Oleanane-type triterpene saponins and cassaine-type diterpenoids from Erythrophleum fordii

Article abstract of DOI:10.1055/s-0030-1270992

Phytochemical investigation of the EtOH extract of the leaves of Erythrophleum fordii led to the isolation of two oleanane-type triterpene saponins (12) and five cassaine-type diterpenoids (48) along with one known methyl 3-hydroxy-erythrosuamate (3). Their structures were established by extensive NMR, as well as ESIMS analyses and acid hydrolysis. Biological evaluation of compounds 38 against five human cancer cell lines revealed that compounds 57 exhibited potent cytotoxic activity with ICvalues ranging from 1.51 to 8.68μM. Georg Thieme Verlag KG Stuttgart·New York·.

Full text of DOI:10.1055/s-0030-1270992

Original Papers 1631
Oleanane-Type Triterpene Saponins
and Cassaine-Type Diterpenoids from
Erythrophleum fordii
Authors
Dan Du, Lei Fang, Jing Qu, Shishan Yu, Shuanggang Ma, Haining Lv, Jing Liu, Yuanyan Liu, Jiaming Wang,
Xiaojing Wang
Affiliation
Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education
and Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing,
Peopleʼs Republic of China
Key words
Abstract
and acid hydrolysis. Biological evaluation of com-
pounds 3–8 against five human cancer cell lines
revealed that compounds 5–7 exhibited potent
cytotoxic activity with IC50 values ranging from
1.51 to 8.68 µM.
"
l Erythrophleum fordii Oliver
!
"
l Leguminosae
Phytochemical investigation of the EtOH extract
of the leaves of Erythrophleum fordii led to the iso-
lation of two oleanane-type triterpene saponins
(1–2) and five cassaine-type diterpenoids (4–8)
along with one known methyl 3β-hydroxy-eryth-
rosuamate (3). Their structures were established
by extensive NMR, as well as ESI‑MS analyses
"
l oleanane‑type triterpene
saponin
l cassaine‑type diterpenoid
"
"
l cytotoxic activity
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/
plantamedica
Introduction
imeter. IR spectra were recorded on a Nicolet
5700 FT‑IR spectrometer by a microscope trans-
mission method. UV spectra were obtained on a
V650 spectrometer. 1D and 2D NMR experiments
were performed on an Inova 500 FT‑NMR spec-
trometer. ESIMS and HR-ESIMS were measured
on an Agilent 1100 Series LC/MSD Trap mass spec-
trometer. Preparative HPLC was performed on a
Shimadazu LC-6AD instrument with a SPD-10A
detector, using an YMC-Pack ODS‑A column
(250 × 20 mm, 5 µm). Analytical HPLC was per-
formed on an Agilent 1100 Series instrument
with a DAD detector, using an YMC-Pack ODS
(100 × 4.6 mm, 5 µm). GC analyses were obtained
using an Agilent N6890 instrument. Macroporous
resin HP 20 (Mitsubishi Chemical Corporation),
polyamide (30–60 mesh; Jiangsu Linjiang Chemi-
cal Reagents Factory), Sephadex LH-20 (Amer-
sham Pharmacia Biotech AB), and ODS (50 µm,
Merck) were used for column chromatography.
Silica gel GF-254 (Qingdao Marine Chemical Fac-
tory) was used for TLC. Solvents (petroleum,
!
Erythrophleum fordii Oliver (Leguminosae) is a
toxic plant which grows in South China, Taiwan,
and Vietnam [1,2]. Its bark has traditionally been
used by the native Chinese for invigoration and
promotion of blood circulation [3]. In previous
phytochemical studies on the genus Erythrophle-
um, alkaloids (cassaine-type diterpenoid amines
and amides containing a perhydrophenanthrene
skeleton), triterpenoids, diterpenoids, and diter-
penoid dimers have been isolated and identified
received
revised
accepted
Sept. 13, 2010
March 10, 2011
March 20, 2011
Bibliography
DOI http://dx.doi.org/
0.1055/s-0030-1270992
1
[4–11]. The present investigation of the leaves of
Published online April 11, 2011
Planta Med 2011; 77:
E. fordii, with particular attention paid to the ter-
penoids and their glycosides, resulted in the isola-
tion of compounds 1–8, including two new triter-
pene glycosides (1–2), five new diterpenoids (4–
1
631–1638 © Georg Thieme
Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
8
), and one previously reported methyl 3β-hy-
Correspondence
Prof. Dr. Shishan Yu
Key Laboratory
droxy-erythrosuamate (3). To our knowledge, it
is the first report of triterpene saponins from the
Erythrophleum genus. The following describes the
structural characterization of compounds 1–8
and the cytotoxic activities of compounds 5–7.
of Bioactive Substances
and Resources Utilization
of Chinese Herbal Medicine
Ministry of Education
and Institute of Materia Medica
Chinese Academy
of Medical Sciences
and Peking Union
Medical College
No. 1 Xian Nong Tan Street
Beijing 100050
CHCl , MeOH, and EtOH) were analytical grade
3
and purchased from Beijing Chemical Company.
The authentic sugars, L-cysteine methyl ester hy-
drochrochloride, and N-trimethylsilylimidazole,
were bought from Fluka.
Materials and Methods
!
General procedures
Peopleʼs Republic of China
Phone: + 861063165324
Fax: + 861063017757
yushishan@imm.ac.cn
Melting points were determined on a XT-5B mi-
cromelting point apparatus and uncorrected. Op-
tical rotations were measured with a P2000 polar-
Du D et al. Oleanane-Type Triterpene Saponins… Planta Med 2011; 77: 1631–1638

1
632 Original Papers
Plant material
Identification of isolated compounds
The leaves of Erythrophleum fordii Oliver (Leguminosae) were
collected from Guangxi Province, China, and identified by Prof.
