Two new triterpenoid saponins from the leaves of Aralia elata

Article abstract of DOI:10.1080/10286020.2013.805207

Phytochemical investigation of the leaves of Aralia elata has led to the isolation of two new compounds 3-O-β-d-glucopyranosyl-(1 → 3)-β-d-glucopyranosyl-(1 → 3)-β-d-glucopyranosyl echinocystic acid (1) and 3-O-β-d-glucopyranosyl-(1 → 3)-β-d-glucopyranosy

Full text of DOI:10.1080/10286020.2013.805207

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Two new triterpenoid saponins from the
leaves of Aralia elata
Zhi-Qiang Ma a , Yan Zhang b , Cheng-Ke Cai a , Qiang Li a & Jian Ni
a
a School of Chinese Materia Medica, Beijing University of Chinese
Medicine , Beijing , 100029 , China
b School of Pharmaceutical Engineering, Shenyang Pharmaceutical
University , Shenyang , 110016 , China
Published online: 25 Jun 2013.
To cite this article: Journal of Asian Natural Products Research (2013): Two new triterpenoid
saponins from the leaves of Aralia elata , Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2013.805207
To link to this article: http://dx.doi.org/10.1080/10286020.2013.805207
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Journal of Asian Natural Products Research, 2013
http://dx.doi.org/10.1080/10286020.2013.805207
Two new triterpenoid saponins from the leaves of Aralia elata
Zhi-Qiang Maa, Yan Zhangb, Cheng-Ke Caia, Qiang Lia and Jian Nia*
aSchool of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029,
China; bSchool of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang
110016, China
(Received 13 November 2012; final version received 10 May 2013)
Phytochemical investigation of the leaves of Aralia elata has led to the isolation of two
new compounds 3-O-b-D-glucopyranosyl-(1 ! 3)-b-D-glucopyranosyl-(1 ! 3)-b-D-
glucopyranosyl echinocystic acid (1) and 3-O-b-D-glucopyranosyl-(1 ! 3)-b-D-
glucopyranosyl-(1 ! 3)-b-D-glucopyranosyl oleanolic acid 28-O-b-D-glucopyrano-
side (2), together with four known compounds 3–6, which were isolated for the first
time from this genus. Structural determination was accomplished by spectroscopic
analysis, particularly by 13C NMR, 2D NMR, and HR-ESI-MS techniques.
Keywords: Aralia elata; triterpenoid saponins; structural identification
1. Introduction
30-CH3), 1.03 (3H, s, 29-CH3), 0.99 (3H, s,
26-CH3), 0.97 (3H, s, 24-CH3), and 0.82
(3H, s, 25-CH3) and one trisubstituted
olefinic proton at d 5.62 (1H, br s, H-12).
Compound 1 displayed three anomeric
proton signals at d 4.85 (1H, d, J ¼ 7.8 Hz,
glc-H-10), 5.25 (1H, d, J ¼ 7.8 Hz, glc-H-
100), and 5.30 (1H, d, J ¼ 7.8 Hz, glc-H-1000)
in the 1H NMR spectrum, and also
exhibited three anomeric carbon signals at
d 106.4 (glc-C-10), 105.8 (glc-C-100), and
105.3 (glc-C-1000) in the 13C NMR spec-
trum. Characteristic chemical shifts at d
55.9 (C-5), 89.0 (C-3), 74.6 (C-16), and
180.1 (C-28) further revealed that com-
pound 1 was an echinocystic acid glycoside
with three sugars attached to C-3 position
[7]. The monosaccharides obtained after
aqueous acid hydrolysis of compound 1
were identified as glucose by thin-layer
chromatography (TLC) in comparison with
authentic samples. The absolute configur-
ation of the monosaccharides was deter-
mined to be D by the GC analysis of chiral
derivatives of the monosaccharides in the
hydrolysate. The relatively large coupling
Aralia elata is widely distributed in the
northeast of China and Korea [1]. Its leaves
had been used as a folk medicine for
rheumatism, diabetes, and as a tonic in
China, Japan, and Russia [2–5]. It has
been reported that triterpene saponins were
the main active principles [6]. In this
paper, we describe the isolation and the
structural elucidation of two new triter-
penoid saponins, along with four known
saponins obtained from the buds of A. elata
(Figure 1). All of them were isolated for
the first time from this genus.
2. Results and discussion
Compound 1 was obtained as a white
amorphous powder, and its Molish and
Liebermann-Burchard reactions were posi-
tive. Its high-resolution time-of-flight mass
spectrum displayed a [M þ Na]þ ion peak
at m/z 981.4980, indicating a molecular
1
formula of C48H78O19Na. The H NMR
spectrum of compound 1 showed seven
characteristic methyls at d 1.84 (3H, s, 27-
CH3), 1.27 (3H, s, 23-CH3), 1.15 (3H, s,
*Corresponding author. Email: njtcm@263.net
q 2013 Taylor & Francis

