Ursane saponins from the stems of Firmiana simplex and their cytotoxic activity

Article abstract of DOI:10.5935/0103-5053.20150113

Three new ursane triterpene saponins, together with twelve known ursane triterpenes were isolated from the stems of Firmiana simplex. The structures of the saponines were elucidated on the basis of spectroscopic and chemical methods. The cytotoxic activity of all compounds was evaluated in vitro against lung adenocarcinoma (A549), ovarian cancer (SK-OV-3), skin melanoma (SK-MEL-2), and colon cancer (HCT-15) human cell lines, using a sulforhodamine (SRB) assay. 23-Hydroxyursolic acid showed cytotoxicity against the tested cell lines with IC₅₀ values ranging from 11.96 to 14.11 μM.

Full text of DOI:10.5935/0103-5053.20150113

http://dx.doi.org/10.5935/0103-5053.20150113
J. Braz. Chem. Soc., Vol. 26, No. 7, 1450-1456, 2015.
Printed in Brazil - ©2015 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Article
A
Ursane Saponins from the Stems of Firmiana simplex and their Cytotoxic Activity
Kyeong Wan Woo,a Sang Un Choi,b Ki Hyun Kima and Kang Ro Lee*,a
aNatural Products Laboratory, School of Pharmacy, Sungkyunkwan University, 2066 Seobu-Ro,
Jangan-ku, Suwon, Gyeonggi-do, Republic of Korea
bKorea Research Institute of Chemical Technology, 305-343 Daejeon, Republic of Korea
Three new ursane triterpene saponins, together with twelve known ursane triterpenes were
isolated from the stems of Firmiana simplex. The structures of the saponines were elucidated on
the basis of spectroscopic and chemical methods. The cytotoxic activity of all compounds was
evaluated in vitro against lung adenocarcinoma (A549), ovarian cancer (SK-OV-3), skin melanoma
(SK-MEL-2), and colon cancer (HCT-15) human cell lines, using a sulforhodamine (SRB) assay.
23-Hydroxyursolic acid showed cytotoxicity against the tested cell lines with IC50 values ranging
from 11.96 to 14.11 μM.
Keywords: Firmiana simplex, Sterculiaceae, ursane triterpene saponin, cytotoxicity
Introduction
(COSY), distortionless enhancement by polarization
transfer (DEPT), heteronuclear multiple quantum coherence
(HMQC), heteronuclear multiple-bond correlation (HMBC)
and nuclear Overhauser effect spectroscopy (NOESY), were
recorded on a Varian UNITY INOVA 700 spectrometer
operating at 700 MHz (1H) and 175 MHz (13C). High
resolution fast atom bombardment mass spectrometry
(HR-FABMS) was conducted using a JEOL JMS700
mass spectrometer. Preparative high performance liquid
chromatography (HPLC) was performed using a Gilson
306 pump with a Shodex refractive index detector. Silica
gel 60 (230-400 mesh, Merck) and reversed-phase (RP)-C18
silica gel (230-400 mesh, Merck) were used for column
chromatography. A Hewlett-Packard gas chromatography
(GC) system 6890 Series was equipped with a 5973 mass
selective detector (MSD). The system was controlled by
the Enhanced Chem Station version B.01.00 program.
The capillary column used for GC was an Agilent J&W
HP-5MSUI (30.0 m × 0.25 mm i.d., 0.25 μm film thickness,
coated with 5% diphenyl and 95% dimethylpolysiloxane).
Thin-layer chromatography (TLC) was performed using
Merck precoated silica gel F254 plates and RP-18 F254s plates.
Spots were detected on TLC under ultraviolet (UV) light
or by heating after spraying with 10% v/v H2SO4 in EtOH.
Firmiana simplex W. F. Wight (synonym Firmiana
platanifolia Schott et Endl), family of Sterculiaceae, known
as “phoenix tree”, is distributed throughout Korea and
China.1,2 Its seeds have been used as a folk medicine to treat
symptoms of diarrhea and stomach disorders.3 Previous
chemical investigations on this plant reported the isolation
of quinones,2 flavonoids,3 and an antipsychotic neolignan.4
In the course of our continuing search for potential lead
compounds from Korean traditional medicinal plants, we
investigated the MeOH extract of F. simplex stems and
isolated three new ursane triterpene saponins (1-3), together
with twelve known ursane triterpenes (4-15) (Figure 1).
All the compounds (1-15) were tested for their cytotoxic
activity against the cultured human tumor cell lines lung
adenocarcinoma (A549), ovarian cancer (SK-OV-3), skin
melanoma (SK-MEL-2), and colon cancer (HCT-15).
