Oleanane-type triterpene glucuronides from the roots of Glycyrrhiza uralensis Fischer

Article abstract of DOI:10.1055/s-0029-1240907

Investigation of characteristic constituents of the roots of Glycyrrhiza uralensis Fischer led to isolation of four new triterpene glucuronides, namely uralsaponins CF (1-4), an artificial product, namely the methyl ester of glycyrrhizin (5), as well as six known triterpene glucuronides (6-11). These new compounds were identified by 1D and 2DNMR spectroscopic analysis. The cytotoxicity of the selected compounds and their aglycones were evaluated against HeLa and MCF-7 cancer cell lines, and the preliminary structure-activity relationship was also elucidated.

Full text of DOI:10.1055/s-0029-1240907

Original Papers 1457
Oleanane-type Triterpene Glucuronides from the
Roots of Glycyrrhiza uralensis Fischer
Authors
Yun-Feng Zheng1,2, Lian-Wen Qi1, Xiao-Bing Cui 2, Guo-Ping Peng2, Yong-Bo Peng1, Mei-Ting Ren1, Xiao-Lan Cheng1,
Ping Li1
1
Affiliations
Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Ministry of Education, Nanjing, China
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing, China
2
Key words
Abstract
1D and 2D NMR spectroscopic analysis. The cyto-
toxicity of the selected compounds and their agly-
cones were evaluated against HeLa and MCF-7
cancer cell lines, and the preliminary structure-
activity relationship was also elucidated.
"
l Glycyrrhiza uralensis Fischer
!
"
l Glycyrrhiza
Investigation of characteristic constituents of the
roots of Glycyrrhiza uralensis Fischer led to isola-
tion of four new triterpene glucuronides, namely
uralsaponins C–F (1–4), an artificial product,
namely the methyl ester of glycyrrhizin (5), as
well as six known triterpene glucuronides (6–
11). These new compounds were identified by
"
l Leguminosae
"
l triterpene glucuronides
"
l uralsaponins C–F
"
l cytotoxic activities
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica
Introduction
!
Materials and Methods
!
Glycyrrhiza uralensis Fischer, named “licorice”, is
one of the largest and most widely distributed
plants belonging to the genus Glycyrrhiza (Legu-
minosae). Its dried roots have been extensively
used in Oriental countries as a medicine and
sweetening agent. Chemical investigations
showed that saponins and flavonoids were two
main types of ingredients in licorice roots [1,2].
Glycyrrhizin is a characteristic saponin constitu-
ent in licorice that has been shown to possess di-
verse biological activities including cytotoxic [3,
4], antiallergic [5], and antiviral [6] ones. How-
ever, there are few reports regarding biological
activities of other saponins with similar struc-
tures. In this work, we have isolated and purified
eleven saponins from the roots of G. uralensis in-
cluding four new oleanane-type triterpene glu-
curonides named uralsaponins C–F (1–4), an arti-
ficial product named methyl ester of glycyrrhizin
(5), as well as six known triterpene glucuronides
(6–11), using an ingenious pre-processing meth-
od for co-application of polyamide and macropo-
rous resin column chromatography. The cytotox-
icity against HeLa and MCF-7 cancer cell lines
was evaluated for selected compounds and their
aglycones, and preliminary structure-activity re-
lationship was also elucidated.
General experimental procedures
IR spectra were recorded in KBr disks on a Ther-
mo Nicolet IR100 spectrophotometer. UV spectra
were performed on a Shimadzu UV-2401 spectro-
1
photometer. H and 13C NMR spectra were deter-
mined on a Bruker ASR-500 NMR spectrometer.
Spectra were generally recorded in pyridine-d5
solutions and the solvent signals used as the in-
ternal standard for chemical shifts. HRESIMS and
ESI‑MS/MS spectra were recorded on an Agilent
1200 HPLC/Q‑TOF mass spectrometer instrument
(Agilent Corp.) in positive ion mode. Column
chromatography was performed on polyamide
(30–60 mesh; Sinopharm Chemical Reagent Co.,
Ltd.) and macroporous (20–40 mesh, D101; Hai-
guang Chemical Reagent Co., Ltd.) resins. Medium
pressure liquid chromatography (MPLC) was car-
ried out on a BUCHI apparatus (BUCHI Chroma-
tography Pump B-688, 6.5 cm × 50 cm, column)
with RP-18 silica gel (25–50 µm; Merck). Semi-
preparative HPLC was performed on Waters 600
received
revised
accepted
Dec. 2, 2009
January 9, 2010
January 27, 2010
Bibliography
DOI http://dx.doi.org/
10.1055/s-0029-1240907
Published online March 1, 2010
Planta Med 2010; 76:
1457–1463 © Georg Thieme
Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
Prof. Ping Li
liquid chromatography with an Econosil C18
2.2 × 25 cm, column. HPLC was performed on an
Agilent 1100 HPLC instrument with UV detector.