2
Z.-Q. Ma et al.
Figure 1. Structures of compounds 1 and 2.
constants (7.8 Hz) for the anomeric pro-
1
tons in the H NMR spectrum suggested
to Molish and Liebermann-Burchard tests.
Its high-resolution time-of-flight mass
spectrum displayed a [M þ Na]þ ion
peak at m/z 1127.5609, indicating a
molecular formula of C54H88O23Na.
Characteristic signals for seven methyls
at d 1.28 (3H, s, 23-CH3), 1.25 (3H, s, 27-
CH3), 1.08 (3H, s, 26-CH3), 0.97 (3H, s,
24-CH3), 0.89 (3H, s, 29-CH3), 0.86 (3H, s,
30-CH3), 0.80 (3H, s, 25-CH3), one
trisubstituted olefinic proton at d 5.41
(1H, br s, H-12), and four anomeric protons
at d 6.33 (1H, d, J ¼ 7.8 Hz, glc-H-1), 5.33
(1H, d, J ¼ 7.8 Hz, glc-H-100), 5.29 (1H, d,
J ¼ 7.8 Hz, glc-H-1000), 4.86 (1H, d,
J ¼ 7.8 Hz, glc-H-10) were shown in the
1H NMR spectrum of 2. And signals of a
pair of olefinic carbons at d 122.8 and
144.2; four anomeric carbons of sugars at d
106.3 (glc-C-10), 105.8 (glc-C-1000), 105.3
(glc-C-100), and 95.7 (glc-C-1); and a
carboxyl carbon at d 176.4 (C-28) were
displayed in the 13C NMR spectrum of 2.
The signals at d 89.0 (C-3) and 55.8 (C-5)
in the 13C NMR spectrum further revealed
that compound 2 was an oleanolic acid
the b-configurations of the glucopyranosyl
moieties. The sugar linkages were deter-
mined on the basis of HMBC spectrum.
A long-range correlation was observed
between a proton signal at d 4.85 (glc-H-
10) and a carbon signal at d 89.0 (C-3) of
the aglycone moiety, while the anomeric
proton signal at d 5.25 (glc-H-100) showed a
correlation with a carbon signal at d 88.3
(C-30) of the inner sugar. Furthermore, the
long-range correlation was also observed
between the anomeric proton signal at d
5.30 (glc-H-1000) and a carbon signal at d
88.5 (C-300) of the middle sugar (Figure 2).
The HMBC correlations mentioned above
confirmed glycosylation at C-3 of agly-
cone with a glc(1 ! 3)-glc(1 ! 3)-glc
moiety. From the above evidences, the
structure of 1 was concluded to be 3-O-b-
D-glucopyranosyl-(1 ! 3)-b-D-glucopyr-
anosyl-(1 ! 3)-b-D-glucopyranosyl echi-
nocystic acid (Figure 1).
Compound 2 was obtained as a white
amorphous powder, with positive reactions
Figure 2. Key HMBC correlations of compounds 1 and 2.