Experimental
General procedures
Optical rotations were obtained on a JASCO P-1020
polarimeter. Infrared (IR) spectra were recorded on a Bruker
Vector 22 IR spectrophotometer. Nuclear magnetic resonance
Plant material
(NMR) spectra including H-1H correlation spectroscopy
1
F. simplex stems (7.0 kg) were collected at Jecheon
*e-mail: krlee@skku.edu
in Chungcheongbuk-do, Korea, in June 2012, and

Vol. 26, No. 7, 2015
Woo et al.
1451
30
29
20
HO
21
HO
19
18
12
22
13
17
O
11
O
25
H
26
1
H
1
28
R
14
5
HO
OR
16
9
8
O
10
2
15
5
27
2
4
R
1
7
H
3
R
O
3
4
H
6
R
R
2
3
R
R
OH
24
23
1
2
3
4
5
O
4 R = α-OH, R = α-OH, R = CH , R = CH , R = β-D-Glc
3
3
HO
HO
O
1
2
3
4
5
5 R = α-OH, R = α-OH, R = CH , R = CH OH, R = β-D-Glc
3
2
HO
HO
OH
1
2
3
4
5
6 R = α-OH, R = α-OH, R = CH , R = CH , R = H
3
3
HO
1
2
3
4
5
7
R = α-OH, R = α-OH, R = CH , R = CH OH, R = H
3
2
1
2
3
4
5
8
R = H, R = α-OH, R = CH OH, R = CH , R = β-D-Glc
2
3
1
2
3
1 R = α-OH, R = CH , R = CH
3
3
1
2
3
4
5
9
R = α-OH, R = β-OH, R = COOH, R = CH , R = β-D-Glc
3
1
2
3
2 R = α-OH, R = CH , R = CH OH
3
2
1
2
3
4
5
10
β
β
R = H, R = -OH, R = CH , R = CH , R = -D-Glc
3 3
1
2
3
3 R = β-OH, R = COOH, R = CH
3
1
2
3
4
5
11 R = α-OH, R = β-OH, R = CH , R = CH OH, R = β-D-Glc
3
2
O
O
O
1
R
HO
HO
3
OR
OH
OH
2
R
HO
HO
OH
OH
OH
1
2
3
14
15
12 R = H, R = β-OH, R = H
1
2
3
13
R = α-OH, R = α-OH, R = β-D-Glc
Figure 1. Structures of compounds 1-15 isolated from Firmiana simplex.
authenticated by one of the authors (K. R. Lee). A
voucher specimen (SKKU-NPL-1209) was deposited at
the herbarium of the School of Pharmacy, Sungkyunkwan
University, Suwon, Korea.
through RP-C18 silica gel semi-preparative HPLC with 40%
CH3CN elution, at a flow rate of 2.0 mL min-1 (Econosil
RP-18 column; 250 × 10 mm; 10 μm particle size; Shodex
refractive index detector) to yield 7 (3 mg, tR = 14.3 min).
Fraction C3-13 (470 mg) was further separated over a silica
gel column with CHCl3:MeOH (20:1, v/v) elution, and
purified through a RP-C18 silica gel semi-preparative HPLC
with 60% CH3CN elution, to yield 6 (6 mg, tR = 17.6 min),
14 (12 mg, tR = 19.1 min), and 15 (7 mg, tR = 21.7 min).
Fraction C3-16 (120 mg) was purified through RP-C18 silica
gel semi-preparative HPLC with 70% CH3CN elution, to
yield 12 (6 mg, tR = 12.1 min). The EtOAc layer (18 g) was
chromatographed on a RP-C18 silica gel (230-400 mesh,
300 g), eluting with a gradient solvent system of MeOH:H2O
(2:3, 3:2, 4:1, and 1:0, v/v) to yield eight subfractions
(E1-E8). Fraction E3 (1.9 g) was separated over a Sephadex
LH-20 column with MeOH:H2O (4:1, v/v), and purified
through RP-C18 silica gel semi-preparative HPLC with 30
and 40% CH3CN elution, to yield 5 (12 mg, tR = 12.1 min),
8 (3 mg, tR = 14.5 min), 10 (14 mg, tR = 16.2 min), and
11 (12 mg, tR = 19.4 min). Fraction E4 (1.0 g) was separated
over a Sephadex LH-20 column with MeOH:H2O (4:1, v/v)
Extraction and isolation
The stems of F. simplex (7.0 kg) were extracted with
80% MeOH under reflux. The filtered MeOH extract was
concentrated under reduced pressure to afford a viscous
concentrate (400 g), which was suspended in water
(800 mL) and solvent-partitioned successively to yield
hexane (24 g), CHCl3 (14 g), EtOAc (50 g), and BuOH
(270 g) extracts. The CHCl3 extract (14 g) was separated
over a silica gel column (230-400 mesh, 500 g) with
hexane:EtOAc:MeOH (5:1:0.5, v/v) to give five fractions
(C1-C5). Fraction C3 (6.5 g) was separated on a RP-C18
silica gel column (230-400 mesh, 150 g) with a gradient
solvent system of MeOH:H2O (2:3, 3:2, 4:1, and 1:0, v/v)
to give sixteen subfractions (C3-1-C3-16). Fraction C3-12
(110 mg) was further separated over a silica gel column
with CHCl3:MeOH (20:1, v/v) elution, and further purified

