Three new triterpenoid saponins from the stems of Ilex asprella

Article abstract of DOI:10.1080/10286020.2018.1449836

Three new triterpenoid saponins, namely asprellinoids A–C (1–3), featuring a sulfate substitution in sugar moiety, were isolated from the stems of Ilex asprella (Hook. et Arn.) Champ. ex Benth. Their structures were elucidated by the spectroscopic data analyses including HR-ESI-MS, IR, and NMR spectra, and chemical method.

Full text of DOI:10.1080/10286020.2018.1449836

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Three new triterpenoid saponins from the stems
of Ilex asprella
He-Xin-Ge Zhang, Bing-Zhao Du, Feng Zhao, Xin-Yao Yang, Shun-Gang Jiao,
Jian-Yong Xing, Zheng-Zhou Han, Peng-Fei Tu & Xing-Yun Chai
To cite this article: He-Xin-Ge Zhang, Bing-Zhao Du, Feng Zhao, Xin-Yao Yang, Shun-Gang Jiao,
Jian-Yong Xing, Zheng-Zhou Han, Peng-Fei Tu & Xing-Yun Chai (2018): Three new triterpenoid
saponins from the stems of Ilex asprella, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2018.1449836
To link to this article: https://doi.org/10.1080/10286020.2018.1449836
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Journal of asian natural Products research, 2018
https://doi.org/10.1080/10286020.2018.1449836
Three new triterpenoid saponins from the stems of Ilex
asprella
He-Xin-Ge Zhanga, Bing-Zhao Dua, Feng Zhaoa, Xin-Yao Yanga, Shun-Gang Jiaoa,
Jian-Yong Xingb, Zheng-Zhou Hanb, Peng-Fei Tua and Xing-Yun Chaia
aModern research center for traditional chinese Medicine, school of chinese Materia Medica, Beijing
university of chinese Medicine, Beijing 100029, china; bsanJiu Medical & Pharmaceutical co., ltd., shenzhen
518029, china
ABSTRACT
ARTICLE HISTORY
received 2 december 2017
accepted 5 March 2018
Three new triterpenoid saponins, namely asprellinoids A–C (1–3),
featuring a sulfate substitution in sugar moiety, were isolated from the
stems of Ilex asprella (Hook. et Arn.) Champ. ex Benth. Their structures
were elucidated by the spectroscopic data analyses including HR-ESI-
MS, IR, and NMR spectra, and chemical method.
KEYWORDS
aquifoliaceae; Ilex
asprella; sulfated saponin;
asprellinoids a–c
1. Introduction
e plant Ilex asprella (Hook. et Arn.) Champ. ex Benth. (Aquifoliaceae), is mainly distrib-
uted in Southern China. Its roots have been widely used as traditional Chinese medicine
for the treatment of wind-heat cold, acute and chronic pharyngitis, and sore throat, and as
a major material in the folk herbal drinking [1,2]. Previous phytochemical investigations
on I. asprella revealed its main chemical constituents of triterpenoid saponins, especially
CONTACT Xing-Yun chai
xingyunchai@yeah.net
the supplementary material for this article is available online at https://doi.org/10.1080/10286020.2018.1449836
© 2018 informa uK limited, trading as taylor & francis Group

2
H.-X.-G. ZHANG ET AL.
those sulfated [3]. As a part of research toward the pharmacological efficacy and quality
evaluation, a chemical investigation on I. asprella stems was carried out. Herein, we describe
the isolation and structural elucidation of three new sulfated triterpenoid saponins and
anti-inflammation bioassays against NO production in RAW 264.7 and Bv-2 cells (Figure 1).
2. Results and discussion
Compound 1 was obtained as a white amorphous powder, with [ꢀ]25 +20 (c 0.1, MeOH).
