, CA,
USA) or a Waters UPLC Premier ESI-QTOF mass spectrometer (Milford,
MA, USA). Semi-preparative HPLC separation was performed on a LC-
3000 system (Beijing Innovation Technology Co., Ltd., Beijing, China)
equipped with a Capcell Pak MG II C18 column (20 mm × 250 mm,
Fig. 5. The inhibitory effect of compounds 1–16 on
the PPARγ expression in transfected 3T3-L1-Lenti-
PPARγ-Luc cells (n = 4, *: comparison of PPARγ
expression with the vehicle control group, #: com-
parison of PPARγ expression with the positive con-
trol, ** P ≤ 0.01, *** P ≤ 0.001). Con and PC re-
presented the vehicle control group (cells treated
with 0.1% DMSO) and the positive control
(T0070907) at 5 μM, respectively. 1–16 represented
compounds 1–16 at 5 μM. Vertical bars represented
the standard error of means (SEM).
6

H. Feng, et al.
Phytochemistry175(2020)112363
5 μm, Shiseido, Tokyo, Japan). An Agilent 1100 series HPLC system
with a Capcell Pak MG II C18 column (4.6 mm × 250 mm, 5 μm,
Shiseido, Tokyo, Japan) was used for HPLC-DAD analysis. MPLC was
performed on a Buchi Sepacore system (Buchi Labortechnik AG,
Switzerland), and a column packed with C-18 bonded silica gel
(45–60 μm, YMC Co., Ltd., Kyoto, Japan). D101 macroporous resin
(Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), silica gel
(100–200 mesh, 200–300 mesh, Qingdao Marine Chemical Co., Ltd.,
Qingdao, China), Sephadex LH-20 (GE Healthcare Bio-Sciences AB,
Uppsala, Sweden) and MCI-gel (CHP20P, 75–100 μm, Mitsubishi
Chemical Industries Co., Tokyo, Japan) were used for open column
chromatography (CC). GC-MS analysis was carried out on a Trace DSQ
gas chromatography-mass spectrometer (ThermoFisher Scientific,
Waltham, MA, USA) equipped with a HP-5 MS capillary column
(Hewlett Packard, 30 m × 0.25 mm, film thickness 0.25 μm).
subjected to silica gel CC eluted with EtOAc, EtOAc-MeOH (50:1 and
10:1), and EtOAc-MeOH-H2O (10:1:0.1, 6:1:0.2, and 4:1:0.4) to give 6
fractions (Frs. 7.1–7.6). Fraction 7.2 (346 mg) eluting with EtOAc-
MeOH (50:1) was separated by Sephadex LH-20 (MeOH), followed by
purification via preparative TLC (silica gel, EtOAc-MeOH-H2O, 10:2:1)
to give 12 (113 mg). Fraction 7.3 (970 mg) eluting with EtOAc-MeOH
(10:1:0.1) was separated by Sephadex LH-20 using PE-CH2Cl2-MeOH
(3:1:1) as the eluting solvent to give two subfractions. The subfraction
7.3.1 was further purified by semi-preparative HPLC (solvent: MeCN-
H2O, 33:67; flow rate: 8 mL/min) to give 1 (tR = 31.39 min; 33 mg), 2
(tR = 39.48 min; 17 mg), 3 (tR = 40.59 min; 20 mg) and 6
(tR = 37.31 min; 13 mg). Similarly, 4 (tR = 11.67 min; 20 mg) was
obtained from the subfraction 7.3.2. Fraction 7.4 (1.2 g) was separated
by Sephadex LH-20 (CH2Cl2-MeOH, 1:1) and semi-preparative HPLC
(solvent: MeCN-H2O, 40:60; flow rate:
8 mL/min) to give 5
(tR = 34.34 min; 101 mg) and 13 (tR = 36.64 min; 30 mg). Fraction 10
(30 g) was subjected to MCI-gel with aqueous methanol (10–90%) to
give 9 subfractions. Then the subfraction 10.8 eluting with MeOH-H2O
(20:80) was separated by a combination of Sephadex LH-20 (MeOH-
H2O, 1:1) and semi-preparative HPLC (solvent: MeCN-H2O, 18:82; flow
rate: 8 mL/min) to give 14 (tR = 19.99 min; 80 mg). The subfraction
10.9 eluting with MeOH-H2O (10:90) was separated by a combination
of Sephadex LH-20 (MeOH) and semi-preparative HPLC (solvent:
MeCN-H2O, 30:70; flow rate: 8 mL/min) to give 15 (tR = 34.31 min;
19 mg) and 16 (tR = 35.49 min; 15 mg).