1452
Ursane Saponins from the Stems of Firmiana simplex and their Cytotoxic Activity
J. Braz. Chem. Soc.
1
and purified by RP-C18 silica gel semi-preparative HPLC
with 40% CH3CN elution, to yield 9 (15 mg, tR = 11.8 min).
Fraction E5 (250 mg) was separated over a Sephadex
LH-20 column with MeOH:H2O (4:1, v/v) and purified
by RP-C18 silica gel semi-preparative HPLC with 50%
CH3CN elution, to yield 4 (5 mg, tR = 15.5 min). Fraction
E6 (230 mg) was separated over a Sephadex LH-20 column
with MeOH:H2O (4:1, v/v), and purified by RP-C18 silica
gel semi-preparative HPLC with 60% CH3CN elution, to
yield 13 (5 mg, tR = 14.7 min). The BuOH extract (30 g)
was separated over a silica gel column (230-400 mesh,
500 g) with CHCl3:MeOH (5:1, v/v), to give six fractions
(B1-B6). Fraction B4 (8.7 g) was chromatographed on a
RP-C18 silica gel eluting with a gradient solvent system
of MeOH:H2O (3:7, 5:5, 7:3, and 1:0, v/v) to yield eight
subfractions (B41-B48). Fraction B45 (200 mg) was
purified by RP-C18 silica gel semi-preparative HPLC with
25% CH3CN elution, to yield 2 (15 mg, tR = 16.1 min).
Fraction B47 (700 mg) was separated over a Sephadex
LH-20 column with MeOH:H2O (4:1, v/v), and purified
by RP-C18 silica gel semi-preparative HPLC with 30%
CH3CN elution, to yield 1 (19 mg, tR = 22.7 min). Fraction
B5 (16.4 g) was chromatographed on a RP-C18 silica gel
eluting with a gradient solvent system of MeOH:H2O
(2:3, 3:2, 4:1, and 1:0, v/v) to yield seven subfractions
(B51-B57). Fraction B54 (700 mg) was separated over a
Sephadex LH-20 column with MeOH:H2O (4:1, v/v) and
purified by RP-C18 silica gel semi-preparative HPLC with
25% CH3CN elution, to yield 3 (90 mg, tR = 18.7 min).
1228, 1167, 1032, 646; H NMR (700 MHz, CD3OD),
see Table 1; 13C NMR (175 MHz, CD3OD), see Table 2;
HRMS-FAB [M–H]– calcd. for C42H65O17: 841.4216; found:
841.4216.
Table 1.1H NMR data in CD3OD for compounds 1-3 (d in ppm (J in Hz))
Position
1
2
3
1
1.60 m, 1.29 m
1.63 m, 1.34 m
2.05 m, 0.93 m
2
3.95 m
3.91 m
4.09 m
3
3.38 m
3.63 d (2.0)
2.91 d (9.0)
4
–
–
–
5
1.28 m
1.58 m
1.05 m
6
1.46 m, 1.40 m
1.42 m, 1.37 m
1.79 m
7
1.59 m, 1.34 m
1.65 m, 1.31 m
1.53 m, 1.40 m
8
–
–
–
9
1.87 m
1.90 m
1.72 m
10
11
12
13
14
15
16
–
–
–
2.05 m, 1.99 m
2.07 m, 2.02 m
2.07 m, 2.01 m
5.33 t (3.5)
5.34 t (3.5)
5.33 t (3.5)
–
–
–
–
–
–
1.84 m, 1.04 m
1.84 m, 1.03 m
1.84 m, 1.05 m
2.64 td (13.0, 4.5), 2.64 td (13.0, 4.0), 2.63 td (13.0, 3.0),
1.64 m
1.66 m
1.66 m
17
18
19
20
21
22
23
–
–
–
2.54 brs
–
2.56 brs
–
2.54 brs
–
1.40 m
1.40 m
1.39 m
1.77 m, 1.28 m
1.79 m, 1.64 m
1.01 s
1.76 m, 1.28 m
1.81 m, 1.62 m
1.76 m, 1.27 m
1.80 m, 1.63 m
1.45 s
3.56 d (11.0),
3.41 m
28-O-[β-D-Glucopyranosyl-(1→6)-β-D-glucopyranosyl]-
2α,3α,19α-trihydroxy-12-en-28-ursolic acid (1)
White gum; [α]2D5 –2.0 (c 0.5, MeOH); IR (KBr)
vmax / cm-1 3385, 2938, 2879, 2843, 1732, 1651, 1454, 1390,
1228, 1205, 1166, 1062, 637; 1H NMR (700 MHz, CD3OD),
see Table 1; 13C NMR (175 MHz, CD3OD), see Table 2;
HRMS-FAB [M–H]– calcd. for C42H67O15: 811.4474; found:
811.4474.