,
Key Laboratory of Modern
Chinese Medicines
China Pharmaceutical
University, Ministry of Education
24 Tongjia Lane
Nanjing 210009
China
Phone: + 862583271379
Fax: + 862583271379
liping2004@126.com
Plant materials
The aerial parts of G. uralensis were collected in
September 2008 in Hangjinqi County, Nei Meng-
gu Province, China. Voucher specimens (GCN
Zheng Y-F et al. Oleanane-type Triterpene Glucuronides… Planta Med 2010; 76: 1457–1463

1458 Original Papers
20080917) were stored in a dry and dark room, deposited at the
solvent was removed. This compound was then dissolved in
MeOH and injected into HPLC‑ESI‑MS for analysis. The product
in the reaction mixture was unambiguously identified as 3-O-
[β-D-glucuronopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-24-
hydroxy-glabrolide by comparing the retention time (tR) and ESI
fragmentation behaviors with those of compound 6.
Key Laboratory of Modern Chinese Medicines (China Pharma-
ceutical University) and identified by Prof. Ping Li, a professor in
the field of pharmacognosy.
Extraction and isolation
The air dried material of G. uralensis (20 kg) was extracted with
50% aqueous ethanol (100 L × 2, each extraction lasted 2 hours).
After solvent removal, the combined residues were passed over a
polyamide resin column (10 kg, 30–60 mesh, 20 × 200 cm) with
flow velocity 150 mL/min, and the effluent was chromato-
graphed on a macroporous resin column (12 kg, 20–40 mesh,
20 × 200 cm) to afford crude saponin fractions using EtOH‑H2O
(60:40, 40 L, flow rate 120 mL/min) as an eluent. After concen-
tration under vacuum, the residue was preliminarily analyzed
by HPLC on a ZORBAX C18 column (250 × 4.6 mm × 5.0 µm). The
mobile phases consisted of 0.1% trifluoracetic acid water (A) and
methanol (B) using a gradient program of 60–65% B at 0–15 min
and 65–80% B at 15–30 min at a flow rate of 1.0 mL/min. The de-
tection wavelength was set at 254 nm. The retention times of the
target compounds in HPLC were as follows: compound 1 at
9.0 min, 2 at 8.7 min, 3 at 7.8 min, 4 at 9.8 min, 6 at 8.3 min, 7 at
10.9 min, 8 at 12.5, 9 at 18.5 min, 10 at 12.9 min, and 11 at
21.1 min. Based on the preliminary data from HPLC, the residue
(285 g) was then separated by MPLC on a RP-18 silica gel column
(800 g, 25–50 µm, 6.5 × 50 cm) with MeOH‑H2O-AcOH (40:60:1;
50:50:1, 60:40:1, each 4 L) to yield five fractions: fraction A was
eluted with a ratio of 40:60:1 (2 L), fraction B was eluted with a
ratio from 40:60:1 to 50:50:1 (3 L), fraction C was eluted with a
ratio of 50:50:1 (2 L), fraction D was eluted with a ratio from
50:50:1 to 60:40:1 (2 L), and fraction E was eluted with a ratio
of 50:50:1 (3L). Fraction B (about 65 g) was submitted to re-
peated chromatography over RP-18 silica gel column (400 g, 25–
Two-phase acid hydrolysis and determination of
aglycones of 2, 7, 9, and 11
Two-phase acid hydrolysis of glycyrrhizin (11) (30 mg) was car-
ried out in a solution of 10% hydrochloric acid (50 mL) and CHCl3
(25 mL) under reflux for 6 h [7]. After cooling, the CHCl3 layer was
washed with H2O (3 × 2 mL) and concentrated in vacuo to afford
the aglycone (glycyrrhetic acid, 13 mg). This compound was iden-
tified by HRESIMS experiment (pseudomolecular ion [M + H]+ at
m/z 471.3468, C30H46O4), and its purity was tested to be higher
than 95% by HPLC‑UV area normalization method (mobile phase:
CH3OH‑H2O‑TFA with a ratio of 80:20:0.05 and detection wave-
length 254 nm). The aglycones of 2, 7, and 9 (12 mg, 13 mg, and
13 mg, respectively) were obtained using the same method (the
aglycone of 2, m/z 499.3051 [M + H]+, C30H42O6, purity > 95%;
the aglycone of 7, m/z 469.3322 [M + H]+, C30H44O4, purity
> 95%; the aglycone of 9, m/z 486.3345 [M + H]+, C30H46O5, purity
> 95%).
