Triterpenoids with Antiplatelet Aggregation Activity from the Roots of Ilex pubescens

Article abstract of DOI:10.1055/s-0042-123708

Two new triterpenes and five new triterpene saponins, named ilexpusons A-G (1 - 7), as well as eight known compounds were isolated from Ilex pubescens. The structures of the new compounds were established by a combination of chemical and spectroscopic methods, including HRESIMS, ¹ H-NMR, ¹³ C-NMR, ¹ H- ¹ H COSY, HSQC, HMBC, and NOESY. Additionally, the biological activity of compounds 1 - 15 against adenosine diphosphate-induced platelet aggregation in rabbit plasma was determined. Among the tested compounds, 1, 2, 5, 6, 8, 13, 14, and 15 exhibited significant inhibition of platelet aggregation in vitro.

Full text of DOI:10.1055/s-0042-123708

Original Papers
Triterpenoids with Antiplatelet Aggregation Activity from the Roots
of Ilex pubescens
Authors
Correspondence
Qinglong Tan1,3*, Maosong Qiu *, Di Cao , Tianqin Xiong ,
1
1
1
Zhongxiang Zhao
1
1
1
1
2
Lei Zhang , Lian Zhou , Li Rong , Jinsong Zhou , Jing Jin ,
Zhongxiang Zhao1
School of Chinese Materia Medica, Guangzhou University of Chinese
Medicine
No. 232, Waihuan East Road, Higher Education Mega Center,
Guangzhou, Guangdong Province, 510006, China
Phone: + 862039358325, Fax: + 862039358253
zzx37@163.com
Affiliations
1
2
3
School of Chinese Materia medica, Guangzhou University of
Chinese Medicine, Guangzhou, China
School of Pharmaceutical Sciences, Sun Yat-Sen University,
Guangzhou, China
Supporting information available online at
Second affiliation hospital, Guangzhou University of Chinese
Medicine, Guangzhou, China
http://www.thieme-connect.de/products
Key words
ABSTRACT
Aquifoliaceae, Ilex pubescens, triterpenoids, ilexpusons A–G,
Two new triterpenes and five new triterpene saponins, named ilexpu-
sons A–G (1–7), as well as eight known compounds were isolated from
Ilex pubescens. The structures of the new compounds were established
by a combination of chemical and spectroscopic methods, including
HRESIMS, 1H‑NMR, 13C‑NMR, 1H- H COSY, HSQC, HMBC, and NOESY.
Additionally, the biological activity of compounds 1–15 against aden-
osine diphosphate-induced platelet aggregation in rabbit plasma was
determined. Among the tested compounds, 1, 2, 5, 6, 8, 13, 14, and
antiplatelet aggregation
received
revised
September 10, 2016
December 3, 2016
December 8, 2016
1
accepted
Bibliography
DOI http://dx.doi.org/10.1055/s-0042-123708
Published online | Planta Med © Georg Thieme Verlag KG Stuttgart ·
New York | ISSN 0032‑0943
15 exhibited significant inhibition of platelet aggregation in vitro.
Correspondence
Jing Jin
School of Pharmaceutical Sciences, Sun Yat-Sen University
No. 132, Waihuan East Road, Higher Education Mega Center,
Guangzhou, Guangdong Province, 510006, China
Phone: + 862039943034, Fax: + 862039943000
jinjing@mail.sysu.edu.cn
2
9]. A phytochemical investigation of the roots of I. pubescens was
Introduction
thus performed as part of our continuous work on the discovery of
new antiplatelet aggregation agents from Ilex species. This led to
the isolation of seven new triterpenoids, named ilexpusons A–G
(1–7), together with eight known triterpenoids (8–15) (▶ Fig. 1).
In this paper, we address the isolation and structural elucidation of
these triterpenoids (1–15), as well as their antiplatelet aggrega-
tion activities in vitro.
Ilex pubescens Hook et Arn. (Aquifoliaceae) is an evergreen shrub
with a wide distribution in Southern China, and the root, called
“
Mao Dong Qing”, has been a well known folk medicine in China
for the treatment of cardiovascular disease, cerebral thrombosis,
and hyperlipidemia [1]. Previous research revealed that I. pubes-
cens is rich in triterpenoids, possessing the bioactivities of inhib-
iting platelet aggregation, preventing thrombosis, enlarging
blood vessels, improving microcirculation, reducing cardiac ische-
mia, and enhancing anoxia resistance [2–19]. In addition, other
species of Ilex L. are a rich resource of triterpenoids and flavo-
noids, with similar pharmacological activities as I. pubescens [20–
Results and Discussion
Air-dried roots of I. pubescens were percolated with MeOH, and
then the extract was concentrated under reduced pressure to
leave a residue, which was suspended in H O and successively ex-
2
tracted with EtOAc and n-BuOH. The EtOAc residue was chroma-
*
Co-first authors.
tographed on silica gel, sephadex LH-20, ODS, and semiprepara-
Tan Q et al. Triterpenoids with Antiplatelet… Planta Med

Original Papers
and 1 was established as 19α-hydroxy-3-oxo-24-norurs-12-en-28-
oic acid.