Songji Wei (Guangxi College of Chinese Traditional Medicine) in
August 2007. A voucher specimen (NO. 07089) was deposited in
the Herbarium of the Department of Medicinal Plants, Institute
of Materia Medica, Chinese Academy of Medical Sciences.
3β‑O-{β-D-xylopyranosyl-(1 → 4)-[β-D-xylopyranosyl-(1 → 2)]-β-D-
glucopyranosyl}-2α-hydroxyolean-12-en-28-O-[β-D-glucopyrano-
syl-(1 → 6)-β-D-glucopyranosyl-(1 → 2)-α-L-rhamnopyranosyl] es-
ter (1): White powder; m.p. 248–249°C; [α] + 19.9 (c 0.13,
D
MeOH); UV (MeOH): λmax (log ε) = 204 (3.96) nm; IR: νmax = 3375,
20
2930, 1724, 1641, 1455, 1431, 1369, 1160, 1075, 1044, 972, 921,
−
1 1
8
97, 825, 627 cm ; H NMR (500 MHz, CD OD) data of the agly-
3
Extraction and isolation
con δ 0.74 (3H, s, CH -26), 0.84 (3H, s, CH -24), 0.86 (3H, s, CH -
3 3 3
The air-dried and powdered leaves (5.2 kg) were refluxed three
times with 95% EtOH (3 h for each time). The combined EtOH ex-
tract was evaporated under reduced pressure to yield a dark
brown residue (806 g, 15.5% yielded from the dried leaves),
which was dissolved in MeOH and then applied to the top of the
diatomite column (15 × 120 cm, 2000 g) and eluted successively
with petroleum, EtOAc, and MeOH to yield three fractions (A–C,
29), 0.91 (3H, s, CH -30), 0.96 (3H, s, CH -25), 1.05 (3H, s, CH -
3 3 3
23), 1.11 (3H, s, CH -27), 2.86 (1H, dd, J = 14.0, 3.0 Hz, H-18),
3
2.91 (1H, d, J = 9.0 Hz, H-3), 3.68 (1H, ddd, J = 12.0, 9.0, 3.0 Hz, H-
1
3
2), 5.30 (1H, br s, H-12); C NMR (125 MHz, CD OD) data of the
3
aglycon δ 17.2 (25-CH ), 17.5 (24-CH ), 18.3 (26-CH ), 19.3 (6-
3
3
3
CH ), 23.8 (16-CH ), 24.0 (30-CH ), 24.6 (11-CH ), 26.4 (27-
2
2
3
2
CH ), 28.5 (23-CH ), 28.5 (15-CH ), 31.6 (20-C), 33.4 (29-CH ),
3
3
2
3
9
0 g, 45 g, and 532 g, respectively) after removal of the solvents.
33.8 (22-CH ), 33.9 (7-CH ), 34.6 (21-CH ), 37.7 (10-C), 40.7 (8-
2 2 2
n
Chemical screening including HPLC/UV/ESI analysis of these
three extracts indicated the presence of triterpene saponins and
diterpenoids in fractions A and C. Fraction A (90 g) was directly
chromatographed over a polyamide column (30–60 mesh,
C), 41.7 (4-C), 42.9 (18-CH), 43.0 (14-C), 46.8 (19-CH ), 47.4 (1-
2
CH ), 48.6 (17-C), 48.8 (9-CH), 56.7 (5-CH), 67.9 (2-CH), 96.5 (3-
2
1
CH), 124.3 (12-CH), 144.8 (13-C), 177.3 (28-CO); H NMR
(500 MHz, CD OD) data and C NMR (125 MHz, CD OD) data for
sugar moieties see l Table 1; HR-ESIMS: m/z = 1391.6459 [M +
Na]+ (calcd. for C64H104NaO31, 1391.6453); ESIMS (positive-ion
mode): m/z = 1391 [M + Na] ; ESIMS (negative-ion mode): m/z =
1367 [M – H] ; ESIMS‑MS (positive-ion mode) MS : m/z = 921
[M + Na – 162 – 162 – 146] , MS : m/z = 789 [M + Na – 162 – 162
– 146 – 132] , 657 [M + Na – 162 – 162 – 146 – 132 – 132] ;
1
3
3
3
"
1
0 × 120 cm, 1200 g) eluted with 30% EtOH (10 L) and 60% EtOH
(10 L) to yield two corresponding fractions A1 (11.5 g) and A2
(18.3 g) after removing solvents. The fraction A1 (11.5 g) eluted
+
−
2
by 30% EtOH was subjected to Sephadex LH-20 (4 × 85 cm,
00 g) eluting with CH Cl : MeOH (2 L) to give three fractions
+
3
3
2
2
+
+
(A1–1 – A1–3). Fraction A1–2 (8.7 g) was submitted to ODS column
2
(50 µm, 4 × 50 cm, 300 g) using gradient mixtures of MeOH – H O
ESIMS‑MS (negative-ion mode) MS : m/z = 897 [M – H – 162 –
2
−
3
−
(10:90, 1.5 L), (20:80, 1.5 L), (30:70, 2 L), (40:60, 2 L), (50:50, 2
162 – 146] , MS : m/z = 765 [M – H – 162 – 162 – 146 – 132] ,
4
−
5
L), (60:40, 2 L), (70:30, 2 L), (80:20, 1.5 L), and (90:10, 1.5 L) as
eluants, to give ten subfractions (A1–2–1 – A1–2–10). Subfraction
A1–2–7 (360 mg) was purified by preparative HPLC using MeCN–
H O (35:65) to yield compound 5 (63 mg; flow rate: 5 mL/min;
tR = 72.0 min). Subfraction A1–2–9 (570 mg) was separated by pre-
MS : m/z = 633 [M – H – 162 – 162 – 146 – 132 – 132] , MS : m/z =
−
471 [M – H – 162 – 162 – 146 – 132 – 132 – 162] .