Journal of Asian Natural Products Research
3
glycoside with four sugars attached to C-3
and C-28 positions [8]. The monosacchar-
ides obtained after aqueous acid hydrolysis
of compound 2 were identified as glucose
by the TLC comparison with authentic
samples. The absolute configuration of the
monosaccharides was determined to be D
by the GC analysis of chiral derivatives of
the monosaccharides in the hydrolysate.
The sugar linkages were determined on
the basis of HMBC spectrum, suggesting
the glycosylation at C-3 of aglycone with
a glc(1 ! 3)-glc(1 ! 3)-glc moiety and
at C-28 with a glucose. The anomeric
configurations of the sugar moieties were
determined to be b for glucoses on the
basis of the JH–H values (7.8 Hz). There-
fore, the structure of 2 was concluded to
be 3-O-b-D-glucopyranosyl-(1 ! 3)-b-D-
glucopyranosyl-(1 ! 3)-b-D-glucopyra-
nosyl oleanolic acid 28-O-b-D-glucopyr-
anoside (Figure 1).
Ltd, Kyoto, Japan), and D101 macroporous
adsorption resin (Shanghai Hualing Resin
Factory, Shanghai, China). TLC was carried
out on precoated silica gel GF254 plates
(Qingdao Marine Chemical Factory, Qing-
dao, China). HPLC separations were per-
formed on a Hitachi 655-15 series pumping
system equipped with a Hitachi L-2490
refractive index detector. The column used
was
YMC-Park
ODS-A
column
(250 £ 10mm ID, S-5 mm, 12nm). GC
separations were performed on an Agilent
7890A gas chromatograph equipped with an
SPH-300A FID detector. The column used
was Agilent 19091J-413 capillary column
(30 m £ 320 mm £ 0.25mm).
3.2 Plant material
The leaves of A. elata were collected from
Xinbin county, Fushun city, Liaoning
Province of China in August 2005 and
were taxonomically identified by Prof. Sun
Qishi of Shenyang Pharmaceutical Uni-
versity. A voucher specimen was depos-
ited at School of Traditional Chinese
Materia Medica (No. 050715).
The known compounds 3, 4, 5, and 6
were identified as 3-O-b-D-glucopyranosyl-
(1 ! 3)-a-L-arabinopyranosyl oleanolic
acid 28-O-b-D-glucopyranoside [9], 3-O-
b-D-glucopyranosyl-(1 ! 3)-b-D-gluco-
pyranosyl-(1 ! 3)-a-L-arabinopyranosyl
echinocystic acid [10], 3-O-b-D-glucopyr-
anosyl-(1 ! 4)-b-D-glucopyranosyl olea-
nolic acid 28-O-b-D-glucopyranoside [11],
and 3-O-b-D-glucopyranosyl-(1 ! 3)-b-D-
glucopyranosyl hederagenin 28-O-b-D-glu-
copyranosyl ester [12] on the basis of the
NMR data.
3.3 Extraction and isolation
The air-dried leaves (5 kg) were first
extracted with 60% ethanol for three
times under reflux. The combined sol-
utions were concentrated under vacuum
and were subjected to macroporous resin
D101 column chromatography, eluting
with EtOH–H2O in a gradient manner.
The solution eluted by 60% ethanol was
evaporated to dryness under vacuum to
afford a residue (400 g). The residue was
chromatographed on silica gel with
CH2Cl2 –MeOH (in gradient) to give
seven fractions (Fr. A–G).
3. Experimental
3.1 General experimental procedures
NMR spectra were carried out on Bruker
¨
ARX-600 spectrometer (Bruker, Fallanden,
Switzerland) with tetramethylsilane as an
internal standard. HR-ESI-MS were
measured with a Bruker Daltonics Inc.
micro-TOF-Q spectrometer. Column chro-
matography was performed on a 200–300
mesh silica gel (Qingdao Marine Chemical
Factory, Qingdao, China), YMC Octade-
cylsilyl-A gel (12 nm S-75 mm, YMC Co.,
Fr. C (31 g) was subjected to column
chromatography over silica gel, eluting
with a gradient of increasing MeOH in
CH2Cl2 (30–100%), to yield 10 fractions
(Fr. C1–C10). Fr. C3 (5.2 g) was subjected
to C18 column chromatography eluted with

4
Z.-Q. Ma et al.
MeOH–H2O in a gradient manner to give
12 fractions (Fr. C3–1–C3–12). Among
these fractions, Fr. C3 –10 (1.5 g) was
further separated by silica gel column
chromatography with a solvent system of
CH2Cl2–MeOH–H2O (v/v/v; 6:4:1) to
afford 1 (10 mg). Fr. E (40 g) was subjected
to column chromatography over silica
gel, eluting with a gradient of increasing
MeOH in CH2Cl2 (30–100%), to afford
nine fractions (Fr. E1–E9). Fr. E5 (2.0 g)
was separated by C8 column chromatog-
raphy with a solvent system MeOH–H2O
to afford 2 (15 mg). Fr. F (38 g) was
subjected to column chromatography
over silica gel, eluting with a gradient of
increasing MeOH in CH2Cl2 (20–100%),
to afford eight fractions (Fr. F1–F8). Fr. F3
was separated by C8 column chromatog-
raphy, eluting with a gradient of increasing
MeOH in H2O (20–100%), to afford 3
(25 mg). Fr. F5 was applied to preparative
HPLC with a solvent system MeOH–H2O
(v/v; 5:1) to afford 4 (10 mg). Fr. F8 was
further separated by silica gel column
chromatography with a solvent system of
CH2Cl2–MeOH–H2O (v/v/v; 8:2:0.25) to
afford 5 (20 mg) and 6 (16 mg).
Table 1. 1H NMR spectral data of 1 and 2 (600 MHz in pyridine-d5).
Aglycon
1
2
Sugar
1
2
3-O-Glc
10
3
3.36 (br d,
J ¼ 11.8 Hz)
5.62 (br s)
3.59 (br d,
J ¼ 10.8 Hz)
1.27 (s)
0.97 (s)
0.82 (s)
0.99 (s)
1.84 (s)
3.34 (dd, J ¼ 4.2,
11.4 Hz)
5.41 (br s)
3.19 (dd, J ¼ 4.2 Hz,
13.2 Hz)
4.85 (d,
J ¼ 7.8 Hz)
4.03
4.86 (d,
J ¼ 7.8 Hz)
3.90
12
18
2
30
4.21
4.07
23
24
25
26
27
1.28 (s)
0.97 (s)
0.80 (s)
1.08 (s)
1.25 (s)
40
50
60
Glc
100
4.06
4.21
4.41
4.20
4.21
4.47
4.50
4.29
5.25 (d,
J ¼ 7.8 Hz)
4.04
5.33 (d,
J ¼ 7.8 Hz)
4.21
29
30
1.03 (s)
1.15 (s)
0.89 (s)
0.86 (s)
200
300
4.21
4.06
4.21
4.46
4.21
4.07
4.43
4.21
400
500
600
Glc
1000
4.27
3.94
5.30 (d,
J ¼ 7.8 Hz)
4.06
5.29 (d,
J ¼ 7.8 Hz)
4.04
2000
3000
3.99
4.16
3.91
4.51
4.51
4.18
4.07
4.44
4000
5000
6000
28-O-Glc
1
4.27
4.41
6.33 (d,
J ¼ 7.8 Hz)
4.27
2
3
4
5
6
4.28
4.35
4.45
4.08
4.01
Note: For the protons of sugar moieties, except for anomeric protons, others are significantly overlapped.