Ilexpuson B (2) was obtained as white amorphous powder and
−
showed a quasi-molecular ion at m/z 663.3753 [M + HCOO]
(
calcd. for 663.3739). The H and 13C NMR data (▶ Tables 1 and
1
2
) of compound 2 were very similar to those of compound 1,
except for an extra D-glucose group at C-28 in 2. Moreover, acid
hydrolysis of 2 yielded D-glucose, identified by GC analysis. There-
fore, the structure of compound 2 was determined to be 19α-hy-
droxy-3-oxo-24-norurs-12-en-28-O-β-D-glucopyranosyl ester.
Ilexpuson C (3) was isolated as white amorphous powder. The
1H and 13C NMR data (▶ Tables 1 and 2) of compound 3 sug-
gested that it was an oleane-type triterpene with an aldehyde
substitution, whose NMR data were very similar to those of ilexpu-
blesnin M [13]. The most notable difference was the chemical
shifts of C-3 (δ 76.5 in 3; δ 87.0 in ilexpublesnin M) and the ab-
▶
Fig. 1 Structures of compounds 1–7.
C
C
tive HPLC to yield seven new triterpenoids (1–7) along with eight
known (8–15) triterpenoids (▶ Fig. 1). The known compounds
were identified as ilexpublesnin S (8) [17], ilexoside A (9) [30], ole-
anolic acid-3-O-6-O-methyl-β-D-glucuronopyranoside (10) [31],
calenduloside E (11) [32], ilexpublesnin M (12) [13], pedunculo-
side (13) [32], ilexgenin B 3-O-β-D-xylpyranoside (14) [32], and
sence of five carbons (δ 107.3, 78.0, 77.9, 75.2, and 73.4) in ilex-
C
publesnin M, suggesting that a hydroxyl was located at C-3 in 3
instead of the glucuronic acid located at C-3 in ilexpublesnin M.
The NOESY correlations of H-3 (δH 3.61, m) with H -23 (δ 1.49,
3
H
s), H-24 (δ 10.43, s) with H-25 (δ 0.82, s), and H-30 (δ 1.12, s)
H
H
H
with H-18 (δ 3.62, br s) and H-19 (δ 3.63, br s) indicated the β-
H
H
ilexsaponin B (15) [33], respectively, by comparison of observed
and reported spectroscopic and mass spectral data.
orientation of the hydroxyl located at C-3 and the aldehyde, and
the α-orientation of the hydroxyl located at C-19 (▶ Fig. 2). On
the basis of above analysis, 3 was elucidated as 3β,19α-dihydroxy-
olea-24-oxo-12-en-28-oic acid.
3
Ilexpuson A (1) was isolated as white amorphous powder, and
had a molecular formula of C29H44O4 as deduced from its HR‑
−
ESI‑MS ([M – H] m/z 455.3160, calcd. for C29H43O , 455.3166).
Ilexpuson D (4) was isolated as white amorphous powder. Its
1D NMR data were parallel to compound 3, except for a difference
for the downfield shift of C-3 to δ 87.0 ppm (vs. δ 76.5 ppm in
4
The IR spectrum showed absorption bonds due to hydroxyl
(
(
3484 cm ), carbonyl (1732 cm−1), and olefinic groups
−
1
C
C
−
1
1647 cm ). Signals of four tertiary methyls [δ 1.00, 1.14, 1.46,
3), the presence of one anomeric proton signal at δH 4.84 (d,
J = 7.6 Hz), and five extra carbon signals attributed to a xylose
H
1
1
.69 (s, 3 H each)], two doublets [δH 1.07 (d, J = 6.5 Hz) and δH
.15 (d, J = 6.3 Hz)], and one olefinic proton [δ 5.61 (t,
moiety (δ 108.2, 75.6, 78.9, 71.4, and 67.5), suggesting that 4
H
C
1
J = 3.2 Hz)] were observed in the H NMR spectrum (▶ Table 1).