3β‑O-{β-D-xylopyranosyl-(1 → 4)-[β-D-xylopyranosyl-(1 → 2)]-β-D-
xylopyranosyl}-2α-hydroxyolean-12-en-28-O-[β-D-glucopyrano-
syl-(1 → 6)-β-D-glucopyranosyl-(1 → 2)-α-L-rhamnopyranosyl] es-
2
20
parative HPLC using MeCN–H O (50:50) to yield compounds 6
D
ter (2): White powder; m.p. 230–231°C; [α] + 22.7 (c 0.13,
2
(
31 mg; flow rate: 6 mL/min; t = 89.7 min) and 7 (15 mg; flow
MeOH); UV (MeOH): λmax (log ε) = 204 (3.83) nm; IR: νmax = 3390,
R
rate: 6 mL/min; t = 62.0 min). Fraction C (532 g) was submitted
to a polyamide column (30–60 mesh, 10 × 150 cm, 2500 g) using
2931, 1724, 1642, 1457, 1430, 1368, 1162, 1049, 972, 921, 896,
R
−
1 1
825, 625 cm ; H NMR (500 MHz, CD OD) data of the aglycon δ
3
40% EtOH (40 L) and 85% EtOH (20 L) as eluents; after evaporation
0.74 (3H, s, CH -26), 0.84 (3H, s, CH -24), 0.86 (3H, s, CH -29),
3
3
3
of the solvent, fractions C1 (360 g) and C2 (15 g) were obtained.
Fraction C1 (360 g) was fractionated over macroporous resin
0.91 (3H, s, CH -30), 0.95 (3H, s, CH -25), 1.04 (3H, s, CH -23),
3 3 3
1.11 (3H, s, CH -27), 2.86 (1H, dd, J = 14.0, 3.0 Hz, H-18), 2.89
3
(
10 × 150 cm, 1800 g) with H O (20 L), 35% EtOH (20 L) and 80%
(1H, d, J = 9.0 Hz, H-3), 3.64 (1H, ddd, J = 12.0, 9.0, 3.0 Hz, H-2),
2
1
3
EtOH (20 L) to furnish three fractions C1–1 (250 g), C1–2 (170 g),
and C1–3 (20 g), respectively. Fraction C1–3 (20 g) was chro-
matographed over Sephadex LH-20 (4 × 100 cm, 400 g) eluting
with MeOH (2 L) to give three fractions (C1–3–1 – C1–3–3). Fraction
C1–3–1 (15.7 g) was submitted to ODS column (50 µm, 4 × 50 cm,
5.30 (1H, br s, H-12); C NMR (125 MHz, CD OD) data of the
3
aglycon δ 17.2 (25-CH ), 17.4 (24-CH ), 18.3 (26-CH ), 19.3 (6-
3
3
3
CH ), 23.8 (16-CH ), 24.0 (30-CH ), 24.6 (11-CH ), 26.4 (27-
2
2
3
2
CH ), 28.4 (23-CH ), 28.6 (15-CH ), 31.6 (20-C), 33.4 (29-CH ),
3
3
2
3
33.8 (22-CH ), 33.9 (7-CH ), 34.6 (21-CH ), 38.8 (10-C), 40.7 (8-
2
2
2
3
00 g) eluted with a system of MeOH–H O (10:90, 20:80, 30:
C), 41.7 (4-C), 42.9 (18-CH), 43.0 (14-C), 46.8 (19-CH ), 47.5 (1-
2
2
7
0, 40:60, 50:50, 60:40, 70:30, 80:20 and 90:10, each 1.5 L)
CH ), 48.6 (17-C), 48.8 (9-CH), 56.7 (5-CH), 67.7 (2-CH), 96.2 (3-
2
1
to afford eleven subfractions (C1–3–1–1 – C1–3–1–11). Compound 3
33 mg; flow rate: 6 mL/min; t = 92.5 min) was obtained from
CH), 124.3 (12-CH), 144.9 (13-C), 177.3 (28-CO); H NMR
1
3
(
(500 MHz, CD OD) data and C NMR (125 MHz, CD OD) data for
3 3
R
"
subfraction C1–3–1–2 (1.3 g) using a preparative HPLC eluted with
MeCN–H O (25:75). Compounds 4 (25 mg; flow rate: 6 mL/min;
sugar moieties see l Table 1; HR-ESIMS: m/z = 1361.6344 [M +
Na]+ (calcd. for C63H102NaO , 1361.6348); ESIMS (positive-ion
2
30
+
t = 73.2 min) and 8 (17 mg; flow rate: 6 mL/min; t = 28.5 min)
mode) m/z: 1361 [M + Na] ; ESIMS (negative-ion mode) m/z:
R
R
−
2
were isolated from subfraction C1–3–1–5 (0.5 g) using a prepara-
tive HPLC eluted with MeCN–H O (33:67). Compounds 1
1337 [M – H] ; ESIMS‑MS (positive-ion mode) MS m/z: 891 [M
+
3
+ Na – 162 – 162 – 146] , MS : m/z = 759 [M + Na – 162 – 162 –
146 – 132] ; ESIMS‑MS (negative-ion mode) MS : m/z = 867 [M
– H – 162 – 162 – 146] , MS : m/z = 735 [M – H – 162 – 162 –
146 – 132] , MS : m/z = 603 [M – H – 162 – 162 – 146 – 132 –
2
+
2
(19 mg; flow rate: 6 mL/min; t = 32.3 min) and 2 (12 mg; flow
R
+
3
rate: 6 mL/min; t = 37.2 min) were acquired by preparative
HPLC eluted with MeCN–H O (30:70) from subfraction C
R
−
4
2
1–3–1–6
−
5
(0.2 g). The purities of these compounds ranged from 97.6 to
132] , MS : m/z = 471 [M – H – 162 – 162 – 146 – 132 – 132 –
−
9
9.8% as determined by HPLC.