24
25
26
27
28
29
30
1’
0.89 s
1.02 s
0.81 s
1.05 s
-
0.98 s
0.79 s
0.80 s
0.81 s
1.36 s
1.37 s
1.35 s
–
–
–
1.22 s
1.23 s
1.22 s
0.95 d (7.0)
5.31 d (8.0)
3.36 m
3.42 m
3.44 m
3.52 m
0.96 d (6.5)
5.31 d (8.0)
3.35 m
3.42 m
3.44 m
3.52 m
0.95 d (7.0)
5.32 d (8.0)
3.34 m
3.44 m
3.44 m
3.52 m
4.13 m,
2’
28-O-[β-D-Glucopyranosyl-(1→6)-β-D-glucopyranosyl]-
2α,3α,19α,23-tetrahydroxy-12-en-28-ursolic acid (2)
White gum; [α]2D5 –1.0 (c 0.6, MeOH); IR (KBr)
vmax / cm-1 3376, 2939, 2835, 1731, 1600, 1453, 1382, 1265,
1164, 1032, 637; 1H NMR (700 MHz, CD3OD), see Table 1;
13C NMR (175 MHz, CD3OD), see Table 2; HRMS-FAB
[M–H]– calcd. for C42H67O16: 827.4424; found: 827.4423.
3’
4’
5’
6’
4.17 dd (12.0, 2.0), 4.13 dd (12.0, 2.0),
3.78 dd (12.0, 5.0) 3.78 dd (12.0, 5.0) 3.78 dd (12.0, 5.0)
1’’
2’’
3’’
4’’
5’’
6’’
4.37 d (8.0)
4.37 d(8.0)
4.37 d (8.0)
3.23 t (8.5)
3.38 m
3.23 dd (9.0, 8.0) 3.23 dd (9.0, 8.0)
3.38 m
3.32 m
3.26 m
3.38 m
3.31 m
3.27 m
28-O-[β-D-Glucopyranosyl-(1→6)-β-D-glucopyranosyl]-
2α,3β,19α-trihydroxyurs-12-ene-24,28-dioic acid (3)
White gum; [α]2D5 + 0.2 (c 0.9, MeOH); IR (KBr)
vmax / cm-1 3366, 2935, 2839, 1732, 1695, 1454, 1380, 1263,
3.32 m
3.26 m
3.87 dd (12.0, 2.0), 3.87 dd (12.0, 2.0), 3.87 dd (12.0, 2.0),
3.69 dd (12.0, 5.0) 3.69 dd (12.0, 5.0) 3.70 dd (12.0, 5.0)

Vol. 26, No. 7, 2015
Woo et al.
1453
Table 2.13CNMR data in CD3OD for compounds 1-3 (d in ppm)
layer appeared to be glucose by co-TLC comparison
(CHCl3:MeOH:H2O = 2:1:0.2, Rf value: 0.2) with a
glucose standard (Aldrich), which was confirmed by gas
chromatography-mass spectrometry (GC-MS) as follows.