The 13C NMR spectrum (▶ Table 2) exhibited twenty-nine carbon
signals, including six methyls, two olefinic carbons, and two car-
bonyl signals. Analyzing the above data suggested that 1 was a
norursane-type triterpene. The HMBC correlations from one
methyl group at δH 1.07 (3 H, d, J = 6.5 Hz) to C-3/C-4/C-5, and
the NOESY correlation from the same methyl group to H-5 further
suggested that 1 had a 24-norursane-type skeleton (▶ Fig. 2). De-
tailed analysis of the NMR spectroscopic data of 1 with 6β,19α-di-
hydroxy-3-oxo-24-norurs-12-en-28-oic acid [34] indicated their
structural similarity. The main differences between them were
the absence of a hydroxyl in 1 and the chemical shift of C-6 (δC
was an O-xyloside and that the glycosidic linkage was through
the hydroxyl group at C-3. This was verified by the HMBC
(▶ Fig. 2) correlations of δH 4.84 (Xyl H‑1)/C-3 (δ 87.0). In addi-
C
tion, the cross-peaks observed in the NOESY experiment also sup-
ported the proposed relative configuration of 4 (▶ Fig. 2). Acid hy-
drolysis of 4 afforded 3 and D-xylose after identification by the
analysis of TLC and GC. Based on the abovementioned evidence,
the structure of compound 4 was established as 3-O-β-D-xylopy-
ranosyl-3β,19α-dihydroxyolea-24-oxo-12-en-28-oic acid.
Ilexpuson E (5) had the molecular formula C35H54O . Acid hy-
9
drolysis of 5 afforded D-xylose, which was identified by TLC and
GC analyses comparison with authentic samples. Extensive 2D
NMR spectroscopic analysis (▶ Tables 1 and 2) indicated that
compound 5 possessed the same aglycone as that of ilexpubles-
nin D [13], but differed in the sugar moiety in the site of C-3. The
position of the xylose was confirmed to be at C-3 on the basis of
the long-range correlation between the anomeric proton of the
xylose (δH 4.80, d, J = 13.5 Hz) and C-3 (δc 87.1) in the HMBC
spectrum (▶ Fig. 3). Subsequently, compound 5 was elucidated
as 3-O-β-D-xylopyranosyl-3β,19α-dihydroxyurs-24-oxo-12-en-28-
oic acid.
2
2
2.7 in 1; δC 68.8 in 6β,19α-dihydroxy-3-oxo-24-norurs-12-en-
8-oic acid), suggesting that the hydroxyl group located at C-6
was inexistent in 1. The NOESY correlations of H -23 [δ 1.07 (d,
3
H
J = 6.5 Hz)]/H-5 (δ 1.02, m)/H‑9 (δ 1.79, m) proved the α-orien-
H
H
tation of these protons, while correlations of H-4 (δ 2.22, m)/H -
H
3
2
5 (δ 1.00, s)/H -26 (δ 1.15, s) indicated they were β-oriented.
H 3 H
From the perspective of biosynthesis, the configuration of H-18 in
ursane-type triterpenoids should be β-oriented. Also, the NOESY
correlations of H-29 (δH 1.46, s)/H-18 (δH 3.07, br s)/H‑20 (δH
1
.52, m) verified the β-orientation of them and further revealed
Ilexpuson F (6) was isolated as white amorphous powder
and displayed a quasi-molecular ion peak at m/z 663.3751 [M –
H] (calcd. for 663.3749), in agreement with the molecular for-
that the hydroxyl located at C-19 and the H -30 (δ 1.14, s) were
assigned as α-oriented. The signal assignments were completed
by analysis of H- H COSY, HSQC, HMBC, and NOESY correlations,
3
H
−
1
1
mula C36H56O11. The IR experiment showed the presence of
Tan Q et al. Triterpenoids with Antiplatelet… Planta Med

▶
Table 1 1H NMR spectroscopic data of compounds 1–7 [500 MHz, pyridine-d5, δ (ppm), (J in Hz)].