Journal of Asian Natural Products Research
5
3.3.1 3-O-b-D-Glucopyranosyl-(1 ! 3)-
b-D-glucopyranosyl-(1 ! 3)-b-D-
glucopyranosylechinocystic acid
spectral data, see Table 2; HR-ESI-MS:
m/z 1127.5609 [M þ Na]þ (calcd for
C54H88NaO23, 1127.5614).
White amorphous powder (MeOH); 1H
NMR (600 MHz, C5D5N) spectral data,
see Table 1; 13C NMR (150 MHz, C5D5N)
spectral data, see Table 2; HR-ESI-MS:
m/z 981.4980 [M þ Na]þ (calcd for
C48H78O19Na, 981.5030).
3.4 Sugar analysis
Each saponin (5 mg) was heated in 2 M HCl
(10 ml) at 958C for 4 h. The reaction
mixture was extracted with CHCl3
(5 ml £ 3). Each remaining aqueous layer
was concentrated to dryness to give a
residue and dissolved in pyridine (2 ml),
and then L-cysteine methyl ester hydro-
chloride (5 mg) was added to the solution.
The mixture was heated at 608C for 2 h, and
chlorotrimethylsilane (0.3 ml) was added,
followed by heating at 608C for 2 h. After
that, the supernatant was analyzed by GC
under the following conditions: column
3.3.2 3-O-b-D-Glucopyranosyl-(1 ! 3)-
b-D-glucopyranosyl-(1 ! 3)-b-D-
glucopyranosyloleanolic acid
28-O-b-D-glucopyranoside
White amorphous powder (MeOH); 1H
NMR (600 MHz, C5D5N): spectral data,
see Table 1; 13C NMR (150 MHz, C5D5N)
Table 2. 13C NMR spectral data of 1 and 2 (150 MHz in pyridine-d5).
Aglycon
1
2
Sugar
1
2
1
2
3
4
5
6
7
8
38.8
26.6
89.0
39.6
55.9
18.6
33.4
39.9
47.2
37.1
23.9
122.4
145.2
42.2
36.2
74.6
48.9
41.5
47.4
31.1
36.2
32.9
28.2
17.1
15.6
17.6
27.3
180.1
33.4
24.8
38.7
26.5
89.0
39.5
55.8
18.5
33.1
39.9
48.0
37.0
23.8
122.8
144.2
42.1
28.2
23.6
47.0
41.7
46.2
30.8
34.0
32.5
28.1
17.0
15.5
17.5
26.1
176.4
33.1
23.4
3-O-Glc
10
106.4
74.8
88.3
69.8
78.3
62.2
106.3
74.5
88.5
69.7
77.9
62.5
2
30
40
50
60
Glc
9
100
105.8
74.4
88.5
69.8
78.3
62.6
105.3
74.2
88.2
69.8
78.2
62.2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
200
300
400
500
600
G0l0c0
1
105.3
75.7
78.7
71.7
77.9
62.6
105.8
75.5
78.6
71.6
78.2
62.1
2000
3000
4000
5000
6000
28-O-Glc
1
2
3
4
5
6
95.7
74.1
78.9
71.1
79.3
62.5

6
Z.-Q. Ma et al.
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then up to 80–2808C, with a rate of
258C/min, keeping at 2808C for 5 min, and
the carrier gas was N2 (1.4 ml/min), split
ratio 1/20, injection temperature: 2508C,
injection volume: 1 ml. The absolute
configurations of the monosaccharides
were confirmed to be all D-glucose in
comparison of the retention times of
monosaccharide derivatives with those of
standard samples: D-glucose (14.489 min).
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