The sugars obtained from the hydrolysis of compounds
1-3 were dissolved in anhydrous pyridine (0.1 mL)
and L-cysteine methyl ester hydrochloride (2 mg) was
Position
1
1
2
3
42.6
67.3
80.2
39.5
49.7
19.4
34.1
41.5
48.3
39.5
24.9
129.7
139.8
42.9
29.8
26.3
49.3
55.0
73.8
43.0
27.3
38.5
29.4
22.6
17.2
17.8
24.9
178.7
24.7
16.7
95.9
73.6
78.3
71.1
77.9
69.6
104.8
75.2
78.3
71.6
78.1
62.8
42.0
67.3
78.4
42.3
43.9
19.2
33.7
41.1
48.1
38.9
24.8
129.2
139.5
42.6
29.5
26.6
49.3
54.9
73.4
42.9
27.2
38.1
71.4
17.8
17.5
17.7
24.8
178.7
27.5
16.6
95.8
73.8
78.2
71.0
77.9
69.4
104.7
75.2
78.1
71.6
78.0
62.8
48.5
69.2
84.3
49.9
57.6
21.5
34.3
41.2
48.0
39.7
25.0
129.6
139.7
42.9
29.7
26.6
49.2
55.0
73.7
42.8
27.1
38.3
24.9
180.7
15.5
17.6
24.6
178.6
27.2
16.7
95.8
73.8
78.2
71.0
77.7
69.7
104.7
75.2
78.2
71.5
78.0
62.8
2
3
4
5
o
added. The mixture was stirred at 60 C for 1.5 h. After
6
the reaction mixture was dried in vacuo, the residue was
trimethylsilylated with 1-trimethylsilylimidazole (0.1 mL)
for 2 h. The mixture was partitioned between hexane and
H2O (0.3 mL each). The H2O layer was neutralized by
passage through an Amberlite IRA-67 column (Rohm and
Haas) and was repeatedly evaporated to give D-glucose,
identified by co-injection of the hydrolyzate with standard
silylated samples, giving a GC-MS single peak at 9.712 min.
Compounds 2 (2 mg) and 3 (5 mg) were treated using the
same method to give 2α,3α,19α,23-tetrahydroxyurs-12-
en-28-oic acid (2a) and 2α,3β,19α-trihydroxy-urs-12-ene-
24,28-dioic acid (3a).
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1’
2α,3α,19α-Trihydroxyurs-12-en-28-oic acid (1a)
Colorless gum; 1H NMR (700 MHz, pyridine-d5) d 5.55
(brs, 1H, CH), 4.27 (dt, 1H, J 10.0, 3.5 Hz, CHOH), 3.72 (d,
1H, J 2.5 Hz, CHOH), 3.11 (ddd, 1H, J 13.5, 13.0, 4.5 Hz,
CH2), 3.01 (s, 1H, CH), 2.29 (ddd, 1H, J 13.5, 13.0, 4.0 Hz,
CH2), 1.59 (s, 3H, CH3), 1.38 (s, 3H, CH3), 1.22 (s, 3H,
CH3), 1.07 (d, 3H, J 6.0 Hz, CH3), 1.05 (s, 3H, CH3), 0.94
(s, 3H, CH3), 0.85 (s, 3H, CH3).
2α,3α,19α,23-Tetrahydroxyurs-12-en-28-oic acid (2a)
1
Colorless gum; H NMR (700 MHz, CD3OD) d 5.31
2’
(brs, 1H, CH), 3.89 (ddd, 1H, J 12.0, 5.0, 3.0 Hz, CHOH),
3.62 (d, 1H, J 3.0 Hz, CHOH), 3.55 (d, 1H, J 11.0 Hz,
CHOH), 3.41 (d, 1H, J 11.0 Hz, CHOH), 2.58 (td, 1H,
J 13.0, 4.0 Hz, CH2), 2.53 (s, 1H, CH), 1.37 (s, 3H, CH3),
1.21 (s, 3H, CH3), 1.04 (s, 3H, CH3), 0.94 (s, 3H, CH3),
0.94 (d, 3H, J 7.0 Hz, CH3), 0.88 (s, 3H, CH3).
3’
4’
5’
6’
1’’
2’’
3’’
4’’
5’’
6’’
2α,3β,19α-Trihydroxy-urs-12-ene-24,28-dioic acid (3a)
1
Colorless gum; H NMR (700 MHz, CD3OD) d 5.51
(brs, 1H, CH), 4.69 (m, 1H, CHOH), 3.34 (d, 1H, J 9.0 Hz,
CHOH), 3.02 (m, 1H, CH2), 2.33 (dd, 1H, J 13.0, 4.0 Hz,
CH2), 1.68 (s, 3H, CH3), 1.65 (s, 3H, CH3), 1.39 (s, 3H,
CH3), 1.11 (s, 3H, CH3), 1.06 (d, 3H, J 6.0 Hz, CH3), 1.05
(s, 3H, CH3).