Uralsaponin C (1): white amorphous powder; UV (MeOH) λmax
(log ε) 250.2 (4.14) nm; IR (KBr) νmax 3461, 2962, 1724, 1652,
1045 cm−1; 1H NMR and 13C NMR (C5D5 N, 500 MHz), see l Table
"
1; HRESIMS (+), m/z 825.4261 [M + H]+, (calcd. for C42H65O16
,
825.4267).
Uralsaponin D (2): white amorphous powder; UV (MeOH) λmax
(log ε) 249.2 (4.14) nm; IR (KBr) νmax 3462, 2976, 1745, 1655,
1055 cm−1; 1H NMR and 13C NMR (C5D5 N, 500 MHz), see l Table
"
1; HRESIMS (+), m/z 873.3499 [M + Na]+, (calcd. for C42H58O18Na,
873.3515).
50 µm, 4.5 × 50 cm) eluted with
a CH3CN‑H2O-AcOH (from
20:80:1 to 35:65:1) gradient system to obtain four subfrac-
tions: fraction B1 was obtained using 1 L CH3CN‑H2O-AcOH from
20:80:1 to 25:75:1, fraction B2 was obtained using 2 L
CH3CN‑H2O-AcOH from 25:75:1 to 30:70:1, fraction B3 was
obtained using 2 L CH3CN‑H2O-AcOH with a ratio of 30:70:1,
and fraction B4 was obtained using 2 L CH3CN‑H2O-AcOH from
30:70:1 to 35:65:1. Compounds 2 (80 mg) and 7 (250 mg) were
crystallized from fractions B1 and B4, respectively. Fraction B3
was further fractionated by RP-18 silica gel column chromatogra-
phy (CH3CN‑H2O-AcOH, 30:70:1) to afford 1 (10 mg) and 4
(14 mg). Compounds 3 (15 mg) and 6 (20 mg) were obtained by
semipreparative HPLC (CH3CN‑H2O-AcOH, 35:65:1) from B2.
Separation of fraction C by RP-18 silica gel column chromatogra-
phy yielded 8 (18 mg) and 10 (15 mg) using CH3CN‑H2O-AcOH
(30:70:1). Compound 11 (900 mg) was crystallized from frac-
tion E. Compound 5 (15 mg) was obtained by RP-18 silica gel col-
umn chromatography using CH3OH‑H2O-AcOH with a ratio of
65:35:1 and then by semipreparative HPLC using CH3OH‑H2O-
AcOH with a ratio of 65:35:1 from fraction E. Isolate 9 (200 mg)
was obtained from fraction D by repeated RP-18 silica gel column
chromatography (CH3OH‑H2O-AcOH, 60:30:1).
Uralsaponin E (3): white amorphous powder; UV (MeOH) λmax
(log ε) 249.6 (4.21) nm; IR (KBr) νmax 3425, 2946, 1763, 1654,
1
1053 cm−1; H NMR (C5D5 N, 500 MHz) δ5.68 (1H, s, H-12), 5.40
(1H, overlapped, H-1′′), 4.99 (1H, d, J = 7.0 Hz, H-1′), 4.57 (1H,
overlapped, H-4′′), 4.55 (1H, overlapped, H-5′′), 4.50 (1H, over-
lapped, H-5′), 4.48 (1H, overlapped, H-4′), 4.36 (1H, m, H-3′′),
4.27 (1H, overlapped, H-3′), 4.25 (1H, overlapped, H-2′), 4.20
(1H, overlapped, H-2′′), 4.23 (1H, overlapped, H-22), 4.16, 3.84
(1H each, d, J = 11.0 Hz, H-29), 3.32 (1H, m, H-3), 2.94, 0.98 (1H
each, m, H-1), 2.52, 2.37 (1H each, m, H-19), 2.36 (1H, overlapped,
H-9), 2.32 (1H, overlapped, H-18), 2.29, 2.02 (1H each, over-
lapped, H-2), 1.95, 1.54 (1H each, m, H-21), 1.89, 1.10 (1H each,
overlapped, H-16), 1.63, 1.06 (1H each, overlapped, H-15), 1.47,
1.19 (1H each, overlapped, H-7), 1.45, 1.22 (1H each, overlapped,
H-6), 1.35 (3H, s, H-23), 1.31 (3H, s, H-27), 1.19 (3H, s, H-24), 1.14
(3H, s, H-25), 0.95 (3H, s, H-26), 0.92 (3H, s, H-24), 0.69 (1H, br d,
13
"
J = 11.5, H-5); C NMR (C5D5 N, 500 MHz), see l Table 2; HRE-
SIMS (+), m/z 859.3719 [M + Na]+, (calcd. for C42H60O17Na,
859.3723).
