Three new triterpenoid saponins from Ilex pubescens

Article abstract of DOI:10.1080/10286020.2012.738674

Three new triterpene saponins, ilexsaponins D-F (1-3), were isolated from the roots of Ilex pubescens. Their structures were elucidated as 3-O-β-D-glucopyranosyl(1 → 3)-α-L-arabinopyranosyl urs-12,18-diene-24,28-dioic acid 28-O-β-D-glucopyranoside (1), 3-

Full text of DOI:10.1080/10286020.2012.738674

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Three new triterpenoid saponins from
Ilex pubescens
Ling Li a , Yu-Xin He a , Mei-Ling Gou a & Chun Dai a
a Bioengineering College, Xihua University , Chengdu , 610039 ,
China
Published online: 08 Nov 2012.
To cite this article: Ling Li , Yu-Xin He , Mei-Ling Gou & Chun Dai (2012) Three new triterpenoid
saponins from Ilex pubescens , Journal of Asian Natural Products Research, 14:12, 1169-1174, DOI:
10.1080/10286020.2012.738674
To link to this article: http://dx.doi.org/10.1080/10286020.2012.738674
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Journal of Asian Natural Products Research
Vol. 14, No. 12, December 2012, 1169–1174
Three new triterpenoid saponins from Ilex pubescens
Ling Li, Yu-Xin He*, Mei-Ling Gou and Chun Dai
Bioengineering College, Xihua University, Chengdu 610039, China
(Received 24 June 2012; final version received 7 October 2012)
Three new triterpene saponins, ilexsaponins D–F (1–3), were isolated from the roots of
Ilex pubescens. Their structures were elucidated as 3-O-b-D-glucopyranosyl(1 ! 3)-a-L-
arabinopyranosyl urs-12,18-diene-24,28-dioic acid 28-O-b-D-glucopyranoside (1), 3-O-
b-D-glucopyranosyl(1 ! 3)-a-L-arabinopyranosyl urs-12,18-diene-24,28-dioic acid (2),
and 3-O-b-D-glucopyranosyl(1 ! 3)-a-L-arabinoyranosyl-30-hydroxyurs-12,19-diene-
24,28-dioic acid 28-O-b-D-glucopyranoside (3) on the basis of the chemical and
spectroscopic evidence.
Keywords: Ilex pubescens; triterpene saponin; ilexsaponins D–F
1. Introduction
data (Table 1). The 1H NMR spectrum
indicated six methyl group signals at dH
1.72, 1.17, 1.21, 1.08, 1.73 (s, 3H each), and
1.06 (3H, d, J ¼ 7.0 Hz) correlated with the
carbons at dC 24.8, 14.6, 18.7, 22.0, 19.5,
and 18.6 in the HSQC spectrum, respect-
ively. There were two carboxyl carbons at
dC 174.7 and 180.6 and two pairs of olefinic
carbon signals at dC 126.7, 138.8, 133.8,
and 136.0 in the 13C NMR spectrum. An
olefinic proton at dH 5.71 (brt) correlated
with the carbon at dC 126.7 in the HSQC
spectrum (see Table 1) indicated the
presence of an ursane-type triterpene
aglycon. The UV absorbance at 225 nm
suggested that compound 1 contains a
heteroannular-conjugated diene system
[8]. The second double bond located at C-
18/C-19 was assigned by the interactions of
the olefinic proton at dH 5.71 (brt,
J ¼ 3.0 Hz), methyl protons H-29 (dH
1.73) with the carbon signals C-18 (dC
133.8), and methyl protons H-29 (dH 1.73),
H-30 (dH 1.06) with C-19 (dC 136.0) in the
HMBC spectrum. In addition, the HMBC
correlations of H-23 (dH 1.72), H-3
The roots of Ilex pubescens Hook et Arn.
(native name, Mao-Dong-Qing) are a
traditional Chinese medicine (TCM) com-
monly used in South China for the treatment
of cardiovascular disease and hypercholes-
terolemia [1]. Previous phytochemical
investigation of its roots and leaves led to
the isolation of glaberides, triterpene sapo-
nins, and phenolics, specifically vomifoliol
[2–7]. Asapartofourprogramsearchingfor
bioactive saponins from TCMs, we present
in this report the isolation and structural
elucidation of three new triterpene saponins
(1–3) (Figure 1) from the roots of
I. pubescens.
2. Results and discussion
Compound 1 was obtained as a white
amorphous powder. It showed positive
Liebermann–Burchard and Molish reac-
tions. The molecular formula was assigned
as C47H72O19 on the basis of the negative-
ion HR-ESI-MS at m/z 939.4598 [M–H]2,
confirmed by its 1H and 13C NMR spectral
*Corresponding author. Email: heyuxin66@tom.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2012.738674
http://www.tandfonline.com