Acid hydrolysis of 1-3 and sugar determination
Compound 1 (2 mg) was shaken with 1 mL of 1 mol L-1
o
HCl for 1 h at 90 C. After cooling, the hydrolyzate was
extracted with CHCl3 and the extract was evaporated
in vacuo to yield 2α,3α,19α-trihydroxyurs-12-en-28-oic
acid (1a), which was identified by comparing its 1H NMR
data with those reported in literature. The sugar in water
Cytotoxicity assays
A sulforhodamine (SRB) bioassay was used to
determine compound cytotoxicity against cultured human

1454
Ursane Saponins from the Stems of Firmiana simplex and their Cytotoxic Activity
J. Braz. Chem. Soc.
tumor cell linesA549, SK-OV-3, SK-MEL-2, and HCT-15.5
The assays were performed at the Korea Research Institute
of Chemical Technology. Doxorubicin was used as a
positive control. The IC50 values of doxorubicin against
A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines were
0.029, 0.036, 0.001, and 2.041 μM, respectively.
23), 24.9 (C-27), 24.7 (C-29), 22.6 (C-24), 17.8 (C-26),
17.2 (C-25), and 16.7 (C-30), two olefinic carbon signals
at dC 139.8 (C-13) and 129.7 (C-12), two oxygenated
methine carbon signals at dC 80.2 (C-3) and 67.3 (C-2),
eight methylene carbon signals at dC 42.6 (C-1), 38.5
(C-22), 34.1 (C-7), 29.8 (C-15), 27.3 (C-21), 26.3 (C-16),
24.9 (C-11), and 19.4 (C-6), four methine carbon signals
at dC 55.0 (C-18), 49.7(C-5), 48.3 (C-9), and 43.0 (C-20),
one carbonyl carbon at d 178.7 (C-28), six quaternary
carbon signals at dC 73.8 (C-19), 49.3 (C-17), 42.9 (C-14),
41.5 (C-8), and 39.5 (C-4), and two anomeric carbons at
dC 104.8 (C-1’’) and 95.9 (C-1’).
The NMR data of 1 were very similar to those of
the ursane 4,6 with the exception of an additional sugar
moiety in 1. The linkage of the disaccharide moiety to
the pentacyclic scaffold, and the attachment position
between the two sugar units were assigned from the
following HMBC correlations: dH 4.37 (d, J 8.0 Hz,
H-1’’) to dC 69.6 (C-6’), and dH 5.31 (d, J 8.0 Hz, H-1’) to
dC 178.7 (C-28) (Figure 2).
The relative stereochemistry of the aglycone was
assigned from the NOESY cross-peaks of H-2/H-25,
H-3/H-24, H-5/H-9, H-9/H-27, H-24/H-25, and H-25/H-26
(Figure 3).6
The coupling constant (J 8.0 Hz) of the anomeric protons
of H-1’and H-1’’suggested a β-orientation.7 Acid hydrolysis
of 1 gave 2α,3α,19α-trihydroxyurs-12-en-28-oic acid (1a),
identified by comparison of its 1H NMR spectrum data with
of previously reported values,8 and D-glucose, which was
identified by GC-MS.9 Thus, compound 1 was determined to
be 28-O-[β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl]-
2α,3α,19α-trihydroxy-12-en-28-ursolic acid.
Results and Discussion
The stems of F. simplex were extracted with 80%
aqueous MeOH. Chemical investigation of the extract
using successive column chromatography over silica gel
and Sephadex LH-20, and preparative HPLC resulted
in the isolation and identification of three new ursane
triterpene saponins (1-3), together with twelve known
ursane triterpenes (4-15). Their structures were elucidated
as follows.
Compound 1 was obtained as a colorless gum, and
its molecular formula C42H67O15 was inferred from the
negative HR-FABMS ion at m/z 811.4474 [M–H]–. The
IR absorption bands at 3385 and 1732 cm-1 implied the
presence of hydroxyl and carboxylic functionalities. The
1H NMR spectrum (Table 1) of 1 showed the signals for
an olefinic proton at dH 5.33 (t, 1H, J 3.5 Hz, H-12), two
oxygenated methine protons at dH 3.95 (m, 1H, H-2) and
3.38 (m, 1H, H-3), one methine proton at dH 2.54 (brs, 1H,
H-18), six tertiary methyl protons at dH 1.36 (s, 3H, H-27),
1.22 (s, 3H, H-29), 1.02 (s, 3H, H-25), 1.01 (s, 3H, H-23),
0.89 (s, 3H, H-24) and 0.79 (s, 3H, H-26), one secondary
methyl proton at dH 0.95 (d, 1H, J 7.0 Hz, H-30), and two
anomeric protons at dH 5.31 (d, 1H, J 8.0 Hz, H-1’) and
4.37 (d, 1H, J 8.0 Hz, H-1’’). The 13C NMR (Table 2),
DEPT, and HMQC spectral data revealed forty-two signals,
which included seven methyl carbon signals at dC 29.4 (C-
Compound 2 was obtained as a colorless gum, and its
molecular formula C42H67O16 was inferred from the negative
HO
HO
HO
O
O
O
HO
HO
HO
O
O
O
O
HO
O
O
HO
HO
OH
OH
OH
O
OH
OH
O
O
O
HO
O
O
HO
HO
O
HO
HO
HO
OH
OH
OH
HO
HO
HO
HO
HO
HO
HO
HO
HO
2
3
1
COSY
HMBC
Figure 2. Key 1H-1H COSY and HMBC (H → C) correlations of compounds 1-3.