Uralsaponin F (4): white amorphous powder; UV (MeOH) λmax
(log ε) 250.2 (4.12) nm; IR (KBr) νmax 3437, 2975, 1724, 1656,
1
1049 cm−1; H NMR (C5D5 N, 500 MHz) δ5.89 (1H, s, H-12), 5.60
Chemical conversion
(1H, d, J = 7.5 Hz, H-1′′), 4.94 (1H, d, J = 7.5 Hz, H-1′), 3.39 (1H, dd,
J = 4.5, 12.0 Hz, H-3), 2.34 (1H, s, H-9), 1.94 (3H, s, OOCCH3), 0.79,
0.94, 1.07, 1.21, 1.36, 1.37 (3H each, s, 6 × CH3); 13C NMR (C5D5 N,
500 MHz), see l Table 2; HRESIMS (+), m/z 897.4113 [M + H] ,
(calcd. for C44H65O19, 897.4115).
A 5 mL MeOH solution containing 2 mg of compound 4 was
treated with 5 mL 2% NaOH, and then heated at 60°C for 1 h. After
cooling, the reaction mixture was neutralized with 10% hydro-
chloric acid and then extracted with 5 mL n-butanol. Approxi-
mately 1 mg of the compound 6 was obtained after the n-butanol
+
"
Zheng Y-F et al. Oleanane-type Triterpene Glucuronides… Planta Med 2010; 76: 1457–1463

Original Papers 1459
Zheng Y-F et al. Oleanane-type Triterpene Glucuronides… Planta Med 2010; 76: 1457–1463

1460 Original Papers
Table 2 13C NMR chemical shifts of compounds 3–6 (C5D5 N, 500 MHz).
Pos.
3
4
5
6
Pos.
3
4
5
6
δC, mult.
δC, mult.
δC, mult.
δC, mult.
δC, mult.
δC, mult.
δC, mult.
δC, mult.
1
2
39.5 CH2
26.4 CH2
89.2 CH
39.9 qC
39.7 CH2
26.3 CH2
89.3 CH
44.3 qC
39.5 CH2
26.7 CH2
89.2 CH
38.4 qC
39.5 CH2
26.0 CH2
89.6 CH
44.4 qC
24
25
26
27
28
29
30
16.9 CH3
16.8 CH3
18.7 CH3
22.3 CH3
24.0 CH3
63.2 CH2
178.5 qC
63.0 CH2
16.3 CH3
18.2 CH3
23.8 CH3
21.5 CH3
29.1 CH3
178.9 qC
16.5 CH2
16.8 CH3
18.8 CH3
23.6 CH3
27.9 CH3
28.7 CH3
179.2 qC
63.4 CH2
16.5 CH3
20.3 CH3
22.3 CH3
23.9 CH3
21.3 CH3
179.7 qC
3
4
5
55.4 CH
17.6 CH2
33.1 CH2
45.1 qC
55.6 CH
18.0 CH2
32.7 CH2
43.3 qC
55.5 CH
17.6 CH2
32.1 CH2
45.6 qC
56.0 CH
18.5 CH2
33.5 CH2
44.9 qC
6
7
8
9
62.0 CH
37.3 qC
61.7 CH
36.6 qC
62.1 CH
37.3 qC
61.9 CH
37.1 qC
1′
2′
3′
4′
5′
6′
105.1 CH
84.3 CH
77.6 CH
73.1 CH
77.8 CH
172.5 qC
104.2 CH
80.6 CH
77.4 CH
72.8 CH
77.2 CH
172.2 qC
105.0 CH
84.7 CH
76.6 CH
73.0 CH
77.4 CH
172.6 qC
104.7 CH
81.7 CH
77.7 CH
73.0 CH
77.9 CH
172.2 qC
10
11
12
13
14
15
16
17
18
19
20
21
22
199.1 qC
130.0 CH
164.6 qC
44.4 qC
199.3 qC
128.5 CH
168.5 qC
44.1 qC
199.5 qC
128.7 CH
169.6 qC
43.5 qC
198.9 qC
130.0 CH
164.4 qC
45.0 qC
25.3 CH2
26.0 CH2
48.9 qC
25.3 CH2
26.2 CH2
34.8 qC
26.6 CH2
26.8 CH2
48.7 qC
25.2 CH2
25.9 CH2
35.0 qC
1′′
106.6 CH
76.7 CH
77.2 CH
73.4 CH
79.1 CH
172.8 qC
104.5 CH
75.4 CH
77.3 CH
72.9 CH
77.5 CH
172.1 qC
170.2 qC,
20.7 CH3
106.9 CH
77.3 CH
77.7 CH
73.0 CH
77.8 CH
170.2 qC
105.4 CH
75.8 CH
77.7 CH
73.1 CH
77.8 CH
172.2 qC
2′′
44.6 CH
33.6 CH2
36.4 qC
45.4 CH
40.3 CH2
39.1 qC
44.1 CH
41.7 CH2
39.9 qC
44.4 CH
40.8 CH2
42.1 qC
3′′
4′′
5′′
36.6 CH2
84.1 CH
35.7 CH2
77.1 CH
31.6 CH2
33.0 CH
38.1 CH2
84.0 CH
6′′
22-Acetyls
23
28.1 CH3
22.7 CH3
28.7 CH3
23.0 CH3
6′′-OCH3
51.9 CH3
Methyl ester of glycyrrhizin (5): white amorphous powder; UV
(MeOH) λmax (log ε) 249.2 (4.14) nm; IR (KBr) νmax 3401, 2945,
Results and Discussion
!