4.2. Chemicals
Fetal bovine serum (1659859), pancreatin (1806021), and peni-
cillin-streptomycin (15140-122) were purchased from ThermoFisher
Scientific (San Jose, CA, USA). Dulbecco's Modified Eagle Medium
(DMEM, AC11223317), dimethyl sulfoxide (DMSO, 60313ES60), di-
thiothreitol (DTT, D47340), D-luciferin potassium salt (D37410), and 3-
(4,5-dimethylthyazol-2-yl)-2,5-diphenzyl-tetrazolium bromide (MTT,
M03593) were purchased from Yeasen Biotech Co., Ltd. (Shanghai,
China). T0070907 was purchased from Selleck (Shanghai, China). Tris
(hydroxymethyl)methyl aminomethane (Tris, 154563), coenzyme A
(SLBR2838V), and adenosine triphosphate (ATP, SLBR1590V) were
4.4.1. Ilexcornutoside A (1)
20
Colorless needles (MeOH-H2O, 1:1); mp 265.3–266.5 °C, [α]D
purchased
from
Sigma-Aldrich
(St
Louis,
MO,
USA).
-33.0 (c 0.05, MeOH); IR νmax (KBr) cm−1: 3370, 2947, 1750, 1652,
1563, 1369, 1073; 1H NMR (600 MHz, C5D5N) and 13C NMR (150 MHz,
C5D5N) data, see Tables 1 and 2; ESI-MS m/z 803 [M+Na]+; HR-ESI-
MS m/z 798.4630 [M+NH4]+ (calcd. for C41H68O14N, 798.4634).
Ethylenediaminetetraacetic acid (EDTA, AR, 20140522), NaHCO3 (AR,
F20090928) and MgSO4 (AR, 20150828) were obtained from
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
4.3. Plant material
4.4.2. Ilexcornutoside B (2)
20
Colorless needles (MeOH-H2O, 1:1); mp 271.5–273.2 °C, [α]D
Leaves of Ilex cornuta Lindl. & Paxton (Aquifoliaceae) were pur-
chased from Bozhou Medicinal Materials Market (Anhui, China) in
January (winter), 2011. The material was authenticated by Dr. Lihong
Wu, Institute of Chinese Materia Medica, Shanghai University of
Traditional Chinese Medicine. A voucher specimen (No. 20110113-7)
was deposited at the same Institute.
-41.7 (c 0.05, MeOH); IR νmax (KBr) cm−1: 3358, 2946, 1646, 1552,
1371, 1076; 1H NMR (600 MHz, C5D5N) and 13C NMR (150 MHz,
C5D5N) data, see Tables 1 and 2; ESI-MS m/z 803 [M+Na]+; HR-ESI-
MS m/z 798.4634 [M+NH4]+ (calcd. for C41H68O14N, 798.4634).
4.4.3. Ilexcornutoside C (3)
20
White amorphous powders; [α]D -30.0 (c 0.05, MeOH); IR νmax
4.4. Extraction and isolation
(KBr) cm−1: 3349, 2942, 1648, 1555, 1370, 1077; 1H NMR (600 MHz,
C5D5N) and 13C NMR (150 MHz, C5D5N) data, see Tables 1 and 2; ESI-
MS m/z 803 [M+Na]+; HR-ESI-MS m/z 798.4626 [M+NH4]+ (calcd.
for C41H68O14N, 798.4634).