Position
1
2
3
4
5
6
7
1
1.77 m
1.77 m
1.15 m
2.06 m
1.87 m
–
1.39 m
1.04 m
2.26 m
2.04 m
3.61 m
1.59 m
1.00 m
2.36 m
2.31 m
3.56 m
1.62 m
1.01 m
2.33 m
2.07 m
3.56 m
1.41 m
0.80 m
2.06 m
1.76 m
3.59 m
–
1.39 m
0.81 m
2.86 m
2.15 m
3.58 m
–
1
.17 m
2
2.40 m
2
.31 m
3
4
5
6
–
2.22 m
1.02 m
1.47 m
2.21 m
1.01 m
1.41 m
1.20 m
1.48 m
1.39 m
1.74 m
2.10 m
2.08 m
5.58 t (2.9)
2.47 m
1.232 m
3.11 td
(13.0, 3.7)
2.03 m
2.96 br s
–
–
–
–
1.14 m
1.92 m
1.52 m
1.54 m
1.40 m
1.89 m
1.96 m
1.72 m
5.57 br s
2.26 m
1.20 m
2.13 m
1.10 m
1.83 m
1.43 m
1.45 m
1.30 m
1.85 m
2.00 m
1.93 m
5.56 br s
2.83 m
1.18 m
2.11 m
1.11 m
1.79 m
1.41 m
1.53 m
1.35 m
1.84 m
2.02 m
1.52 m
5.57 br s
2.25 m
1.25 m
2.05 m
0.94 m
1.62 m
1.38 m
1.58 m
1.36 m
1.79 m
1.94 m
1.67 m
5.58 br s
2.31 m
1.29 m
2.05 m
0.94 m
1.66 m
1.40 m
1.51 m
1.33 m
1.80 m
1.95 m
1.63 m
5.54 br s
2.17 m
1.28 m
2.01 m
1
.20 m
1.50 m
.34 m
7
9
1
1.79 m
2.11 m
1
1
2
.09 m
1
1
2
5
5.61 t (3.2)
2.32 m
1
.27 m
3.14 td
13.1, 4.4)
.08 m
1
6
(
2
1.27 m
1.19 m
1.32 m
1.35 m
3.05 br s
–
1.46 m
1
1
2
2
8
9
0
1
3.07 br s
–
3.62 br s
3.63 br s
3.63 br s
3.63 br s
3.02 br s
3.62 br s
3.62 br s
–
–
1.52 m
2.11 m
1.38 m
2.00 m
1.227 m
2.41 m
2.30 m
1.07 d (6.5)
–
–
–
1.48 m
1.45 m
1.33 m
2.13 m
2.03 m
1.59 s
1.49 m
2.02 m
1.93 m
2.15 m
2.04 m
1.55 s
2.14 m
1.29 m
2.18 m
2.02 m
1.49 s
2.14 m
1.16 m
2.17 m
2.02 m
1.61 s
2.04 m
1
.36 m
2.16 m
.10 m
1.92 m
2
2
2.20 m
2
2.05 m
2
2
3
4
1.07 d (6.5)
1.56 s
–
10.43 s
10.37 s
10.36 s
4.36 d (2.8)
4.38 t (9.3)
3.64 t (9.3)
0.78 s
3
.62 d (2.8)
2
2
2
2
3
3
5
1.00 s
1.04 s
0.82 s
0.99 s
1.64 s
1.20 s
1.12 s
0.77 s
0.97 s
0.75 s
0.77 s
6
1.14 s
1.25 s
0.99 s
1.05 s
1.01 s
7
1.69 s
1.65 s
1.66 s
1.72 s
1.77 s
1.70 s
9
1.46 s
1.42 s
1.21 s
1.44 s
1.44 s
1.21 s
0
1.15 d (6.3)
1.09 d (6.5)
28-O-Glc
6.31 d (8.1)
4.25 m
4.32 m
4.36 m
4.07 m
1.12 s
1.12 d (6.7)
Xyl
1.13 d (6.6)
GlcA
1.12 s
-O-
1
Xyl
GlcA
4.84 d (7.6)
4.00 t (8.8)
4.18 t (8.8)
4.25 m
4.41 dd
4.80 d (13.5)
3.98 t (8.8)
4.17 t (8.8)
4.22 m
5.16 d (7.9)
4.13 m
5.17 d (7.8)
4.14 t (9.3)
4.37 t (9.3)
4.64 t (9.7)
4.76 d (9.7)
2
3
4.36 m
4
4.64 t (9.6)
4.75 d (9.6)
5
4.39 dd
(
10.7, 5.2)
(10.7, 5.2)
3
.81 t (10.7)
3.78 t (10.7)
6
4.50 m
4
.44 m
Tan Q et al. Triterpenoids with Antiplatelet… Planta Med

Original Papers
▶
Table 2 13C NMR spectroscopic data of compounds 1–7 (125 MHz, pyridine-d5).