1732, 1651, 1055 cm−1
;
1H NMR(C5D5 N, 500 MHz) δ5.94 (1H, s,
Due to the different adsorption capability of polyamide and mac-
roporous resin for licorice-flavonoids and licorice-saponins, a
method for co-application of polyamide and macroporous resin
column chromatography was carried out to prepare total sapo-
nins, which facilitates the subsequent separation of licorice sapo-
nins. The HPLC of preprocessing samples is shown as Supporting
Information. The resulting extract was separated by MPLC on a
RP-18 silica gel column [MeOH–H2O–AcOH, 40:60:1, 50:50:1,
60:40:1]. Fractions containing the desired compounds were
combined and further purified using MPLC and semipreparative
HPLC with CH3CN–H2O–AcOH (from 20:80:1 to 35:65:1). Con-
sequently, we obtained four new oleanane-type triterpene glu-
curonides named uralsaponins C–F (1–4), and an artificial prod-
uct named the methyl ester of glycyrrhizin (5). Besides, six
known constituents were also obtained, namely 3-O-[β-D-glu-
curonopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-24-hydroxy-
glabrolide (6) [9], licorice-saponin E2 (7) [10], 22β-acetoxy-gly-
cyrrhizin (8) [11], licorice-saponin G2 (9) [10], licorice-saponin
A3 (10) [1] and glycyrrhizin (11) [12]. Their structures are shown
H-12), 5.35 (1H, d, J = 8.0 Hz, H-1′′), 4.98 (1H, d, J = 7.5 Hz, H-1′),
3.31 (1H, dd, J = 4.0, 11.5 Hz, H-3), 3.81 (3H, s, COOCH3), 2.45
(1H, s, H-9), 0.77, 1.08, 1.15, 1.25, 1.32, 1.33, 1.43 (3H each, s,
6 × CH3); C NMR (C5D5 N, 500 MHz), see l Table 2; HRESIMS
(+), m/z 859.4082 [M + Na]+, (calcd. for C43H64O16Na, 859.4087).
13
"
Cellular proliferation assay
The cytotoxicity of compounds was evaluated by MTT assay ac-
cording to a method reported previously [8]. Briefly, the HeLa
(cervical cancer cells) and the MCF-7 (breast cancer cells) were
plated in 96-well plates at 5 or 8 × 103/well in the complete me-
dium, 100 µL/well. After incubation for 24 h, the medium was re-
placed with the medium containing various amounts of com-
pounds (10, 40, 80, 100 µM). Finally, 10 µL of 5 µg/mL MTTwas di-
rectly added to each well. Cells were then incubated at 37°C for
4 h. Formazan was solubilized by 100 µL of DMSO and measured
at 570 nm on the Model E1310 Autoplate reader (Bio-Tek Instru-
ments). Each experiment was performed in triplicate. Results of
three independent experiments were used for statistical analysis.
Half-maximal inhibitory concentration (IC50 value) was calcu-
lated by the Logit method. 5-Fluorouracil (Shanghai Xudong Hai-
pu Pharmaceutical Company, Ltd., purity > 98%) was used as the
positive control.
"
in l Fig. 1.