132] .
Du D et al. Oleanane-Type Triterpene Saponins… Planta Med 2011; 77: 1631–1638

Original Papers 1633
Position
1
Position
2
Table 1 1H and 13C spectroscopic
δH
δC
δH
δC
NMR data for sugar moieties of
compounds 1–2 (500 MHz for
1H NMR and 125 MHz for 13C NMR,
CD3OD)a.
3
-O-sugar moieties
3-O-sugar moieties
D‑Glc 1
4.40 d (8.0)
3.58 dd (9.0, 8.0)
3.68 t (9.0)
3.56 t (9.0)
3.42 m
104.8
81.1
76.7
80.0
76.0
61.3
D‑Xyl 1
4.32 d (7.5)
105.2
77.9
76.5
77.6
64.6
2
3
4
5
6
2
3
4
5
3.53 dd (8.0, 7.5)
3.63 dd (9.0, 8.0)
3.67 m
4.00 dd (10.5, 4.5)
3.29 dd (10.5, 10.0)
3.79 m
3.66 m
D‑Xyl(1 → 2) 1
4.60 d (7.5)
3.14 dd (9.0, 7.5)
3.24 t (9.0)
105.5
74.8
77.8
71.5
67.0
D‑Xyl(1 → 2) 1
4.56 d (7.5)
105.8
74.2
77.9
71.5
67.0
2
3
4
5
2
3
4
5
3.15 dd (8.5, 7.5)
3.25 t (9.0)
3.36 m
3.37 m
3.74 brd (11.5)
3.74 dd (11.0, 5.5)
3.07 t (11.0)
4.26 d (7.5)
3.06 dd (11.0, 10.5)
D‑Xyl(1 → 4) 1
4.28 d (8.0)
105.3
75.0
77.8
70.9
67.1
D‑Xyl(1 → 4) 1
103.9
75.1
77.6
71.0
67.0
2
3
4
5
3.13 t (8.5)
2
3
4
5
3.17 dd (9.0, 7.5)
3.31 dd (9.0, 8.5)
3.44 m
3.25 dd (9.0, 9.5)
3.45 m
3.84 dd (11.5, 4.5)
3.82 dd (10.0, 5.0)
3.18 m
3.17 dd (12.0, 9.5)
2
8-O-sugar moieties
28-O-sugar moieties
L‑Rha 1
6.29 br s
94.0
81.6
72.2
73.7
72.3
18.1
106.9
76.3
77.9
71.2
76.9
69.9
L‑Rha 1
6.29 br s
94.0
81.6
72.2
73.7
72.3
18.1
106.9
76.1
77.7
71.2
76.9
70.0
2
3.75 br s
2
3.70 br s
3
3.62 m
3
3.63 m
4
3.37 m
4
3.39 m
5
3.68 m
5
3.67 m
6
1.17 d (6.0)
4.36 d (7.5)
3.23 dd (8.0, 7.5)
3.29 t (8.0)
3.35 m
6
1.17 d (6.0)
4.36 d (7.5)
3.24 t (8.0)
3.29 t (8.0)
3.34 m
D‑Glc(1 → 2) 1
D‑Glc(1 → 2) 1
2
3
4
5
6
2
3
4
5
6
3.30 m
3.32 m
4.06 brd (9.5)
4.06 brd (10.0)
3.72 brd (11.5)
4.25 d (8.0)
3.17 t (8.5)
3.18 t (8.5)
3.24 t (9.0)
3.31 m
3.70 brd (11.5)
D‑Glc(1 → 6) 1
4.24 d (8.0)
3.17 t (8.5)
3.18 t (8.5)
3.23 t (9.0)
3.30 m
104.7
75.2
77.9
71.2
77.7
62.7
D‑Glc(1 → 6) 1
104.8
75.2
77.8
71.2
77.9
62.8
2
3
4
5
6
2
3
4
5
6
3.81 brd (10.5)
3.81 brd (10.5)
3.60 brd (12.0)
3.60 brd (12.0)
a
J values are in parentheses and reported in Hz; chemical shifts are given in ppm; assignments were confirmed by 1D-TOCSY, HSQC, and
HMBC experiments
Methyl 3β-hydroxy-erythrosuamate (3): White powder; m.p.
Ethyl 3β-hydroxy-erythrosuamate (5): White powder; m.p. 84–
20
20
1
2
1
11–112°C; [α]
D
− 92.4 (c 0.08, EtOH); UV (EtOH): λmax (log ε) =
85°C; [α]
D
− 82.8 (c 0.08, EtOH); UV (EtOH): λmax (log ε) = 223
23 (4.24) nm; IR: νmax = 3459, 2945, 2882, 1717, 1646, 1435,
391, 1259, 1190, 1158, 967, 939, 871 cm ; H NMR (500 MHz,
(4.23) nm; IR: νmax = 3470, 2973, 2944, 1715, 1647, 1458, 1394,
−
1
1
−1 1
1259, 1198, 1157, 1036, 967, 873 cm ; H NMR (500 MHz, CDCl )
3
"
13
"
13
"
CDCl ) data, see l Table 2; C NMR (125 MHz, CDCl ) data, see
data, see l Table 2; C NMR (125 MHz, CDCl ) data, see l Table 3;
3
3
3
"
+
+
l Table 3; HR-ESIMS: m/z = 431.2054 [M + Na] (calcd. for
HR-ESIMS: m/z = 423.2392 [M
+
H] (calcd. for C23
H
O ,
35 7
+
C22H32NaO , 431.2040); ESIMS (positive-ion mode): m/z = 409
423.2377); ESIMS (positive-ion mode): m/z = 423 [M + H] , 445
[M + Na] , 461 [M + K] ; ESIMS (negative-ion mode): m/z = 421
7
+
+
+
+
+
[
M + H] , 431 [M + Na] , 447 [M + K] ; ESIMS (negative-ion mode):
m/z = 407 [M – H] , 443 [M + Cl] .