1170
L. Li et al.
CH2OH
COOR2
COOR2
R1O
R1O
COOH
R1
COOH
R2
R1
R2
1
2
Ara3-Glc
Ara3-Glc
Glc
H
3
Ara3-Glc
Glc
Figure 1. The structures of compounds 1–3.
(dH 3.34) with C-24 (dC 180.6) showed that
the carboxyl carbon at dC 180.6 was located
at C-24, therefore the other carboxyl carbon
at dC 174.7 was attributed to C-28 (see
Figure 2). After extensive NMR (1H NMR,
13C NMR, HMBC, and NOESY) spectral
analysis (Table 1), the aglycon was
established to be a 3b-hydroxyurs-12, 18-
dien-24, 28-dioic acid [9]. ESI-MS showed
the ion peaks at m/z 777 [M–H–162]2, 483
[M–H–162–132–162]2, attributable to
the sequential losses of a hexose, pentose,
and hexose residues. Acid hydrolysis and
gas chromatography (GC) experiment of 1
showed that the sugar moieties are L-
arabinose and D-glucose [10]. H-10 (dH
4.95) of the L-arabinose was correlated
with the C-3 (dC 88.9), and H-100 (dH 5.16)
of the terminal glucose was correlated with
C-30 (dC 81.1) of L-arabinosyl in the HMBC
spectrum. H-1000 (dH 6.28) of the D-glucose
was correlated with the C-28 (dC 174.7) in
the HMBC spectrum. The anomeric con-
figuration of the L-arabinosyl and D-
glucosyl moieties were a and b, respect-
ively, from the 3J1-2 coupling constant and
NOESY experiment [10]. On the basis of
this evidence, compound 1 was character-
ized as 3-O-b-D-glucopyranosyl(1 ! 3)-
a-L-arabinopyranosyl urs-12,18-diene-
24,28-dioic acid 28-O-b-D-glucopyrano-
side, named ilexsaponin D.
Compound 2 was obtained as a white
amorphous powder. It showed positive
Liebermann–Burchard and Molish reac-
tions. The molecular formula was assigned
as C41H62O14 on the basis of the negative-
ion HR-ESI-MS at m/z 777.4033 [M–H]2.
Acid hydrolysis of 2 afforded L-arabinose,
D-glucose, and the aglycon, which is the
same as those of 1. Comparison of the
NMR spectral data for 2 and 1 revealed
that the two compounds are similar except
that signals due to the D-glucopyranoside
linked to C-28 in 1 were absent for 2. This
observation was supported by a relative
downfield shift of C-28 (dC 180.6) of 2 in
the 13C NMR spectrum. Thus, the structure
of 2 was determined as 3-O-b-D-glucopyr-
anosyl (1 ! 3)-a-L-arabinopyranosyl urs-
12,18-diene-24,28-dioic acid, named ilex-
saponin E.
Compound 3 was obtained as a white
amorphous powder. It showed positive
Liebermann–Burchard and Molish reac-
tions. The molecular formula was assigned
as C47H72O20 on the basis of the negative-
ion HR-ESI-MS at m/z 955.4539 [M–H]2.
In the 1H NMR spectrum, the aglycon part
of 3 showed five methyl proton signals dH
1.67, 1.11, 1.19, 1.12, and 1.83 (s, 3H each),
and one olefinic proton signal at dH 5.72
(brt). It also had two pairs of olefinic carbon
signals at dC 128.1, 129.7, 131.1, and 137.6