Uralsaponin C (1), a white and amorphous powder, produced a
protonated ion at m/z 825.4261 (calcd. 825.4267) in the HRE-
SIMS, corresponding to the molecular formula of C42H64O16. The
IR spectrum showed absorption bands at 3461 (OH), 1724 (C = O),
and 1652 (C = C) cm−1. The carbohydrate chain of 1 consisted of
two monosaccharide residues representing the signals of two
anomeric carbons at δC 105.1 and δC 106.9 as deduced from the
13C NMR spectra. This was correlated by the HSQC spectrum with
the corresponding signals of anomeric protons at δH 5.03 (1H, d,
J = 7.5 Hz) and δH 5.41 (1H, d, J = 7.5 Hz). The coupling constants of
the anomeric protons indicated the glycosidic bonds had a β-con-
Supporting information
HPLC charts of preprocessing samples from the roots of G. uralen-
sis, 1D, 2D NMR, and MS spectra of 1–3 and 1D NMR and MS spec-
tra of 4–5 are available as Supporting Information.
Zheng Y-F et al. Oleanane-type Triterpene Glucuronides… Planta Med 2010; 76: 1457–1463

Original Papers 1461
Fig. 1 Chemical structures of compounds 1–11.
figuration [13]. The positive ESIMS of 1 produced a protonated
ion peak at m/z 825.4261 [M + H]+, as well as two fragment ion
peaks at m/z 649.3940 [M – C6H8O6 + H]+ and m/z 473.3623 [M –
2C6H8O6 + H]+, demonstrating that the carbohydrate chain con-
sists of two glucuronopyranoses, which was confirmed by the
signals of two carboxyl carbons at 172.0 and 172.3 ppm, two
anomeric carbons at 105.1 and 106.6 ppm, and ten tertiary car-
bons at 73.1–84.3 ppm. The determination of the linkage sites
was obtained from the HMBC correlations between the proton
signals at δH 5.03 (H-1′) and the carbon resonance at δC 89.2 (C-
3), and the proton signal at δH 5.45 (H-1′′) and the carbon reso-
nance at δC 84.5 (C-2′). Thus, the carbohydrate sequence of 1 was
established as 3-O-β-D-glucuronopyranosyl-(1 → 2)-β-D-glucu-
ronopyranosyl. Complete assignments of the 1H and 13C NMR sig-
nals of the carbohydrate portion were accomplished by HSQC,
H‑H COSY, and HMBC.
Fig. 2 Key COSY and HMBC correlations of compounds 1–2.
The 13C NMR spectrum of 1 exhibited 30 carbon resonances as-
signed to the aglycone moiety consisting of seven methyls, nine
methines (of which one was oxygenated), five methylenes (in-
cluding two oxygenated methylenes and one unsaturated meth-
ylene), and nine quaternary carbons (including one carbonyl and
one unsaturated quaternary carbon). Additionally, the 1H NMR
spectrum showed for the aglycon moiety signals due to a dou-
ble-bond proton at δH 5.79 (1H, s) and seven tertiary methyl
groups at δH 1.06, 1.12, 1.12, 1.19, 1.22, 1.39, 1.39 (each 3H, s).
Thus, compound 1 was considered to be an oleanane-type triter-
pene glucuronide and indicated the presence of a 12(13)-double
bond and a keto group at C-11 in the aglycone moiety. Indeed, the
HMBC correlations between the δH 5.79 (H-12) vinyl protons and
δC 199.4 (C-11), δC 62.0 (C-9), δC 44.0 (C-14) clearly confirmed the
12(13)-position of the double bond and the presence of a 11-keto
group. The presence of an α, β-unsaturated ketone fragment was
confirmed by the UV spectrum of 1 (λmax = 250.2 nm). In addi-
tion, the correlations in the HMBC spectrum from δH 1.12 (H-28)
to δC 74.6 (C-22), and δH 3.73 (H-22) to δC 35.8 (C-20), from δH
1.49 (H-19), δH 1.11 (H-29) to δC 69.9 (C-30), and from δH 5.03
(H-1′), δH 1.39 (H-23), δH 1.22 (H-24) to δC 89.2 (C-3) helped in
assigning one oxygenated methine at C-30, one oxygenated
methylene at C-22, and one oxygenated methylene at C-3, respec-
tively. These correlations and other key H‑H COSY and HMBC cor-
"
relations are shown in l Fig. 2.
The relative configuration of 1 was established on the basis of
NOESY correlations of H-3 with H-1′α and H-5, as well as H-16α
with H-27α and H-22, which revealed that both the substituent
groups of C-3 and C-22 were β-oriented. Consequently, com-
pound
1 was assigned as 3β-O-[β-D-glucuronopyranosyl-
(1 → 2)-β-D-glucuronopyranosyl]- oleanane-11-oxo-12(13)-en-
Zheng Y-F et al. Oleanane-type Triterpene Glucuronides… Planta Med 2010; 76: 1457–1463

1462 Original Papers
Table 3 Cytotoxicity data for the aglycones of isolated compounds from
G. uralensis in selected human cell lines.
Compound
HeLa
MCF-7
Aglycone of 2
Aglycone of 7
Aglycone of 9
Aglycone of 11
5-Fluorouracil
35.3 3.6
86.3 10.0
19.9 2.5
15.5 1.4
11.0 2.1
38.5 2.8
83.3 8.2
20.5 3.6
11.4 3.0
8.7 1.6
* Results are expressed as IC50 values in µM. Cell lines: HeLa human cervical cancer;
MCF-7 human breast adenocarcinoma
Fig. 3 Chemical conversion of compound 4 to compound 6.