−
−
−
−
[M – H] , 458 [M + Cl] .
Methyl 3β-acetoxy-erythrosuamate (4): White powder; m.p. 96–
Ethyl 3β-acetoxy-erythrosuamate (6): White powder; m.p. 85–
20
20
9
7°C; [α]
D
− 82.2 (c 0.08, EtOH); UV (EtOH): λmax (log ε) = 223
86°C; [α]
D
− 88.0 (c 0.08, EtOH); UV (EtOH): λmax (log ε) = 223
(
1
4.19) nm; IR: νmax = 3473, 2947, 1721, 1649, 1435, 1375, 1261,
(4.26) nm; IR: νmax = 3479, 2973, 2945, 1731, 1715, 1646, 1457,
−
1
1
−1 1
238, 1158, 1033, 968, 871 cm ; H NMR (500 MHz, CDCl ) data,
1375, 1260, 1237, 1155, 1032, 968, 871 cm ; H NMR (500 MHz,
3
"
13
"
"
13
see l Table 2; C NMR (125 MHz, CDCl ) data, see l Table 3; HR-
ESIMS: m/z = 473.2167 [M
4
CDCl ) data, see l Table 2; C NMR (125 MHz, CDCl ) data, see
3 3
3
Na]+ (calcd. for C24H34NaO8,
l Table 3; HR-ESIMS m/z: 465.2505 [M + H] (calcd. for
"
+
+
+
73.2145); ESIMS (positive-ion mode): m/z = 451 [M + H] , 473
C25H37O , 465.2482); ESIMS (positive-ion mode) m/z: 465 [M +
8
+
+
+
+
+
[M + Na] , 489 [M + K] ; ESIMS (negative-ion mode): m/z = 449
H] , 487 [M + Na] , 503 [M + K] ; ESIMS (negative-ion mode) m/z:
463 [M – H] , 499 [M + Cl] .
−
−
−
−
[M – H] , 485 [M + Cl] .
Du D et al. Oleanane-Type Triterpene Saponins… Planta Med 2011; 77: 1631–1638

1
634 Original Papers
Position 3
4
5
6
7
8
Table 2 1H NMR spectroscopic
1
2
3
5
6
7
8
9
1
2
4
5
7
8
0
1
2
3
1
2
3
4
5
6
1.89 m, 1.36 m
1.91 m, 1.43 m
2.26 m, 1.74 m
1.89 m, 1.34 m
2.14 m, 1.76 m
1.87 m, 1.41 m
2.23 m, 1.73 m
1.88 m, 1.22 m
1.90 m, 1.23 m
1.81 m, 1.09 m
1.65 m, 1.44 m
data for compounds 3–7
(500 MHz, CDCl3) and compound 8
(500 MHz, CD3OD)a.
2.14 m, 1.76 m
3.35 m
4.67 dd (12.0, 4.5) 3.35 m
4.66 dd (12.0, 4.5) 3.10 dt (12.0, 4.0) 2.15 m, 1.10 m
2.33 s
2.44 s
2.33 s
2.43 s
1.36 d (12.0)
4.60 d (12.0)
1.40 d (11.0)
4.71 d (11.0)
3.92 d (10.5)
1.85 m
3.95 d (10.5)
1.86 m
3.92 d (10.5)
1.85 m
3.94 d (10.0)
1.85 m
2.39 dd (12.5, 1.5) 2.45 dd (13.5, 2.5)
1.68 dt (12.0, 3.0) 1.73 m
1.68 dt (12.0, 3.5) 1.69 m
1.67 m
1.74 dt (13.0, 3.5)
1.98 m, 1.18 m
3.71 m, 2.03 m
2.93 m
1
1
1
1
1
1
2
2
2
2
2.08 m, 1.18 m
3.80 m, 2.04 m
2.81 m
1.93 m, 1.21 m
1.92 m, 1.21 m
3.81 m, 2.01 m
2.81 m
1.89 m, 1.18 m
2.01 m, 1.23 m
3.79 m, 2.05 m
2.97 m
3.83 m, 2.05 m
2.83 m
3.81 m, 2.00 m
2.81 m
5.73 br s
1.19 d (7.0)
1.36 s
5.76 br s
1.21 d (7.0)
1.21 s
5.73 br s
5.73 br s
1.20 d (7.0)
1.20 s
5.70 br s
5.73 br s
1.18 d (6.5)
1.36 s
1.13 d (7.0)
1.70 s
1.09 d (7.0)
1.36 s
0.90 s
0.99 s
0.90 s
0.98 s
0.89 s
0.84 s
3.74 s
3.78 s
3.75 s
3.77 s
3.75 s
3.63 s
3.68 s
3.70 s
4.13 q (7.0)
1.27 t (7.0)
4.14 q (7.0)
1.27 t (7.0)
4.14 q (7.0)
1.27 t (7.0)
′
′
′
′
′
′
5.52 d (8.0)
3.29 m
2.05 s
2.03 s
3.37 m
3.32 m
3.34 m
3.77 dd (11.5, 3.5)
3
.59 dd (12, 5)
aJ values are in parentheses and reported in Hz; chemical shifts are given in ppm; assignments were confirmed by HSQC and HMBC
experiments
Ethyl 3β,6α-dihydroxy-cassamate (7): White powder; m.p. 69–
Table 3 13C NMR spectroscopic data for compounds 3–7 (125 MHz, CDCl3)
and compound 8 (125 MHz, CD3OD).