Leaves of I. cornuta (180 kg) were extracted in triplicate with 2700 L
of 75% aqueous ethanol under reflux for 2.5 h at 80 °C. The alcoholic
extracts were combined, evaporated under reduced pressure, and
freeze-dried to give a crude extract powder (21.84 kg). A part of the
resulting extract (1.21 kg) was suspended in distilled water and sub-
jected to a D101 macroporous resin column eluted with an EtOH-H2O
gradient. The 60% EtOH eluent (171.4 g) was subjected to silica gel CC
eluted successively with petroleum ether (PE) (60–90 °C), PE-CH2Cl2
(1:1), CH2Cl2, CH2Cl2-MeOH (50:1, 25:1, 10:1, 6:1, 3:1 and 1:1) and
MeOH to give 10 fractions (Frs. 1–10). Compound 8 (50 mg) was di-
rectly obtained by precipitating from fraction 2 eluting with PE-CH2Cl2
(1:1). Fraction 5 (5.3 g) eluting with CH2Cl2-MeOH (50:1) was further
fractionated by MPLC with a gradient of MeOH-H2O (from 10:90 to
100:0) to give fractions 5.1–5.8. Compound 9 (15 mg) was obtained by
precipitating from fraction 5.8 (MeOH eluent). Fraction 6 (10.8 g)
eluting with CH2Cl2-MeOH (10:1) was fractionated by MPLC with a step
gradient elution of MeOH-H2O (from 10:90 to 100:0) to give fractions
6.1–6.8. Fraction 6.2 (eluting with MeOH-H2O, 60:40) was chromato-
graphed on Sephadex LH-20 (CH2Cl2-MeOH, 1:1), and then purified by
preparative TLC (silica gel, CH2Cl2-MeOH, 10:1) to give 10 (8 mg) and
11 (33 mg). Compound 7 (20 mg) was obtained as a precipitate from
fraction 6.7 (eluting with MeOH-H2O, 90:10). Fraction 7 (34.4 g) was
4.4.4. Ilexcornutoside D (4)
20
Colorless needles (MeOH-H2O, 1:1); mp 288.1–288.8 °C; [α]D
-43.0 (c 0.05, MeOH); IR νmax (KBr) cm−1: 3347, 2940, 1605, 1547,
1455, 1389, 1073; 1H NMR (600 MHz, C5D5N) and 13C NMR (150 MHz,
C5D5N) data, see Tables 1 and 2; ESI-MS m/z 805 [M+Na]+; HR-ESI-
MS m/z 827.4453 [M-H+HCOOH]- (calcd. for C45H67O16, 827.4429).
4.4.5. Ilexcornutoside E (5)
20
White amorphous powders; [α]D +11.7 (c 0.05, MeOH); IR νmax
(KBr) cm−1: 3372, 2946, 1753, 1370, 1173, 1060; 1H NMR (600 MHz,
C5D5N) and 13C NMR (150 MHz, C5D5N) data, see Tables 1 and 2; ESI-
MS m/z 789 [M+Na]+; HR-ESI-MS m/z 789.4415 [M+Na]+ (calcd.
for C41H64O14Na, 789.4396).
4.4.6. Ilexcornutoside F (6)
20
Colorless needles (MeOH-H2O, 1:1); mp 261.9–262.8 °C, [α]D
-32.3 (c 0.05, MeOH); IR νmax (KBr) cm−1: 3369, 2943, 1757, 1706,
1647, 1363, 1162, 1077; 1H NMR (600 MHz, C5D5N) and 13C NMR
(150 MHz, C5D5N) data, see Tables 1 and 2; ESI-MS m/z 803 [M
7

H. Feng, et al.
Phytochemistry175(2020)112363
Table 2
temperature of 280 °C. Nitrogen gas was used with a constant flow of
1 mL/min. The MS data were acquired using electron impact (EI) at
70 eV and at an electron multiplier voltage of 1271 V in the scan range
of 40–550 m/z. The hydrolysates of compounds 1, 4 and 5 showed
peaks at tR 77.93 and 92.42 (1), 77.93 and 92.42 (4), and 77.92 and
92.44 min (5), respectively, which were identified as L-arabinose and D-
glucose by comparison of their retention times with those of authentic
standards: tR = 92.43 min for D-glucose, tR = 92.51 min for L-glucose,
tR = 78.37 min for D-arabinose, and tR = 77.93 min for L-arabinose.
13C NMR (150 MHz) spectra data of 1–6 (δ in ppm, J in Hz, C5D5N).
Pos.