22β,30-diol. This is the first finding of a hydroxyl group at C-30 in
oleanane-type glucuronides from G. uralensis.
Uralsaponin D (2), as a white and amorphous powder, generated
a [M + Na]+ ion at m/z 873.3499 and a [M + H]+ at m/z 851.3610
corresponding to the molecular formula of C42H58O18 and four-
teen degrees of unsaturation. Two fragment ion peaks at m/z
m/z 467.3134. Based on this evidence, compound 4 was pre-
sumed to be 24-hydroxy-22-acetoxyglycyrrhizin. To further veri-
fy the presumption, a chemical conversion of 4 to 6 was under-
"
taken (l Fig. 3). It was found that treatment of 4 with a 1:1 mix-
ture of 2% aqueous sodium hydroxide and methanol at 60°C
yielded 6, which is in agreement with some previous studies [10].
The molecular formula of 5 was determined as C43H64O16 by
HRESIMS with a pseudomolecular ion [M + Na]+ at m/z 859.4082
(calcd. 859.4087). The signals of 5 in the 13C NMR spectrum were
very similar to those of glycyrrhizin (11), and detailed spectro-
scopic analysis showed that the only difference was in the carbo-
hydrate unit. Signals assigned to COOMe at δC 170.2, δC 51.9, and
675.3438 [M – C6H8O6 + H]+ and m/z 499.3066 [M – 2C6H8O6
+
H]+ suggested the presence of two glucuronopyranosyl moieties.
1
The H and 13C NMR spectra of 2 were similar to those of 1, and
further spectroscopic analysis revealed that the main difference
"
was in the ring-E region, C-17 to C-22, and C-28 to C-30 (l Table
1). Besides five carbonyls and one carbon-carbon double bond of
2, the presence of eight rings was necessary to meet the number
of unsaturation degrees. Since two rings of carbohydrate unit and
five rings of oleanane-type triterpene accounted for seven de-
grees of unsaturation, another ring must have been present.
Moreover, signals at δC 172.1 (C-30), δC 85.4 (C-22), and δH 4.42
(1H, d, J = 5.5, H-22) suggested the presence of a 22 (30)-lactone
ring [7] which was confirmed by the HMBC correlations from δH
2.66 (H-21α) and δH 4.42 (H-22) to δC 175.1 (C-30), together with
H‑H COSY correlation between δH 3.02 (H-21β) and δH 4.42 (H-
22). In addition, the HMBC correlations between δH 3.02 (H-21β)
and δC 172.1 (C-29) indicated that a carboxyl group (δC 172.1) was
δ
H 3.77 (3H, s) showed one of the glucuronopyranosyl was meth-
yl esterified [14] and the fragment ion at m/z 647.3787 [M –
C7H10O6 – Na + H]+ in positive ion ESIMS analysis revealed that
the terminal glucuronopyranose was esterified. Therefore, com-
pound 5 was characterized as 3β-O-[β-D-(6-methyl)-glucurono-
pyranosyl-(1 → 2)-β-D-glucuronopyranosyl]- glycyrrhetic acid.
Considering that compound 5 is absent in the HPLC chromato-
gram of the 50% aqueous ethanol extract of the roots of G. uralen-
sis, it might be an artifact from glycyrrhizin due to the use of
methanol in the column chromatography.
Previous studies demonstrated that glycyrrhizin and its aglycone
had inhibitive effects on the growth of tumor cells [3,4]. Triter-
pene glucuronides 2, 7, 9, 11, and their corresponding aglycones
obtained by two-phase acid hydrolysis [7] were evaluated for cy-
totoxic activities against the HeLa (human cervical cancer) and
MCF-7 (human breast carcinoma) cell lines, using the MTT colori-
metric assays with 5-fluorouracil as the positive control [8]. Oth-
er isolated compounds were not tested for cytotoxic activities
due to limited amounts. Results demonstrated that triterpene
glucuronides showed no cytotoxic activity on tested cancer cell
"
present at C-29 (l Fig. 2). Considering the fact that the NOESY
correlations of 2 were consistent with those of 1, compound 2
was assigned as 3β-O-[β-D-glucuronopyranosyl-(1 → 2)-β-D-glu-
curonopyranosyl]-29- carboxyl-glabrolide.