20
7
0°C; [α]
D
− 36.6 (c 0.08, EtOH); UV (EtOH): λmax (log ε) = 223
(
4.18) nm; IR: νmax = 3453, 2943, 2878, 1072, 1648, 1455, 1391,
−1
1
Position
3
4
5
6
7
8
1154, 1096, 980, 868 cm ; H NMR (500 MHz, CDCl ) data, see
3
13
"
1
2
3
4
5
6
7
8
9
37.1
27.5
78.0
47.6
64.3
207.9
75.7
50.8
46.0
43.1
26.2
23.5
165.1
40.2
113.2
167.2
13.7
25.5
174.1
14.4
51.8
50.9
36.5
23.5
78.7
45.7
64.6
207.9
75.7
50.9
46.4
43.0
26.2
23.7
164.9
40.2
113.4
167.2
13.7
25.7
172.6
14.4
51.8
51.2
37.1
27.6
78.1
47.7
64.3
207.9
75.8
50.8
46.1
43.1
26.2
23.5
164.7
40.2
113.7
166.8
13.7
25.5
174.1
14.4
51.8
59.6
14.2
36.5
23.4
78.7
45.7
64.6
208.0
75.7
51.2
46.4
43.0
26.2
23.7
164.5
40.2
113.8
166.8
13.7
25.7
172.6
14.3
51.2
59.6
14.2
170.4
21.0
37.9
27.8
78.0
50.3
58.1
75.4
209.9
51.0
46.3
37.6
27.3
23.5
163.9
39.4
113.5
166.7
14.8
25.3
178.0
13.5
51.7
59.7
14.2
40.7
20.3
40.7
46.4
59.7
76.9
211.2
52.8
47.9
38.9
28.4
25.1
169.3
41.1
113.4
166.5
15.4
32.1
179.0
14.2
52.0
l Table 2; C NMR (125 MHz, CDCl ) data, see l Table 3; HR-
"
3
Na]+ (calcd. for C23H34NaO7,
ESIMS: m/z = 445.2212 [M
4
+
+
45.2196); ESIMS (positive-ion mode): m/z = 445 [M + Na] , 461
+
−
[M + K] ; ESIMS (negative-ion mode): m/z = 421 [M – H] , 457 [M
−
+
Cl] .
β-D-glucopyranosyl 6α-hydroxy-cassamate (8): White powder;
20
m.p. 146–147°C; [α]
D
+ 43.6 (c 0.07, EtOH); UV (EtOH): λmax
(
log ε) = 223 (4.22) nm; IR: ν
= 3459, 2945, 2879, 1716, 1643,
max
1
−
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
1
2
3
4
5
6
1458, 1155, 1072, 952 cm ; H NMR (500 MHz, CD OH) data,
see l Table 2; C NMR (125 MHz, CD OH) data, see l Table 3;
HR-ESIMS: m/z = 563.2467 [M + Na]+ (calcd. for C27H40NaO11,
3
"
13
"
3
+
5
63.2463); ESIMS (positive-ion mode): m/z = 563 [M + Na] , 579
+
−
[M + K] ; ESIMS (negative-ion mode): m/z = 539 [M – H] , 575 [M
−
+
Cl] .
Acid hydrolysis and determination of the absolute
configuration of the monosaccharides of compounds
1 and 2
Compounds 1 (3 mg) and 2 (3 mg) were hydrolyzed separately
with 2 M HCl for 10 h at 95°C. The reaction mixture was ex-
tracted with EtOAc; the aqueous layer was evaporated under a
′
′
′
′
′
′
170.4
21.0
95.2
73.9
78.0
71.1
78.7
62.3
vacuum and diluted repeatedly with H O and evaporated in va-
2
cuo to furnish a neutral residue. The residue was dissolved in an-
hydrous pyridine (1 mL), to which 2 mg L-cysteine methyl ester
hydrochloride were added. The mixture was stirred at 60°C for
2
h, and, after evaporation in vacuo to dryness, 0.2 mL N-tri-
methylsilylimidazole were added and kept at 60°C for another
h [12,13]. The reaction mixture was partitioned between n-
hexane and H O (2 mL each) and the n-hexane extract analyzed
a
Chemical shifts are given in ppm; assignments were confirmed by HSQC and HMBC
2
experiments
2
Du D et al. Oleanane-Type Triterpene Saponins… Planta Med 2011; 77: 1631–1638

Original Papers 1635
by comparing the retention times of derivatives of sugars ob-
tained from the water layer of the hydrolysis solution with those
of standard samples using GC, which was performed under the
following conditions: capillary column, HP-5 (30 m × 0.25 mm,
with a 0.25 µm film; Dikma); detection, FID; detector tempera-
ture, 280°C; injection temperature, 250°C; initial temperature
coupled with information from the 13C NMR spectrum, seven
methyl carbons (δ = 17.2, 17.5, 18.3, 24.0, 26.4, 28.5, and 33.4)
C
and a pair of olefinic carbons (δ = 124.3 and 144.8) indicated that
C
the aglycon possesses an olean-12-ene skeleton (see Materials
and Methods section). Further features were signals at δ = 3.68
H
(1H, ddd, J = 12.0, 9.0, 3.0 Hz, H-2) and 2.91 (1H, d, J = 9.0 Hz, H-
3), indicative of secondary alcoholic functions. Thus, the aglycon
of 1 was identified as 2α,3β-dihydroxy-olean-12-en-28-oic acid
160°C, then raised to 280°C at 5°C/min, final temperature was
maintained for 10 min; carrier, He gas. Peaks were observed with
tR (min) of 17.8 (D-xylose), 18.5 (L-rhamnose), and 19.6 (D-glu-
cose). D-xylose, L-rhamnose, and D-glucose were obtained in the
ratio 2:1:3 and 3:1:2 from 1 and 2, respectively.