Vol. 26, No. 7, 2015
Woo et al.
1455
niga-ichigoside F2 (5),13 euscaphicacid (6),14 myrianthic
acid (7),15 kakisaponinA (8),16 trachelosperosideA-1 (9),12
pormolic acid-28-O-β-D-glucopyranosyl ester (10),17
niga-ichigoside F1 (11),13 23-hydroxyursolic acid (12),18
2α,3α,24-trihydroxyurs-12-en-28-oic acid-28-O-β-D-
glucopyranosyl ester (13),19 arjunolic acid (14),20 and
2α,3α,23-trihydroxyursa-12,20(30)-dien-28-oic acid
(15).21
HO
O
H
H
H
HO
H
HO
H
OH
H
Compounds 1-15 were evaluated for their cytotoxicity
against A549, SK-OV-3, SK-MEL-2, and HCT-15 human
tumor cell lines using the SRB assay.5 Compound 12 was
cytotoxic against all the tested human cell lines with IC50
values of 11.96, 13.24, 14.11, and 12.27 μM, respectively,
whereas the other compounds were inactive (IC50 > 30 μM).
NOESY
1
Figure 3. Key NOESY correlations of compound 1.
1
HR-FABMS ion at m/z 827.4423 [M–H]–. The H and
13C NMR spectra were close to those of 1 (Tables 1 and 2).
The major differences were the disappearance of a methyl
signal [dH 1.01 (s, 3H, H-23); dC 29.4] in 1, and the presence
of the oxymethylene signal [dH 3.56 (d, 1H, J 11.0 Hz,
H-23a), 3.41 (m, 1H, H-23b); dC 71.4] in 2. This was
confirmed by the HMBC experiment showing correlations
from the oxymethine proton (dH 3.56) to C-3, C-4, C-5, and
C-24. The nature and position of the disaccharide moiety
revealed to be the same as for compound 1, as indicated
by the HMBC correlations H-1’’/C-6’ and H-1’/C-28
(Figure 2). As for compound 1, the relative configuration
of 2 determined by the NOESY spectrum also indicated
an ursane pentacyclic system. Acid hydrolysis of 2 gave
2α,3α,19α,23-tetrahydroxyurs-12-en-28-oic acid (2a) and
D-glucose, which were identified by GC analysis and TLC
comparison with authentic D-glucose.9,10 Thus, compound 2
was determined to be 28-O-[β-D-glucopyranosyl-(1→6)-
β-D-glucopyranosyl]-2α,3α,19α,23-tetrahydroxy-12-en-
28-ursolic acid.
Conclusions
This is the first study investigating the cytotoxic
activities of triterpene derivatives (1-15) isolated from
Firmiana simplex. Among them, compound 12, which
showed a significant cytotoxicity against the human tumor
cell lines, could be a potentially valuable source for the
development of anti-tumor agents.
Supplementary Information
Supplementary data are available free of charge at http://
jbcs.sbq.org.br as a PDF file.
Acknowledgments
This research was supported by the Basic Science
ResearchProgramthroughtheNationalResearchFoundation
of Korea (NRF), funded by the ministry of Education,
Science and Technology (2013R1A1A2A10005315). We
are thankful to the Korea Basic Science Institute (KBSI)
for the measurements of NMR and MS spectra.
Compound 3 was obtained as a colorless gum. The
molecular formula was determined to be C42H65O17 from
the deprotonated molecule [M–H]– at m/z 841.4216 in
the negative-ion HR-FABMS data. The 1H and 13C NMR
data of 3 were very similar to those reported for 9,11
except for the presence of an additional sugar unit The
connectivities of the two sugar units were deduced
by the HMBC cross peaks H-1’’/C-6’ and H-1’/C-28
(Figure 2). The relative stereochemistry of 3 was assigned
by NOESY cross-peaks H-2/H-25, H-3/H-23, H-5/H-9,
H-9/H-27, and H-25/H-26. Acid hydrolysis of 3 yielded
2α,3β,19α-trihydroxyurs-12-ene-24,28-dioic acid (3a)
and D-glucose.9,12 Thus, compound 3 was determined to be
28-O-[β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl]-
2α,3β,19α-trihydroxyurs-12-ene-24,28-dioic acid.