Position
1
2
3
4
5
6
7
1
2
3
4
5
6
7
8
9
40.6
38.0
40.7
38.0
38.3
28.7
76.5
53.8
57.5
19.7
33.6
40.1
47.5
37.7
25.0
38.1
27.5
87.0
54.1
57.9
19.6
33.5
40.0
47.4
37.4
24.9
38.8
27.6
87.1
54.1
58.0
19.7
33.8
40.5
46.9
37.3
24.9
38.8
27.2
89.4
44.7
56.5
19.2
34.1
40.6
47.8
36.9
25.0
38.6
28.7
89.4
44.7
56.5
19.3
34.0
37.1
48.4
36.1
24.6
212.4
45.1
54.0
22.7
32.7
40.4
45.6
37.2
25.0
128.2
140.5
42.7
29.6
26.7
48.7
55.0
73.0
42.6
27.3
38.8
12.4
–
212.5
45.1
54.1
22.7
32.8
40.6
45.6
37.2
24.9
128.6
139.8
42.6
29.5
26.5
49.0
54.9
73.0
42.5
27.0
38.0
12.4
–
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-O-
1
2
3
4
5
6
123.5
145.3
42.5
28.7
29.5
46.4
45.2
81.6
36.1
29.5
34.0
21.8
207.9
16.2
17.7
25.2
181.2
29.2
25.1
123.4
145.2
42.5
28.7
29.4
46.4
45.2
81.6
36.0
29.5
33.9
21.8
206.4
16.3
17.6
25.1
181.2
29.2
25.1
Xyl
128.3
140.4
42.5
29.6
26.8
48.7
55.0
73.1
42.8
27.6
38.8
22.0
206.4
16.6
17.4
25.0
181.0
27.3
17.1
Xyl
128.3
140.2
42.4
29.6
26.7
48.6
54.9
73.0
42.7
27.4
38.8
23.6
63.6
15.6
17.1
24.5
181.0
27.2
17.4
GlcA
106.8
75.7
78.4
73.8
78.4
173.0
123.4
145.2
40.2
29.2
27.1
46.4
45.1
81.5
42.5
25.1
33.8
23.7
63.5
15.4
17.6
25.1
181.2
29.5
25.1
GlcA
106.8
75.7
78.5
73.9
78.4
173.0
13.4
17.6
25.0
181.0
27.5
17.2
13.5
17.8
24.8
177.4
27.3
17.0
28-O-Glc
96.2
74.4
79.3
71.6
79.7
62.7
108.2
75.6
78.9
71.4
67.5
108.2
75.7
79.0
71.5
67.6
hydroxyl (3431 cm ), carbonyl (1687 cm−1), and olefinic
−
1
showed that they are in a β-orientation, while the correlations of
H-3/H-23, H-5/H-9 revealed that they are α-orientated (▶ Fig. 3).
Based on the above evidence, compound 6 was identified as
rotungenic acid 3-O-β-D-glucuronide.
Ilexpuson G (7) had molecular a formula of C36H56O11 by HR‑
ESI‑MS. Acid hydrolysis of 7 yielded D-glucuronic acid identified
by TLC analyses. The NMR spectra of 7 were again similar to those
of ilexsaponin M [13]. Interpretation of the spectroscopic data re-
vealed that the only difference was the presence of a hydroxy-
−
1
(
1636 cm ) groups. Acid hydrolysis of 6 gave a D-glucuronic acid,
1
detected by TLC with authentic samples of the sugar. The H and
13C NMR spectra (▶ Tables 1 and 2) of 6 showed a number of sim-
ilarities to those of ilexpublesnin G [13], with the exception of the
difference that the glucuronic acid was located at C-3 in com-
pound 6 instead of the xylose located at C-3 in ilexpublesnin G,
and the glucose located at C-28 in ilexpublesnin G was not in com-
pound 6. The NOESY correlations of H-25/H-26, H-18/H-29
Tan Q et al. Triterpenoids with Antiplatelet… Planta Med

methyl located at C-4 in 7, instead of an aldehyde group located at
C-4 in ilexsaponin M, proven by the chemical shifts of C-4 (δ 44.7
C
in 7, δ 53.6 in ilexsaponin M) and C-24 (δ 63.5 in 7, δ 205.9 in
C
C
C
ilexsaponin M), and the HMBC correlations of H-24/H-3, H-4, H-
2
3
3 (▶ Fig. 3). Thus, compound 7 was elucidated as spathodic acid
-O-β-D-glucuronide.