1 (20S,
2 (20R,
3 (20S,
4 (20S,
5
6 (20R,
21S)
21R)
21R)
21S)
21S)
1
39.5
27.0
89.1
40.0
56.4
18.7
35.4
41.6
50.1
37.7
33.3
69.6
151.4
43.7
27.9
29.8
45.5
119.9
209.7
52.2
77.8
43.5
28.7
17.0
17.1
19.2
22.5
179.0
29.9
12.2
39.4
26.9
89.1
39.9
56.3
18.7
35.2
41.8
49.9
37.6
33.4
69.4
150.5
43.5
28.0
29.5
44.1
116.8
209.4
52.2
77.6
42.0
28.6
17.0
17.0
18.8
22.4
178.9
29.6
12.2
105.2
81.4
73.7
68.5
65.3
106.4
76.7
78.4
71.8
78.5
62.8
39.4
26.9
89.1
39.9
56.3
18.7
35.2
41.8
49.9
37.6
33.3
69.3
150.5
43.4
28.0
29.5
44.4
117.0
209.2
51.8
77.5
42.1
28.5
17.0
17.0
18.9
22.4
179.1
29.6
13.5
105.2
81.4
73.7
68.6
65.3
106.3
76.7
78.5
71.9
78.5
62.9
39.4
26.9
89.1
39.9
56.4
18.7
35.4
41.6
50.2
37.6
33.4
69.6
150.9
43.7
28.0
30.0
46.0
120.4
68.6
45.8
78.1
44.1
28.5
17.0
17.1
19.2
22.6
179.7
21.8
10.6
105.2
81.5
73.7
68.6
65.3
106.4
76.8
78.5
71.9
78.5
62.9
39.0
26.9
89.3
39.9
56.3
19.1
34.2
40.4
48.7
37.5
24.6
39.5
27.0
89.1
40.0
56.4
18.8
35.5
41.7
50.2
37.7
33.4
2
3
4
5
6
7
4.6. X-ray crystallographic analysis
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Single needle crystals for X-ray diffraction analysis of compounds 1,
2, 4 and 6 were grown in a MeOH-H2O (1:1) mixture. The X-ray crys-
tallographic data of 1, 2, 4 and 6 were measured on a Bruker APEX2
CCD diffractometer with a Cu Kα radiation. Their structures were re-
solved by direct methods using SHELXS-97 or SHELXL-2014. Their
crystallographic data have been deposited in the Cambridge
Crystallographic Data Centre (CCDC) with deposition numbers of CCDC
1886087 (1), 1886088 (2), 1894160 (4), and 1886089 (6) which can be
obtained free of charge from CDCC via 12 Union Road, Cambridge, CB2
1EZ, UK (fax: +44-1223336-033 or e-mail: deposit@ccdc.cam.ac.uk).
Crystal data for 1: C41H64O14 H2O, M = 798.93, colorless crystals,
monoclinic, a = 6.8151(2) Å, b = 16.2081(5) Å, c = 35.5417(11) Å,
α = 90°, β = 90°, γ = 90°, V = 3925.9(2) Å3, space group P212121,
crystal size, 0.100 × 0.200 × 0.200 mm3, T = 178(2) K, Z = 4, μ(Cu
Kα) = 0.845 mm−1, 25801 reflections measured, 7170 independent
reflections (Rint = 0.0497). Final R indices [I > 2σ(I)]: R1 = 0.0492,
wR2 = 0.1202. Final R indices (all data): R1 = 0.0534, wR2 = 0.1234.
And goodness-of-fit on F2 = 1.046, Flack parameter: −0.10(7).
Crystal data for 2: C41H64O14 H2O, M = 798.93, colorless crystals,
monoclinic, a = 14.6013(4) Å, b = 7.1506(2) Å, c = 20.2726(6) Å,
α = 90°, β = 96.4610(10)°, γ = 90°, V = 2103.18(10) Å3, space group
P1211, crystal size, 0.100 × 0.200 × 0.200 mm3, T = 302(2) K, Z = 2,
μ(Cu Kα) = 0.788 mm−1, 42968 reflections measured, 8110 in-
dependent reflections (Rint = 0.0388). Final R indices [(I > 2σ(I)]:
R1 = 0.0526, wR2 = 0.1501. Final R indices (all data): R1 = 0.0564,
wR2 = 0.1549. And goodness-of-fit on F2 = 1.031, Flack parameter:
0.04(6).