Its molecular formula C36H56O13S, with nine degrees of unsaturatioDn, was determined by
a negative HR-ESI-MS ion at m/z 727.3369 [M–H]−. e 1H NMR data (Table 1) showed
seven methyls at δH 0.79 (3H, s), 0.84 (3H, s), 0.93 (3H, d), 0.94 (3H, s), 1.07 (3H, s), 1.19
(3H, s) and 1.33 (3H, s), a typical axial H-3 at δH 3.22 (1H, dd, J = 4.0, 11.5 Hz), an anomeric
sugar proton at δH 4,45 (1H, d, J = 7.5 Hz) and one olefinic proton at δH 5.28 (1H, br s). Its
13C NMR data (Table 1) showed 30 resonances assignable to a triterpene skeleton and the
remaining six carbon resonances from one hexosyl unit. Resonances of a double bond at
δC 129.5 and 139.9, the typical signals of C-12 and C-13 of a Δ12-ursane skeleton, and an
anomeric carbon at δC 106.2 were observed also. In addition, two oxygenated carbons at δC
90.9 and 73.6 could be observed in the 13C NMR spectrum. e 1H and 13C NMR data of 1
are similar to those of (3β)-19-hydroxy 28-oxours-12-en-3-yl β-D-glucopyranosiduronic
acid n-butyl ester except for the sugar moiety [4]. According to the molecular weight of 1,
the S–O vibration (1233 cm−1) in IR spectrum and the C-3′ data (δC 85.2), the 3′-OH in
D-glucuronic acid was replaced by a sulfinic acid group [5]. HMBC correlations from H-1′
(δH 4.45, d, J = 7.5 Hz) to C-3 (δC 90.9) determined its glycosylation site at the 3-O-position,
and the correlations from H-1′ (δH 4.45, d, J = 7.5 Hz) to C-3′ (δC 85.2) and from H-4′ (δH
3.59-3.64 m) to C-6′ (δC 176.3) showed the carbon connection of sugar moiety (Figure 2).
us, the structure of compound 1 was determined and named as asprellinoid A.
Compound 2 was obtained as a yellow amorphous powder, with[ꢀ]2D5 +20 (c 0.1, MeOH).
Its molecular formula C42H66O18S, with 10 degrees of unsaturation, was determined by a
negative HR-ESI-MS ion at m/z 889.3897 [M–H]−. Its 1H and 13C NMR data (Table 1) are
similar to 1, except that 2 has six glycosylation signals more than 1. HMBC correlations
(Figure 2) from H-1′ (δH 4.49, d, J = 7.5 Hz) to C-3 (δC 91.1) and from H-1′′ (δH 5.28-
5.32 m) to C-28 (δC 178.6) established its glycosylation sites. e 28-O- glycoside moiety
was supposed to be D-glucose by comparison with the 13C NMR data of monepaloside F
[6]. HPLC profile comparison with authentic sample, acidic hydrolysis of 2 gave D-glucose.
Figure 1. chemical structures of compounds 1–3.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Table 1. the 1h nMr and 13c nMr spectral data (500/125 Mhz) of 1–2 (cd3od) and 3 (c5d5 n, J in hz).
1
2
3
No.