Uralsaponin E (3) exhibited a pseudomolecular ion at m/z
859.3719 ([M + Na]+, calcd. 859.3723) in the HRESIMS, in agree-
ment with the molecular formula of C42H60 O17, the same as 3-O-
[β-D-glucuronopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-24-
hydroxy-glabrolide (6). Detailed comparison of the NMR data of 3
with those of 6 suggested that the hydroxyl and methyl groups at
C-24 and C-29, respectively, in 6 were transposed in 3. The HMBC
correlations from δH 3.83 (H-29) and δH 4.27 (H-22) to δC 178.5
(C-30) confirmed this deduction. Accordingly, 3 was identified
as 3β-O-[β-D-glucuronopyranosyl-(1 → 2)-β-D-glucuronopyra-
nosyl]-29- hydroxy-glabrolide.
Uralsaponin F (4) produced a protonated ion at m/z 897.4113 by
HRESIMS. The 13C NMR spectra of 4 were similar to those of 3-O-
[β-D-glucuronopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-24-
hydroxy-glabrolide (6) [9], and further spectroscopic analysis re-
vealed that the only difference was in the region of C-22. The sig-
nals of a CH3COO (δC 20.7, δC 170.2) in 4 rather than a lactone ring
at C-22 in 6 were observed, and confirmed by the ESI‑MS/MS ex-
periment, in which the elimination of 60.0210 Da was assigned to
a neutral fragment of the CH3COOH from ion m/z 527.3344 to ion
"
lines with IC50 > 100 µM. As shown in l Table 3, the aglycones of
2, 9, and 11 showed potently cytotoxic activities against HeLa
with IC50 values at 35.3 3.6, 19.9 2.5, and 15.5 1.4 µM, as well
as cytotoxic activities against MCF-7 at 38.5 2.8, 20.5 3.6, and
11.4 3.0 µM, respectively.
In the cytotoxic assay, compounds 2, 7, 9, and 11 with the carbo-
hydrate unit displayed no inhibitive effects on the growth of tu-
mor cells, whereas their corresponding aglycones exhibited po-
tent anticancer activity. Comparing the activity of the aglycone
of glycyrrhizin (11) with that of licorice-saponin E2 (7), it seemed
that the presence of a 22(30)-lactone ring significantly decreased
the cytotoxic activity. Similarly, the aglycone of uralsaponin D (2)
was shown to have weaker anticancer activity than the aglycone
of licorice-saponin G2 (9) owing to the presence of a lactone ring
at position 22(30). Interestingly, the aglycon of 11 showed ap-
Zheng Y-F et al. Oleanane-type Triterpene Glucuronides… Planta Med 2010; 76: 1457–1463

Original Papers 1463
6 Wolkerstorfer A, Kurz H, Bachhofner N, Szolar OH. Glycyrrhizin inhibits
proximately 2-fold stronger anticancer activity than the aglycon
of 9. That might be because an additional CH2OH group is likely to
reduce the lipophilicity of the compound, thus resulting in a de-
crease in the cancer cell membrane permeability to it.
influenza A virus uptake into the cell. Antiviral Res 2009; 83: 171–178
7 Zhang RY, Zhang JH, Wang MT. Studies on the saponins from the roots of
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8 Wang Y, Che CM, Chiu JF, He QY. Dioscin (saponin)-induced generation
of reactive oxygen species through mitochondria dysfunction: a pro-
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9 Huang Q, Qiao XB, Xu XJ. Potential synergism and inhibitors to multiple
target enzymes of Xuefu Zhuyu decoction in cardiac disease therapeu-
tics: a computational approach. Bioorg Med Chem Lett 2007; 17:
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10 Kitagawa I, Hori K, Sakagami M, Zhou JL, Yoshikawa M. On the constitu-
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11 Jinwei L, Nakajima J, Kimura N, Saito K, Seo S. Oleanane-type triterpene
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12 Li W, Sha Y, Chen LX, Qiu F, Wu LJ. NMR data assignments of two 18-epi-
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13 Antonov AS, Avilov SA, Kalinovsky AI, Anastyuk SD, Dmitrenok PS, Kali-
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Acknowledgements
!
The authors greatly appreciate the financial support from the Na-
tional Science and Technology Major Project “Creation of Major
New Drugs” from China (No. 2009ZX09502–020).
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Zheng Y-F et al. Oleanane-type Triterpene Glucuronides… Planta Med 2010; 76: 1457–1463

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