123.3 69.6
145.5 151.3
42.5
29.6
28.9
46.5
45.3
81.7
36.2
29.7
33.7
28.6
17.1
15.8
17.9
25.2
43.7
28.0
30.0
45.8
120.0
209.4
52.1
77.8
43.8
28.6
17.2
17.1
19.2
22.6
181.9 179.2
25.3
29.3
29.7
13.6
Ara -1′ 105.2
Ara -2′ 81.5
Ara -3′ 73.8
Ara -4′ 68.7
Ara -5′ 65.4
105.3 105.3
81.5
73.8
68.7
65.4
81.6
73.8
68.7
65.4
Glc-1″
Glc-2″
Glc-3″
Glc-4″
Glc-5″
Glc-6″
106.4
76.8
78.5
71.9
78.6
62.9
106.4 106.5
76.8
78.6
71.9
78.6
63.0
76.9
78.6
72.0
78.6
63.0
Crystal data for 4: C41H66O14 H2O, M = 800.95, colorless crystals,
monoclinic, a = 6.8095(3) Å, b = 16.2815(7) Å, c = 36.1959(16) Å,
α = 90°, β = 90°, γ = 90°, V = 4013.0(3) Å3, space group P212121,
crystal size, 0.850 × 0.150 × 0.120 mm3, T = 173(2) K, Z = 4, μ(Cu
Kα) = 0.527 mm−1, 28328 reflections measured, 7594 independent
reflections (Rint = 0.0319). Final R indices [I > 2σ(I)]: R1 = 0.0369,
wR2 = 0.0981. Final R indices (all data): R1 = 0.0380, wR2 = 0.0990.
And goodness-of-fit on F2 = 1.038, Flack parameter: 0.08(6).
+Na]+; HR-ESI-MS m/z 798.4638 [M+NH4]+ (calcd. for C41H68O14N,
798.4634).
4.5. Acidic hydrolysis and GC-MS analysis of compounds 1, 4 and 5
Compounds 1, 4 and 5 (1 mg each) were individually dissolved in
2 mL of 2 M trifluoroacetic acid and heated at 120 °C for 1.5 h. The
hydrolysate was evaporated to dryness in vacuo and the residue was
dried overnight in a vacuum desiccator over P2O5. The residue was
suspended in an anhydrous methanol solution of (S)-1-amino -2-pro-
panol (11.1%, 25 μL), and then mixed with anhydrous methanol solu-
tions of glacial acetate acid (20%, 25 μL) and NaBH3CN (3%, 25 μL).
The resulting mixture was heated at 65 °C for 1.5 h. The reaction
mixture was cooled down to room temperature and then evaporated,
followed by being dissolved, twice in an anhydrous methanol solution
of glacial acetate acid (16.7%, 2.0 mL) and three times in anhydrous
methanol (2.0 mL). The residue was treated with 200 μL of pyridine and
200 μL of acetic anhydride at 100 °C for 45 min. Deionized water (3 mL)
was added to terminate the reaction. The reaction mixture was allowed
to cool to room temperature and extracted with chloroform (3 × 3 mL).
The chloroform layer was washed with water (3 × 2 mL), dried over
anhydrous sodium sulfate, and subjected to GC-MS analysis (Cases
et al., 1995). Both the injector and detector temperatures were 280 °C.
The column temperature was initially set at 140 °C, programmed to rise
at 2 °C/min to 220 °C for 40 min, then at 6 °C/min to a final holding
Crystal data for 6: C41H64O14 H2O, M = 798.93, colorless crystals,
monoclinic, a = 6.7914(2) Å, b = 16.1709(3) Å, c = 35.5642(8) Å,
α = 90°, β = 90°, γ = 90°, V = 3905.77(16) Å3, space group P212121,
crystal size, 0.210 × 0.090 × 0.080 mm3, T = 173(2) K, Z = 4, μ(Cu
Kα) = 0.541 mm−1, 25306 reflections measured, 6845 independent
reflections (Rint = 0.0343). Final R indices [I > 2σ(I)]: R1 = 0.0336,
wR2 = 0.0885. Final R indices (all data): R1 = 0.0359, wR2 = 0.0897.
And goodness-of-fit on F2 = 1.055, Flack parameter: −0.10(6).