1H
13C
1H
13C
1H
13C
1
2
3
4
5
6
7
8
0.97–0.99, 1.61–1.63 m
1.53–1.55, 1.73–1.75 m
39.8
26.8
90.9
40.1
57.0
19.4
34.2
41.1
48.8a
37.8
24.7
129.5
139.9
42.5
29.6
26.6
0.97–1.00, 1.60–1.63 m
1.63–1.67, 1.90–1.92 m
3.20 dd 3.5, 11.5
–
0.79–0.82 m
1.32–1.35, 1.49–1.53 m
1.28–1.29, 1.53–1.56 m
–
1.63–1.66 m
–
1.71–1.73, 1.94–1.98 m
5.30 br s
39.8
26.8
91.1
40.1
56.9
19.4
34.1
41.2
49.0a
37.8
24.7
129.7
139.5
42.6
29.6
26.5
0.83–0.86, 1.43–1.46 m
1.75–1.80, 1.96–1.99 m
38.7
26.8
88.9
39.7
56.1
18.9
33.6
40.3
48.5
37.4
24.4
123.7
145.1
42.5
29.5
28.7
3.22 dd 4.0, 11.5
3.18 d 7.0
–
–
0.79–0.82 m
1.41–1.42, 1.64–1.66 m
1.28–1.29, 1.54–1.56 m
0.70–0.80 m
1.29–1.33, 1.47–1.51 m
1.31–1.36, 1.47–1.52 m
–
–
9
1.70–1.73 m
1.77–1.83 m
10
11
12
13
14
15
16
–
–
1.10–1.12, 2.00–2.02 m
1.94–1.98, 2.00–2.06 m
5.28 br s
5.56 s
–
–
–
–
–
–
0.98–1.02, 1.76–1.84 m
1.53–1.56 m, 2.57 dt
4.0, 7.0
0.96–0.98, 1.87–1.92 m
1.86–1.88 m, 2.60 dt
4.0, 8.5
1.26–1.30, 2.11–2.15 m
2.01–2.04 m, 2.84 br s
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1′
–
48.7a
55.1
73.6
43.0
27.1
39.0
28.5
17.0
16.0
17.5
24.8
182.4
27.3
16.6
106.2
74.3
85.2
72.3
76.9
176.3
–
48.7a
54.9
73.6
42.6
27.2
38.2
28.5
17.0
16.0
17.6
24.7
178.6
27.1
16.6
106.2
74.1
84.9
72.1
76.6
175.2
95.7
73.8
78.1
71.1
78.4
62.4
–
3.63 br s
3.63 br s
46.4
45.2
81.6
36.0
29.5
34.0
29.2
17.2
15.7
17.8
25.2
181.2
28.4
25.2
106.0
74.6
81.9
74.4
64.4
2.50 s
–
1.31 s
2.51 s
–
1.36–1.39 m
1.21–1.97, 1.67–1.70 m
1.65–1.69, 1.72–1.76 m
1.06 3h s
–
1.22–1.26, 1.73–1.76 m
1.63–1.65, 1.73–1.76 m
1.07 3h s
1.27–1.31, 2.14–2.19 m
2.01–2.07, 2.15–2.21 m
1.20 3h s
0.84 3h s
0.94 3h s
0.79 3h s
1.33 3h s
0.83 3h s
0.95 3h s
0.77 3h s
1.32 3h s
0.84 3h s
0.93 3h s
1.03 3h s
1.68 3h s
–
–
–
1.19 3h s
1.20 3h s
0.93 3h d 6.5
4.49 d 7.5
3.42–3.46 m
4.30 t 9.0
3.63–3.68 m
3.72–3.77 m
–
1.20 3h s
1.12 3h s
4.78 d 5.5
3.98 br s
5.37 t 7.5
0.93 3h d 6.5
4.45 d 7.5
3.45 t 8.5
2′
3′
4′
5′
4.28 t 8.5
3.59–3.64 m
3.64–3.67 m
–
5.08 br s
3.84 t 10.0, 4.84 br s
6′
1″
2″
3″
4″
5″
6″
5.28–5.32 m
3.32–3.35 m
3.41–3.43 m
3.37–3.42 m
3.35–3.37 m
3.67–3.71, 3.77–3.83 m
aoverlapped.
Figure 2. Key hMBc and cosY correlations of compounds 1 and 3.

4
H.-X.-G. ZHANG ET AL.
e characteristic absorption (1241.68 cm−1) due to an S–O band stretching vibration in its
IR spectrum, supporting the existence of a sulfinic acid group in 2 [5]. us, the structure
of 2 was elucidated and named as asprellinoid B.
Compound 3 was obtained as a white amorphous powder, with[ꢀ]2D5 +60 (c 0.1, MeOH).