1
13
(maslinic acid) by comparison of H and C NMR spectroscopic
data of 1 obtained from the 2D NMR spectrum with those re-
ported in the literature [15]. Glycosidation of the alcohol group
function at C-3 was indicated by the downshift (~ 12 ppm), and
the carboxyl at C-28 was indicated by the upshift (~ 2 ppm), ob-
served for these carbon resonances in 1, if compared to the corre-
sponding signals in non-glycosidated model compounds [16].
Acid hydrolysis of 8
The hydrolysis and derivation method of the residue and the GC
analysis were the same as those described above. Peaks were ob-
1
"
served with t (min) of 19.6 (D-glucose).
For the sugar portion, the H NMR spectrum (l Table 1) of 1 dis-
R
played signals for six anomeric proton signals at δ = 4.24 (d,
H
Cytotoxicity assays
The cytotoxicity assay against HCT-8 (human colon cancer), Bel-
J = 8.0 Hz), 4.28 (d, J = 8.0 Hz), 4.36 (d, J = 7.5 Hz), 4.40 (d,
J = 8.0 Hz), 4.60 (d, J = 7.5 Hz), and 6.29 (br s) and one methyl dou-
7402 (human hepatoma cancer), BGC-823 (human gastric can-
blet δ = 1.17 (d, J = 6.0 Hz), which gave HSQC correlations with
H
cer), A549 (human lung epithelial), and A2780 (human ovarian
cancer) cells (IC50) was assessed using the MTT method as de-
scribed in the literature [14]. Camptothecin (Sigma; 95% purity;
HPLC grade) was used as the positive control.
six anomeric carbon signals at δ = 104.7, 105.3, 106.9, 104.8,
C
105.5, and 94.0 and one methyl carbon at δ = 18.1, respectively,
C
suggesting the occurrence of six monosaccharide units including
one deoxyhexose unit. The chemical shifts of all the individual
protons of the six sugar units were attributed on the basis of 1D
1
3
Supporting information
Original spectra for compounds 1–8 (l Figs. 1 and 2) are available
TOCSY spectral analysis, and the C chemical shifts of their rela-
tive attached carbons were clearly assigned from the HSQC spec-
"
"
as Supporting Information.
trum (l Table 1). These data showed the presence of three β-glu-
copyranoses (Glc, Glc I, Glc II), two β-xylopyranoses (Xyl I, Xyl II),
and one α-rhamnopyranose (Rha). The configurations of the sug-
ar units were determined to be D for glucose and xylose and L for
rhamnose by GC analysis of trimethylsilylated derivatives of the
sugars in the hydrolysate of 1 (see Materials and Methods sec-
tion) [12,13]. The sugar sequences of 1 were established by
ESIMS‑MS in the negative ion mode. The ESIMS of 1 exhibited a
deprotonated molecular ion peak [M – H]− at m/z = 1367. The
abundance of the diagnostic fragment ion peak at m/z = 897 [M
Results and Discussion
!
Compound
1
exhibited
a
pseudomolecular ion at m/z =
+
1391.6459 [M + Na] in the HR-ESIMS, consistent with the molec-
1
3
ular formula C64H104O31. The C NMR spectrum of 1 showed 64
signals, of which 30 were assigned to a triterpenoid moiety and
34 to the saccharide portion. The seven tertiary methyl protons
−
2
(
δ = 0.74, 0.84, 0.86, 0.91, 0.96, 1.05, and 1.11) and a character-
H
– H – 162 – 162 – 146] in ESIMS suggested that the first sugar
chain consists of two glucopyranosyls, and one rhamnopyranosyl
1
istic olefinic proton [δ = 5.30 (1H, br s)] in the H NMR spectrum
H
Fig. 1 Chemical structures of compounds 1 and 2.
Du D et al. Oleanane-Type Triterpene Saponins… Planta Med 2011; 77: 1631–1638

1
636 Original Papers
−
1
32 – 132 – 132] corresponding to the successive loss of three
xylopyranosyls. In particular, the HMBC correlations between H-
(δ 4.32) of Xyl I and C-3 (δ 96.2) of the aglycon, H-1 (δ 4.56) of
1
Xyl II and C-2 (δ 77.9) of Xyl I, and between H-1 (δ 4.26) of Xyl III
and C-4 (δ 77.6) of Xyl I supported the linkage sites of the sugar
chain attached to C-3. Moreover, the configuration of the sugar
units were determined as reported for compound 1. Hence, the
structure of 2 was established as 3β-O-{β-D-xylopyranosyl-
(1 → 4)-[β-D-xylopyranosyl-(1 → 2)]-β-D-xylopyranosyl}-2α-hy-
droxyolean-12-en-28-O-[β-D-glucopyranosyl-(1 → 6)-β-D-gluco-
pyranosyl-(1 → 2)-α-L-rhamnopyranosyl] ester.