4.7. Inhibitory effect on the PPARγ expression in 3T3-L1-Lenti-PPARγ-Luc
cells
4.7.1. Proliferation assay
The PPARγ inhibitory activity was evaluated in the transfected 3T3-
L1-Lenti-PPARγ-Luc cells, which was constructed with Lentivirus
PPARγ Luciferase Reporter. The 3T3-L1 cells were obtained from
Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences. The cells were cultured in DMEM supplemented with 10%
fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in a
8

H. Feng, et al.
Phytochemistry175(2020)112363
humidified 5% CO2 incubator and seeded into 96-well plates at a
concentration of 4 × 103 cells/well. Subsequently, cells were grown to
40–50% confluence and treated with 5 μM of testing samples for 48 h.
After incubation, the medium was removed and 100 μL of MTT solution
(0.5 mg/mL in culture medium) was added to each well. The cells were
incubated for another 4 h, and 100 μL of DMSO was added to each well.
The optical density of each well was measured at 490 nm on a Synergy
H4 microplate reader (BioTek, Winooski, VT, USA). The cell viability
data were presented as a percentage of the vehicle control cells. This
experiment was repeated at least two times with three replicates for
each treatment.
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China Medical Science and Technology Press, Beijing.
Fotie, J., Bohle, D.S., Leimanis, M.L., Georges, E., Rukunga, G., Nkengfack, A.E., 2006.
Lupeol long-chain fatty acid esters with antimalarial activity from Holarrhena flor-
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Antimicrobial activity of oleanolic and ursolic acids: an update. Evid. Based
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4.7.2. Luciferase assay
The 3T3-L1-Lenti-PPARγ-Luc cells were cultured in DMEM supple-
mented with 10% FBS and 1% penicillin-streptomycin at 37 °C in a
humidified 5% CO2 incubator and seeded into 96-well plates at a
concentration of 4 × 103 cells/well. Then the cells were treated with
5 μM of test compounds 1–16 dissolved in 0.1% DMSO for 48 h, and
lysed by 50 μL of 1 × RIPA lysis buffer. The supernatant of each lysate
was transferred to another 96-well plate, and the luciferase reaction
was started by addition of the luciferase assay buffer (a mixture of
200 μL Tri-HCl (pH 7.8), 10 μL of 1 M NaHCO3, 50 μL of 0.5 M MgSO4,
2 μL of 0.5 M EDTA, 15.6 mg DTT, 460 μg coenzyme A, 0.7 mg po-
tassium luciferin, and 1.4 mg ATP was prepared and diluted to 10 mL
with deionized water). Luminescence was measured in a Synergy H4
microplate reader (BioTek, Winooski, VT, USA). This experiment was
repeated at least two times with four replicates for each treatment.
T0070907, a well-known PPARγ inhibitor, was used as a positive con-
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4.8. Statistics
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Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Wang, Y.W., Xie, C.J., 2015. The effects and mechanism of ilexsaponin on blood lipid in
hyperlipemia model rats. Anhui Med. Pharm. J. 19, 242–244.
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Acknowledgements
This work was financially supported by the National Key R&D
Program of China (No. 2019YFC1711000) and the National Natural
Science Foundation of China (No. 81573582).
Zeng, C.Z., Liu, Z.X., Li, J.L., 2011. Research on purification technology of saponins from
Folium Ilicis Cornutae by macroporous resin and its antioxidative activity on oil. J.
Chin. Cereals Oils Assoc. 26, 45–49.
Zhang, J., Yu, R., Wu, X., Ye, Y.H., Zhou, Y.W., 2008. Studies on the chemical constituents
of Ilex cornuta. Nat. Prod. Res. Dev. 20, 821–823.
Appendix A. Supplementary data
Zhang, Y., Li, L.J., Zhang, P., Pi, H.F., Ruan, H.L., Wu, J.Z., 2011. Three new E-secoursane
triterpenoid saponins from the leaves of Ilex dunniana. Hel. Chim. Acta 94,
2207–2214.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2020.112363.
Zhou, S.X., Yao, Z.R., Li, J., Tu, P.F., 2012. Flavonoids from the leaves of Ilex cornuta.
Chin. J. Nat. Med. 10, 84–87.
Zhou, Y., Zeng, K.W., Zhang, J.Y., Li, N., Chai, X.Y., Jiang, Y., Tu, P.F., 2014. Triterpene
saponins from the roots of Ilex pubescens. Fitoterapia 97, 98–104.
Zuo, W.J., Mei, W.L., Zeng, Y.B., Wang, H., Dai, H.F., 2011. Research advances of the
chemical constituents and pharmacological activities of Ilex cornuta. Lindl. et Paxt.
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