Its molecular formula C35H56O11S, with eight unsaturation, was determined by the negative
HR-ESI-MS ion at m/z 683.3471 [M–H]−. e 1H NMR data (Table 1) of 3 showed signals
due to seven methyls at δH 0.84 (3H, s), 0.93 (3H, s), 1.03 (3H, s), 1.12 (3H, s), 1.20 (6H, s)
and 1.68 (3H, s), a typical signal at δH 3.18 (1H, d, J = 7.0 Hz) ascribable to an axial H-3,
an anomeric sugar proton at δH 4.78 (1H, d, J = 5.5 Hz) and one olefinic proton at δH 5.56
(1H, s). e 13C NMR data of 3 (Table 1) exhibit 30 resonances assignable to a triterpene
skeleton and the remaining 5 carbon resonances from one pentose sugar unit. ere are
two C=C carbons at C-12 (δC 123.7) and C-13 (δC 145.1), and an anomeric carbon at
1
13
C-1′ (δC 106.0) defined. e H and C NMR data of 3 are similar to those of a known
19-α-hydroxyoleanolic acid 3-O-β-D-glucuronopyranoside expect for the sugar part [7].
Acidic hydrolysis of 3 gave L-arabinose, which was confirmed by HPLC comparison with
an authentic sample. According to the molecular weight and the S–O (1241.68 cm−1) in IR
spectrum, a hydroxy hydrogen in the L-arabinose was replaced by the sulfonic acid [8]. e
connection of the carbons in sugar moiety was also confirmed through a COSY data between
H-1′ (δH 4.78, d, J = 5.5 Hz) and H-2′ (δH 3.98), H-2′ (δH 3.98) and H-3′ (δH 5.37), H-3′ (δH
5.37) and H-4′ (δH 5.08), H-4′ (δH 5.08) and H-5′ (δH 3.84, 4.84) (Figure 2). e HMBC
spectrum showed correlations from H-1′ (δH 4.78, d, J = 5.5 Hz) to C-3 (δC 88.9), deter-
mined its glycosylation sites. us compound 3 was elucidated, named as asprellinoid C.
Considering that a significant in vivo anti-inflammatory effect of I. asprella aqueous
extract (publish elsewhere) that was pharmacologically evaluated using xylene-induced
ear edema in mice and carrageenan-induced paw edema in rats, these isolated triterpenoid
saponins were evaluated the nitrogen oxide (NO) production inhibition lipopolysaccharide
(LPS)-stimulated RAW 264.7 macrophages and Bv-2 cells in vitro, however, compounds 1–3
did not inhibit NO production in these cells at concentration of 40 μM. Related biological
assay is needed.
3. Experimental
3.1. General experimental procedures
Optical rotations were determined on a Rudolph Autopol IV automatic polarimeter
(Rudolph Research Analytical, Hackettstown, U.S.A.). IR spectra were measured on a
ermo Nicolet Nexus 470 FT-IR spectrophotometer using KBr pellets (Nicolet Company,
Madison, U.S.A.). NMR spectra were obtained on a Varian-500 spectrometer (Varian Inc.,
Santa Clara, U.S.A.). HR-ESI-MS were recorded by a Shimadzu LC-MS-IT-TOF (Shimadzu,
Tokyo, Japan). Column chromatography (CC) separations were performed using mac-
roporous resins (Mitsubishi Chemical Corp., Tokyo, Japan), silica gel (Qingdao Haiyang
Chem. Co. Ltd., Qingdao, China), and Sephadex LH-20 (GE Healthcare, Sweden). in
layer chromatography (TLC) was conducted on silica gel GF254 (Qingdao Haiyang Chem.
Co. Ltd., China) and RP C18 plates (Merck, Darmstadt, Germany). Analytical HPLC was
performed on a LC-20A chromatography system composed of a diode array detector (DAD)
(Shimadzu, Tokyo, Japan), Agilent XDB C18 column (5 μm, 4.6 × 250 mm). in-layer

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
chromatography (TLC) was performed on pre-coated silica gel GF254 (Qingdao Haiyang
Chem. Co. Ltd, China).