Journal of Asian Natural Products Research
1171
Table 1. 1H and 13C NMR spectral data for 1–3 (500 MHz for 1H and 125 MHz for 13C, in C5D5N,
d in ppm, J in Hz).a
1
2
3
Position
1
dH
dC
dH
dC
dH
dC
39.2
1.03 m
1.81 m
1.21 m
2.00 m
39.1
1.00 m
1.82 m
1.20 m
2.00 m
39.7
1.06 m
1.70 m
1.98 m
2
3
28.3
88.9
28.7
88.9
28.2
88.7
3.34 dd (14.0, 4.0)
3.33 dd (14.0, 4.
0)
3.31 dd (14.0, 4.0)
4
5
6
48.2
57.1
20.8
48.7
57.0
20.8
48.0
57.0
20.7
1.04 m
2.00 m
2.14 m
1.52 m
2.58 m
1.07 m
1.98 m
2.10 m
1.55 m
2.44 m
1.06 m
1.99 m
2.09 m
1.53 m
7
35.3
35.3
34.5
8
9
39.4
47.6
37.7
23.8
126.7
138.8
45.0
28.9
39.5
47.4
37.7
23.8
126.3
138.9
45.1
28.9
39.7
47.8
37.8
24.0
128.1
137.6
43.7
28.8
1.52 m
1.55 m
1.57 m
10
11
12
13
14
15
2.00 m
5.71 brt (3.0)
2.02 m
5.73 brt (3.0)
1.98 m
5.72 brt (3.0)
1.25 m
2.42 m
1.50 m
2.55 m
1.24 m
2.41 m
1.49 m
2.55 m
1.23 m
2.22 m
2.03 m
16
35.4
35.6
23.6
17
18
19
20
21
49.9
133.8
136.0
34.6
49.9
133.7
136.0
34.7
47.3
51.8
131.1
129.7
24.4
3.63 s
2.14 m
1.25 m
2.07 m
1.68 m
2.14 m
1.72 s
2.14 m
1.23 m
2.07 m
1.68 m
2.13 m
1.71 s
26.8
26.8
2.56 m
22
31.0
31.2
33.0
1.85 m
2.14 m
1.67 s
23
24
25
26
27
28
29
30
24.8
180.6
14.6
18.7
22.0
174.7
19.5
18.6
24.9
180.4
14.5
18.8
22.1
180.6
19.5
18.7
24.7
180.9
14.4
18.3
22.2
176.2
16.8
63.0
1.17 s
1.21 s
1.08 s
1.18 s
1.21 s
1.09 s
1.11 s
1.19 s
1.12 s
1.73 s
1.06 d (7.0)
1.76 s
1.07 d (7.0)
1.83 s
4.39, 4.46 d (6.5)
3-O-sugar-Ara
10
20
30
40
50
104.8
4.95 d (5.5)
4.57 m
4.23 m
4.34 m
3.76 m
4.26 m
105.0
73.4
81.3
68.5
64.7
4.96 d (5.5)
4.55 m
4.23 m
4.33 m
3.76 m
4.25 m
104.8
73.4
81.1
68.3
64.9
4.93 d (5.5)
4.55 m
4.20 m
4.35 m
3.74 m
4.27 m
73.4
81.1
68.3
64.9
Glc
100
106.0
76.4
78.1
71.5
78.1
5.16 d (7.5)
4.07 m
4.17 m
4.31 m
3.80 m
106.2
76.7
78.3
71.7
78.3
5.20 d (7.5)
4.07 m
4.16 m
4.33 m
3.83 m
106.0
76.4
78.1
71.5
78.1
5.11 d (7.5)
4.05 m
4.16 m
4.32 m
3.77 m
200
300
400
500