Previous phytochemical investigations have revealed that I. pu-
bescens is abundant with the oleanane- and ursane-type triterpe-
noids, and the glycosyl is connected with C-3 or C-28 in general. In
the present study, 15 oleanane- and ursane-type triterpenoids
were isolated from the roots of I. pubescens, which confirms the
above conclusion. It is noteworthy that the hydroxy located at C-
3
is oxidized to carbonyl in compounds 1 and 2, representing the
first example in I. pubescens. This phenomenon is rare in the Aqui-
foliaceae family and might have crucial chemotaxonomic signifi-
cance. Besides, compounds 3–5, containing the rare 24-aldehyde
group, could be considered as special components.
As a part of an ongoing search for antithrombotic agents from
natural plants, the antiplatelet aggregation activities of com-
pounds 1–15 isolated from the roots of I. pubescens were eval-
uated in vitro. The IC50 values obtained are shown in ▶ Table 3.
Detailed analysis of those compoundsʼ structure with their anti-
platelet aggregation activity indicated that the ursane-type triter-
penoids might do better than the oleanane-type ones in the anti-
platelet aggregation. With respect to the potent antiplatelet
aggregation activities of the isolated triterpenoids, further studies
are required to confirm efficacy in vivo and the mechanism of anti-
platelet aggregation effects.
▶ Fig. 2 Key 1H- H COSY, HMBC, and NOESY correlations of com-
pounds 1–4.
1
Materials and Methods
General experimental procedures
Optical rotations were measured with a Perkin-Elmer precisely
model 341 polarimeter (Perkin-Elmer). IR spectra were recorded
on a Nicolet Nexus 300 FTIR spectrometer (Thermo Nicolet). 1D
and 2D NMR were performed on a Bruker AVANCE DRX-500 NMR
spectrometer (Bruker) in pyridine-d5 using tetramethylsilane
(
TMS) as the internal standard. HRESIMS spectra were determined
+
on an AB Sciex TripleTOF 5600 apparatus or an LTQ-Orbitrap Elite
spectrometer (Thermo Fisher Scientific). Column chromatogra-
phy was carried out with silica gel (100–200, 200–300, and 300–
4
00 mesh, Qingdao Marine Chemical Co.), C18 reversed-phase sil-
ica gel (YMC), and Sephadex LH-20 (GE-Healthcare Bio-Sciences
AB). Further purifications were performed by semipreparative
HPLC on an RP‑C18 column (5 µm, 250 × 10 mm) with a Wufeng
LC-100 apparatus (Wufeng) at a flow rate of 3.0 mL/min, and tem-
perature of 30°C. Thin-layer chromatography employed pre-
coated Silica gel plates GF254 (Qingdao Marine Chemical Co.).
The spray reagent for sugars was phenyl amine-diphenylamine
followed by heating. GC analysis was carried out on a GC 9800
gas chromatograph using an AT.SE‑30 column (30 m × 0.32 mm
×
0.5 µm; Kechuang): detection by FID; temperature for injector
and detector, 250°C, initial temperature was maintained at
80°C for 1 min and then raised to 250°C at 10°C/min; carrier
Fig. 3 Key 1H- H COSY, HMBC, and NOESY correlations of com-
pounds 5–7.
1
▶
1
gas, N . All of the solvents and chemicals were of analytical grade
2
and were purchased from Tianjin Baishi Chemical Industry Co. Ltd.
Tan Q et al. Triterpenoids with Antiplatelet… Planta Med

Original Papers
semipreparative HPLC (MeCN‑H O, 40:60) to obtain compounds
2
▶
Table 3 Antiplatelet aggregation activity of the active com-
6
(15 mg, tR 10.08 min), 7 (7 mg, tR 14.25 min), 13 (41 mg, tR
pounds.
1
1.82 min), and 14 (23 mg, t 16.33 min). Fr. G (3.2 g) was puri-
R
fied on an ODS column eluting with a MeOH‑H O gradient (20–
2
Compound
IC50/µM
16.0
23.8
15.7
25.3
19.1
19.7
23.9
22.4
65.2
95% Confidence interval
12.1–20.5
3
0–40–50–60–70–80%) to yield compound 15 (42 mg).
1
2
5
6
8
20.4–27.3
Isolates
10.4–22.2
20
Ilexpuson A (1): white amorphous powder; [α]
D
+ 57.0 (c 1.0,
21.1–29.1
MeOH); IR (KBr) νmax 3446, 2978, 2934, 1732, 1686, 1647,
1457, 1384, 1172, 1073, 1041 cm ; 1H NMR (500 MHz, pyri-
−
1
15.6–22.8
1
3
13
14
15
15.8–23.9
dine-d ) and C NMR (125 MHz, pyridine-d ) spectroscopic data,
5 5
see ▶ Tables 1 and 2; HR‑ESI‑MS (negative ion mode) m/z:
17.6–31.3
−
4
55.3160 [M – H] (calcd. for C29H43O : 455.3166).