3.2. Plant material
e stems of Ilex asprella were collected from Meizhou City Grass Lake Planting Base,
Guangdong Province, China, in Sep 2016. e plant was identified by Prof. Zheng-Zhou Han
(SANJIU Medical & Pharmaceutical Co.). A voucher specimen (2016GM901) is deposited
at the Modern Research Center for TCM, Beijing University of Chinese Medicine, China.
3.3. Extraction and isolation
e dried and powdered stems (19.0 kg) were extracted with water (2 × 190 L) for 2 h.
Afer removal of water under reduced pressure, the aqueous extract (4.0 kg) was subjected
to macroporous resin column chromatography (CC) with a gradient of aqueous ethanol
(EtOH-H2O). Fraction from 95% EtOH (1.5 kg) was chromatographed on silica gel, eluting
with a gradient of CH2Cl2-MeOH (10:1→0:1) to yield 12 fractions (Fr. I–Fr. XII). Fr. VIII
(27.1 g) was chromatographed on silica gel, eluting with a gradient of EtOAc-MeOH-H2O-
Formic acid (60:9:3.5:0.7–0:1:0:0) to yield nine subfractions (Frs. VIII1–VIII9), then Fr. VIII3
(2.1 g) was applied to Sephadex LH-20 CC (MeOH) to yield compound 1 (208 mg), and Fr.
VIII5 (3.2 g) was applied to Sephadex LH-20 CC (MeOH) to yield compound 2 (317 mg).
Fr. VII (34.0 g) was subjected to silica gel CC, using a gradient of EtOAc-MeOH-Water-
Formic acid (90:10:0.8:0.1) to yield 10 fractions (Frs. VII1–VII10) and from Fr. VII8 (4.3 g)
compound 3 (465 mg) was obtained by precipitation in MeOH.
3.3.1. Asprellinoid A (1)
White amorphous powder,[ꢀ]2D5 +20 (c 0.1, MeOH). IR (KBr) vmax: 3444, 2936, 2878, 1694,
1619, 1449, 1414, 1389, 1233, 1157, 1069, 1021, 998 cm–1. 1H and 13C NMR spectral data:
see Table 1. HR-ESI-MS :m/z 727.3369 [M–H]− (calcd for C36H55O13S, 727.3371).
3.3.2. Asprellinoid B (2)
Yellow amorphous powder,[ꢀ]2D5 +20 (c 0.1, MeOH). IR (KBr) vmax: 3436, 2934, 1731, 1620,
1455, 1417, 1389, 1259, 1228, 1163, 1073, 1025, 997 cm–1. 1H and 13C NMR spectral data:
see Table 1. HR-ESI-MS: m/z 889.3897 [M–H]− (calcd for C42H65O18S, 889.3910).
3.3.3. Asprellinoid C (3)
White amorphous powder,[ꢀ]2D5 +60 (c 0.1, MeOH). IR (KBr) vmax: 3456, 2943, 2877, 1696,
1636, 1459, 1389, 1241, 1173, 1051, 1019, 914 cm–1. 1H and 13C NMR spectral data: see Table
1. HR-ESI-MS: m/z 683.3471 [M–H]− (calcd for C35H55O11S, 683.3472).
3.4. Acid hydrolysis
Compounds 1–3 were disposed with HCl (2 mol/L) and refluxed at 80 °C for 4 h, respec-
tively. e reaction mixture was diluted with water and extracted exhaustively with CHCl3.
Afer drying under reduced pressure, the residue and authentic samples were dissolved in
anhydrous pyridine and then mixed with a pyridine solution of L-cysteine methyl ester

6
H.-X.-G. ZHANG ET AL.
hydrochloride reaction for 1 h at 60 °C. en o-tolyl isothiocyanate was added, and the
mixture was warmed at 60 °C for another 1 h [9–13].
3.5. NO production inhibitory assays
e procedures used for in vitro anti-inflammatory activity against NO production in BV-2
and RAW 264.7 were same as those previously reported [14].
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is work was supported by the National Natural Science Foundation of China [grant number
81774001], [grant number 81473426].
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