1172
L. Li et al.
Table 1 – continued
1
2
3
Position
600
dH
dC
dH
dC
dH
dC
62.5
4.38 m
4.41 m
62.6
4.38 m
4.42 m
62.5
4.37 m
4.42 m
20800-O-sugar-Glc
1000
2000
3000
4
95.8
74.1
78.8
71.1
79.2
62.2
6.28 d (8.0)
4.18 t (8.5)
4.25 t (8.9)
4.31 t (9.2)
4.00 m
95.8
74.1
78.9
71.3
79.0
62.4
6.35 d (8.0)
4.22 m
4.30 m
4.42 m
4.12 m
5000
6000
4.37 m
4.42 m
4.41 m
4.54 m
1
a The assignments were based upon H NMR, 13C NMR, HSQC, HMBC, and NOESY spectra.
in the 13C NMR spectrum. UV absorbance
at 203 nm of 3 suggested that there was no
conjugation between the two double bonds.
The double bond was located at C-12/C-13
(dC 128.1 and 137.6) according to the
characteristic proton signal at dH 3.63 (1H,
s, H-18) and carbon signal dC 51.8 (C-18)
[9]. The correlations of H-29 (dH 1.83) with
C-19 (dC 131.1) and C-20 (dC 129.7) in the
HMBC experiment suggested the second
double bond was located at C-19/C-20. The
hydroxymethyl C-30 was attached to C-20
based on the following HMBC correlations
of H-30 (dH 4.39 and 4.46) with C-19 (dC
131.1), C-20 (dC 129.7), and C-21 (dC
24.4). In addition, the two carbonyls at C-
24 and C-28 were assigned by the HMBC
correlations of H-23 (dH 1.67), H-3 (dH
3.31) with C-24 (dC 180.9) and H-18 (dH
3.63) with C-28 (dC 176.2). The aglycon
moiety was further confirmed to be olean-
12, 19-diene-24, 28-dioic acid by compari-
son with the literature data [9]. Acid
hydrolysis of 3 also afforded L-arabinose
and D-glucose. Detailed NMR spectral
comparison of the sugar moiety for 3 with
1 (see Table 1) demonstrated that both
compounds possess same sugar sequence.
Consequently, compound 3 was elucidated
to be 3-O-b-D-glucopyranosyl(1 ! 3)-a-
L-arabinoyranosyl-30-hydroxyurs-12,19-
diene-24,28-dioic acid 28-O-b-D-gluco-
pyranoside, named ilexsaponin F.
Figure 2. Selected HMBC correlations for compounds 1 and 3.

Journal of Asian Natural Products Research
1173
3. Experimental
He, Xihua University. A voucher specimen
(No. 20100810) has been deposited in
the herbarium of Bioengineering College
of Xihua University.
3.1 General experimental procedures
Optical rotations were measured on a
JASCO P-1020 digital polarimeter
(JASCO Corporation, Tokyo, Japan).
Spots were visualized by spraying 10%
H2SO4–EtOH followed by heating. UV
spectra were obtained on a TU-1901
spectrometer (Beijing Purkinje General
Instrument Co., Ltd, Beijing, China). IR
spectra were obtained on a Perkin-Elmer
577 spectrometer (PerkinElmer, Waltham,
MA, USA). NMR spectra were recorded on
an Inova 500 spectrometer (Varian, Palo
Alto, CA, USA), operating at 500 MHz for
1H and 125 MHz for 13C. ESI-MS data were
recorded on an MS Agilent 1100 series LC/
MSD Trap Mass spectrometer (Agilent
Technologies Inc., Santa Clara, CA, USA)
and HR-ESI-MS data were obtained on a
G1969A TOF-MS instrument (Agilent
Technologies Inc.). Precoated silica gel
GF254 plates (Qingdao Haiyang Chemical
Co., Qingdao, China) were employed for
thin layer chromatography. Column chro-
matography was carried out on silica gel
(Qingdao Haiyang Chemical Co.) and
macroporous resin D101 (pore size B 13–
14 nm, 26–60 mesh; Tianjin, China). High
performance liquid chromatography
(HPLC) separation was carried out on an
ODS column (YMC-pack ODS-A,
250 £ 10 mm, i.d. 5 mm, YMC, Kyoto,
Japan) with an Alltech evaporative light
scattering detector (Alltech, Dearfield, IL,
USA). GC analysis was carried out on an
Agilent 6890N gas chromatograph (Agi-
lent, Waldbronn, Germany) using a HP-5
capillary column (28 m £ 0.32 mm i.d.),
with an FID detector (detector temperature:
2608C; column temperature: 1808C; carrier
gas: N2; flow rate: 40 ml/min).
3.3 Extraction and isolation
The roots of I. pubescens (5 kg) were
refluxed two times with 70% (v/v) EtOH,
each for 2 h. After concentrated in vacuo,
the residue was suspended in water,
and partitioned with ethyl acetate and
n-butanol successively. The n-butanol
fraction (114 g) was further chromato-
graphed over a macroporous resin D101
column eluted initially with water, then
with 30%, 50%, and 70% EtOH to give
fractions A–C. Fraction B (34 g) was
subjected to a silica gel column using
CHCl3 –MeOH–H2O
(90:10:0.1–
70:30:5) as an eluent to give fractions
B1–B9. Fraction B3 (2.9 g) was subjected
to ODS open column chromatography
(MeOH–H2O 40:60 to 80:20) to afford
fractions
B
3-1 –B3-4
.
Fraction B3-1
(600 mg) was subjected to prep-HPLC
(MeOH–H2O, 40:60, UV detection at
210 nm, affording 1 (18 mg, tR 16.7 min),
2 (12 mg, 20.7 min), and 3 (5 mg,
18.5 min), respectively.
3.3.1 Ilexsaponin D (1)
22
White amorphous powder; ½aꢀD þ 32
(c ¼ 0.12, MeOH); UV (MeOH) lmax
225 nm; IR (KBr) nmax (cm21): 3432,
1
1724, 1681. H NMR (C5D5N, 500 MHz)
and 13C NMR (C5D5N, 125 MHz) spectral
data, see Table 1; ESI-MS: m/z 939 [M–
H]2; HR-ESI-MS m/z 939.4598 [M–H]2
(calcd for C47H71O19, 939.4595).
3.3.2 Ilexsaponin E (2)
30
White amorphous powder; ½aꢀD þ 34
3.2 Plant material
(c ¼ 0.12, MeOH); UV (MeOH) lmax
The roots of I. pubescens were collected
inGuangzhou, GuangdongProvince, China,
in June 2010, and identified by Prof. Y.-X.
225 nm; IR (KBr) nmax (cm21): 3432,
1
1725, 1680. H NMR(C5D5N, 500 MHz)
and 13C NMR(C5D5N, 125 MHz) spectral