4
18.7–25.4
2
0
Ilexpuson B (2): white amorphous powder; [α]
D
+ 43.0 (c 1.0,
Ozagrel a
48.9–196.2
MeOH); IR (KBr) νmax 3403, 2933, 2878, 1725, 1702, 1635,
a
Ozagrel was used as a positive control
−1 1
1
456, 1381, 1100, 1070, 594 cm ; H NMR (500 MHz, pyridine-
d5) and 13C NMR (125 MHz, pyridine-d ) spectroscopic data, see
5
▶
Tables 1 and 2; HR‑ESI‑MS (negative ion mode) m/z: 663.3753
−
[
M + HCOO] (calcd. for C36H55O11: 663.3739).
2
0
The antiplatelet aggregation activities of all of the compounds pu-
rity were greater than 95%.
Ilexpuson C (3): white amorphous powder; [α]
D
− 23.3 (c 0.3,
MeOH); IR (KBr) ν
3437, 2936, 2877, 1701, 1638, 1385,
max
−
1
1
H
NMR (500 MHz, pyridine-d5) and 13C NMR
1
046 cm ;
Plant material
(
125 MHz, pyridine-d ) spectroscopic data, see ▶ Tables 1 and 2;
5
−
The roots of I. pubescens were purchased from Zhixin Medcine
Health Co., Ltd., Guangdong Province, China, in 2013. The plant
material was authenticated by Prof. Zhong-Xiang Zhao of Guang-
zhou University of Chinese Medicine, China. A voucher specimen
HR‑ESI‑MS (negative ion mode) m/z: 485.3275 [M – H] (calcd. for
C30H45O : 485.3272).
5
2
0
Ilexpuson D (4): white amorphous powder; [α]
D
+ 53.0 (c 1.0,
MeOH); IR (KBr) ν
3421, 2974, 2939, 1686, 1653, 1384,
max
1203, 1047 cm ; H NMR (500 MHz, pyridine-d ) and 13C NMR
−
1
1
(
MDQ-201305) was deposited in the School of Chinese Materia
5
Medica, Guangzhou University of Chinese Medicine, China.
(125 MHz, pyridine-d ) spectroscopic data, see ▶ Tables 1 and 2;
HR‑ESI‑MS (negative ion mode) m/z: 617.3686 [M – H] (calcd. for
5
−
Extraction and isolation
C35H53O : 617.3695).
9
2
0
The dried and powdered roots of I. pubescens (20 kg) were perco-
lated with 95% MeOH (90 L) at room temperature. The extracts
were combined and concentrated under reduced pressure to
Ilexpuson E (5): white amorphous powder; [α]
D
− 35.1 (c 0.4,
MeOH); IR (KBr) ν
3427, 2930, 2877, 1688, 1644, 1385,
max
1203, 1135, 1047 cm ; H NMR (500 MHz, pyridine-d ) and 13C
−
1
1
5
leave a residue (715 g), which was then suspended in H O (3.0 L)
and partitioned in EtOAc (3.0 L × 3) and n-butanol (3.0 L × 3). The
NMR (125 MHz, pyridine-d ) spectroscopic data, see ▶ Tables 1
2
5
and 2; HR‑ESI‑MS (negative ion mode) m/z: 617.3708 [M – H]−
EtOAc extract (179 g) was chromatographed on silica gel column
(calcd. for C35H53O : 617.3695).
9
2
0
with gradient mixtures of CHCl -MeOH (30:1, 15:1, 10:1, 5:1,
Ilexpuson F (6): white amorphous powder; [α]
D
+ 10.0 (c 0.4,
3
3
1
:1, 2:1, 1:1) elution to yield nine fractions (Frs. A–I, A: 7.5 g, B:
.3 g, C: 72.4 g, D: 43.7 g, E: 5.2 g, F: 12.1 g, G: 3.2 g, H: 6.6 g and
MeOH); IR (KBr) ν
max
3431, 2970, 2934, 1687, 1636, 1384,
1073, 1048 cm ; H NMR (500 MHz, pyridine-d ) and 13C NMR
−
1
1
5
I: 15.6 g, respectively). Fr. B (1.3 g) was subjected to Sephadex LH-
0, eluting with CHCl -MeOH (1:1), and then chromatographed
(125 MHz, pyridine-d ) spectroscopic data, see ▶ Tables 1 and 2;
HR‑ESI‑MS (negative ion mode) m/z: 663.3751 [M – H] (calcd. for
5
−
2
3
on silica gel column eluted with petroleum ether-acetone (8:1,
:1, 3:1, 2:1, 1:1) to afford compounds 1 (10 mg) and 3
5 mg). Fr. E (5.2 g) was fractionated by Sephadex LH-20 (CHCl3-
C36H55O11: 663.3749).