References
1. Zhang, L. X.; Song, J. H.; Shen, J. T.; Tan, G. J.; Li, S. S.;
Ding, F.; J. Phytopathol. 2013, 161, 128.
2. Bai, H.; Li, S.; Yin, F.; Hu, L.; J. Nat. Prod. 2005, 68, 1159.
3. Seetharaman, T. R.; Fitoterapia 1990, 61, 373.
4. Son,Y. K.; Lee, M. H.; Han,Y. N.; Arch. Pharmacal Res. 2005,
28, 34.
5. Skehan, P.; Storeng, R.; Scudiero, D.; Monks,A.; Mcmahon, J.;
Vistica, D.;Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R.;
J. Natl. Cancer Inst. 1990, 82, 1107.
Compounds 4-15 were identified by comparing their
1H NMR, 13C NMR, and MS spectra with the literature
data. They were determined to be kaji-ichigoside F1 (4),6
6. Yean, M. H.; Kim, J. S.; Hyun,Y. J.; Hyun, J. W.; Bae, K.; Kang,
S. S.; Nat. Prod. Sci. 2012, 43, 107.

1456
Ursane Saponins from the Stems of Firmiana simplex and their Cytotoxic Activity
J. Braz. Chem. Soc.
7. Woo, K. W.; Moon, E. J.; Park, S. Y.; Kim, S. Y.; Lee, K. R.;
16. Chen, G.; Xue, J.; Xu, S. X.; Zhang, R. Q.; J. Asian Nat. Prod.
Res. 2007, 9, 347.
Bioorg. Med. Chem. Lett. 2012, 22, 7465.
8. Lee, I. K.; Kim, D. H.; Lee, S.Y.; Kim, K. R.; Choi, S. U.; Hong,
J. K.; Lee, J. H.; Park,Y. H.; Lee, K. R.; Arch. Pharmacal Res.
2008, 31, 1578.
17. Wu, Z. J.; Ouyang, M. A.; Wang, C. Z.; Zhang, Z. K.; Shen,
J. G.; J. Agric. Food Chem. 2007, 55, 1712.
18. An, R. B.; Na, M. K.; Min, B. S.; Lee, H. K.; Bae, K. H.; Nat.
Prod. Sci. 2008, 14, 249.
9. Lee, I. K.; Choi, S. U.; Lee, K. R.; Chem. Pharm. Bull. 2012,
60, 1011.
19. Jung, H.A.; Chung, H.Y.; Jung, J. H.; Choi, J. S.; Chem. Pharm.
Bull. 2004, 52, 157.
10. Kim, K. H.; Choi, S. U.; Lee, K. R.; J. Nat. Prod. 2009, 72,
1121.
20. Acebey-Castellon, I. L.;Voutquenne-Nazabadioko, L.; Huong,
D. T. M.; Roseau, N.; Bouthagane, N.; Muhammad, D.;
Le Magrex Debar, E.; Gangloff, S. C.; Litaudon, M.; Sevenet, T.;
Nguyen, V. H.; Lavaud, C.; J. Nat. Prod. 2011, 74, 163.
21. Choi,Y. H.; Zhou, W.; Oh, J.; Choe, S.; Kim, D. W.; Lee, S. H.;
Na, M. K.; Bioorg. Med. Chem. Lett. 2012, 22, 6116.
11. Wang, B. G.; Shen, X. M.;Yang, L.; Jia, Z. J.; Phytochemistry
1997, 46, 559.
12. Abe, F.; Yamauchi, T.; Chem. Pharm. Bull. 1987, 35, 1748.
13. Um, B. H.; Pouplin, T.; Lobstein,A.; Weniger, B.; Litaudon, M.;
Anton, R.; Fitoterapia 2001, 72, 591.
14. Woo, K. W.; Han, J. Y.; Choi, S. U.; Kim, K. H.; Lee, K. R.;
Nat. Prod. Sci. 2014, 20, 71.
Submitted: January 14, 2015
Published online: May 5, 2015
15. Wandji, J.; Tillequin, F.; Mulholland, D. A.; Shirri, J. C.;
Tsabang, N.; Seguin, E.; Verite, P.; Libot, F.; Fomum, Z. T.;
Phytochemistry 2003, 64, 845.