1174
L. Li et al.
data, see Table 1; ESI-MS: m/z 777
[M–H]2; HR-ESI-MS m/z 777.4033
[M–H]2 (calcd for C41H61O14, 777.4066).
were confirmed by comparison with those
of authentic standards [11].
Acknowledgments
This work was financially supported by the
Youth Fund of Sichuan Province Office of
Education (08Z6012), the Chun Hui Project of
Ministry of Education (Z2010103), and the
Innovation Fund of Postgraduate, Xihua
University (YCJJ201243).
3.3.3 Ilexsaponin F (3)
30
White amorphous powder; ½aꢀD þ 40
(c ¼ 0.12, MeOH). UV (MeOH) lmax
203 nm; IR (KBr) nmax (cm21): 3432,
1
1725, 1683. H NMR (C5D5N, 500 MHz)
and 13C NMR (C5D5N, 125 MHz) spectral
data, see Table 1; ESI-MS: m/z 955
[M–H]2; HR-ESI-MS m/z 955.4539
[M–H]2 (calcd for C47H71O20, 955.4544).
References
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and Technological Press, Shanghai,
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3.4 Acid hydrolysis of 1–3
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C.C. Zhu, J.Y. Yang, and Z.X. Zhao,
Fitoterapia 81, 788 (2010).
[3] K. Hidaka, M. Ito, Y. Matsuda, H. Kohda,
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Each saponin (5 mg) was heated in 5 ml of
10% HCl–dioxane (1:1) at 808C for 4 h.
After the dioxane was removed, the
solution was extracted with EtOAc
(3 ml £ 3) to yield the aglycon and the
sugar, respectively. The sugar components
in the aqueous layer obtained after acid
hydrolysis of 1–3 were analyzed by GC
analysis of the methyl sugar peracetates.
The aqueous layer was evaporated and
dissolved in anhydrous pyridine (100 ml),
0.1 M L-cysteinemethyl ester hydrochlo-
ride (200 ml) was added, and the resultant
was warmed at 608C for 1 h. The
trimethylsilylation reagent HMDS–
TMCS (hexamethyldisilazane–trimethyl-
chlorosilane–pyridine, 2:1:10; Acros
Organics, Geel, Belgium) was added and
warmed at 608C for 30 min. To the
mixture, the thiazolidine derivatives were
analyzed by GC for sugar identification.
The retention times of L-arabinose (tR,
5.32 min) and D-glucose (tR, 12.39 min)