2
0
5
(
Ilexpuson G (7): white amorphous powder; [α]
D
− 13.6 (c 0.7,
−
1
MeOH); IR (KBr) ν
3441, 2940, 1689, 1635, 1384, 1065 cm ;
max
1
H NMR (500 MHz, pyridine-d ) and 13C NMR (125 MHz, pyridine-
MeOH, 1:1) and then chromatographed on a reversed silica gel
5
column eluted with MeOH‑H O (20–30–40–50–60–70–80–90–
d5) spectroscopic data, see ▶ Tables 1 and 2; HR‑ESI‑MS (negative
ion mode) m/z: 663.3744 [M – H]− (calcd. for C36H55O11
:
2
1
00%) to give eight subfractions, E1–E8. Subfraction E5 (89 mg)
was purified by semipreparative HPLC (MeCN‑H O, 48:52) to
663.3749).
2
yield compounds 2 (6 mg, tR 9.04 min), 4 (11 mg, tR 8.06), 5
Analysis of the sugar moiety
(
13 mg, tR 8.67 min), 8 (14 mg, tR 6.87 min), and 9 (21 mg, tR
4.26 min). Subfraction E7 (46 mg) was fractionated by semipre-
parative HPLC (MeCN‑H O, 45:55) to give 10 (43 mg, t
1
Compounds 2 and 4–7 (1 mg) were heated in 3 mL of 9% HCl-
dioxane (1:1) at 90°C for 5 h in a water bath. After partitioning
between CHCl and H O, the aqueous phase was evaporated to
2
R
1
4.36 min), 11 (19 mg, t 9.26 min), and 12 (80 mg, t 7.69 min).
R R
3
2
Fr. F (12.1 g) was chromatographed on an ODS, eluting with a
dryness under vacuum, and then analyzed by TLC over silica gel
(CHCl -MeOH‑H O, 8:5:1) by comparison with authentic sam-
MeOH‑H O gradient (40–50–60–70–80%) to divide into seven
2
3
2
subfractions, F1–F7. Subfraction F5 (134 mg) was purified by
ples. For the sugars of 2, 4–5, and 6–7, the R of glucose, xylose,
f
Tan Q et al. Triterpenoids with Antiplatelet… Planta Med

and glucuronic acid by TLC was 0.49, 0.81, 0.44, respectively.
Furthermore, the residue of sugars was dissolved in anhydrous
pyridine (100 µL) and L-cysteine methyl ester hydrochloride
Acknowledgements
(
0.1 mol/L) was added. The mixture was stirred at 60°C for 1 h,
This work was financially supported by grants from the National
Natural Science Foundation of China (Nos. 81673565 and
81270054) and the program for Outstanding Young Teachers in
Higher Education Institutions of Guangdong Province (No.
Yq2013045). It was also supported by the Science and Tech-
nology Planning Project of Guangdong Province (No.
2016A020226031) and the Guangzhou Science Technology and
Innovation Commission (No. 201607010334).
then 150 µL of HMDS-TMCS (hexamethyldisilazane-trimethyl-
chlorosilane, 3:1) were added, and the mixture was stirred at
6
analyzed by GC [35]. The standard sugars gave peaks at t (min)
1
1
0°C for another 30 min. Finally, the thiazolidine derivative was
R
1.33 for D-xylose, 11.66 for L-xylose, 14.15 for D-glucose, and
4.46 for L-glucose, respectively.
Animals and in vitro evaluation of antiplatelet
aggregation activity
Conflict of Interest
Adult New Zealand white rabbits weighing 2.0–2.5 kg were ob-
tained from the Experimental Animals Center of Guangzhou Uni-
versity of Chinese Medicine. All animal care and experimental
protocols used in the study were approved and performed in
accordance with the guidelines provided by the Animal Ethics
Committee of the Guangzhou University of Chinese Medicine
The authors declare that no competing financial interests.
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