Compound 3 showed the molecular formula of C22H32O deduced
7
+
from its HR-ESIMS (m/z = 431.2054) [M + Na] , which agreed with
1
13
methyl 3β-hydroxy-erythrosuamate. Because just few H and
C
NMR data of compound 3 were reported in the literature [18], de-
1
13
Fig. 2 Chemical structures of compounds 3–8.
tailed H and C NMR data were also described and assigned in
"
this paper (l Tables 2 and 3). In the IR spectrum, bands due to
hydroxyl (3459 cm ) and carbonyl (1717 cm ) groups were
clearly seen. The H NMR spectrum showed characteristic signals
−1
−1
1
is ester linked to the aglycon [17]. Further fragments at m/z = 765
of cassaine-type diterpenoid, i.e., three methyls and two me-
−
[
1
M – H – 162 – 162 – 146 – 132] , 633 [M – H – 162 – 162 – 146 –
thoxy groups [δ = 1.19 (3H, d, J = 7.0 Hz, H-17), 1.36, 0.90, 3.74,
H
−
−
32 – 132] , and 471 [M – H – 162 – 162 – 146 – 132 – 132 – 162]
3.68 (each 3H, s, H-18, 20, 21, 22)] and an olefinic proton [δ =
H
n
13
in ESIMS , corresponding to the elimination of two xylopyrano-
syls and one glucopyranosyl, allowed us to establish the gluco-
pyranose as inner sugar for the latter chain. The location of the
trisaccharide chains at C-3 and C-28 were unambiguously defined
by the HMBC experiment, which showed long-range correlations
between H-1 (δ 4.40) of Glc and C-3 (δ 96.5) of the aglycon, H-1 (δ
5.73 (1H, brs, H-15)]. Its C NMR and DEPT spectra revealed a
pair of olefinic carbons (δ = 113.2, 165.1) and another three car-
C
bonyl carbons (δ = 167.2, 174.1, 207.9) [4,10]. Furthermore, evi-
C
dence for a hydroxy group at C-3β, and the 6-keto-7β-hydroxy
group and the methyl esterified at C-16 were provided by de-
tailed analyses of HSQC, HMBC, and NOE correlations. Therefore,
methyl 3β-hydroxy-erythrosuamate has structure 3, as shown in
4.60) of Xyl I and C-2 (δ 81.1) of Glc, H-1 (δ 4.28) of Xyl II and C-4
"
(
δ 80.0) of Glc, H-1 (δ 6.29) of Rha and C-28 (δ 177.3) of the agly-
l Fig. 2.
con, H-1 (δ 4.36) of Glc I and C-2 (δ 81.6) of Rha, H-1 (δ 4.24) of
Glc II, and C-6 (δ 69.9) of Glc I, as shown in l Fig. 3. Hence, on
The molecular formula of 4 was assigned as C24H34O based on the
8
"
+
[M + Na] peak at m/z = 473.2167 in the HR-ESIMS. Strong IR bands
−
1
−1
the basis of this evidence, the structure of 1 was proposed as 3β-
O-{β-D-xylopyranosyl-(1 → 4)-[β-D-xylopyranosyl-(1 → 2)]-β-D-
glucopyranosyl}-2α-hydroxyolean-12-en-28-O-[β-D-glucopyra-
nosyl-(1 → 6)-β-D-glucopyranosyl-(1 → 2)-α-L-rhamnopyranosyl]
due to hydroxyl (3473 cm ) and carbonyl groups (1721 cm )
1
were clearly seen. Its H NMR spectrum showed signals for one
olefinic proton, two oxymethines, four methyls, and two me-
1
3
thoxys. Its C NMR and DEPT spectra indicated signals due to
six methyls, four methylenes, seven methines, and seven quater-
nary carbons. Careful analysis of the NMR data indicated that 4
possessed a cassaine-type diterpenoid skeleton closely related to
that of 3. The presence of a 6-keto-7-hydroxy group was con-
"
ester (l Fig. 1).
Compound 2 exhibited a pseudomolecular ion peak at m/z
+
1
361.6344 [M + Na] in the HR-ESIMS, ascribable to the molecular
1
13
formula C63H102O30. The H and C NMR signals of 2 assigned
from the 2D NMR spectra were almost superimposable on those
of 1 except for the characteristic signals of a xylopyranosyl moi-
ety instead of a glucopyranosyl moiety linked at C-3. Accordingly,
the ESIMS experiment (negative-ion mode) gave a quasi-molecu-
firmed by the important connectivities of H-5 (δ = 2.44) with C-
H
6 (δ = 207.9) and H-7 (δ = 3.95) with C-8 (δ = 50.9) found in the
C
H
C
HMBC spectrum. The position of exo-α,β-unsaturated methyl es-
ter moiety connected to C-13, was based on HMBC correlations of
H-15 (δ = 5.76) with C-12 (δ = 23.7), C-14 (δ = 40.2) and C-16
−
lar ion peak [M – H] at m/z = 1337 indicating a molecular weight
H
C
H
of 1338 for compound 2. Further diagnostic fragment ion peaks in
the ESIMS‑MS spectrum were observed at m/z = 867 [M – H – 162
(δ = 167.2), and H-22 (δ = 3.70) with C-16 (δ = 167.2). Com-
C H C
pared to 3, two extra resonances at δ = 21.0 and 170.4 assigned
C
−
−
–
162 – 146] , 735 [M – H – 162 – 162 – 146 – 132] , 603 [M – H –
to an acetyl group were observed. The HMBC correlations of H-3
−
162 – 162 – 146 – 132 – 132] and 471 [M – H – 162 – 162 – 146 –
(δ = 4.67, dd, J = 12.0, 4.5 Hz) with carbonyls C-1′ (δ = 170.4) of
H
C
Fig. 3 Significant HMBC correlations of the sugar
chains of 1.
Du D et al. Oleanane-Type Triterpene Saponins… Planta Med 2011; 77: 1631–1638

Original Papers 1637
acetyl established that the acetyl group esterified the hydroxy
group at C-3. The relative configuration of 4 was deduced from
its NOE spectrum. Specifically, correlations were clearly observed
between H-3 (δ = 4.67), H-5 (δ = 2.44) and H -18 (δ = 1.21), H-7
Table 4 Cytotoxicity of compounds 5–7 against five human cancer cell lines.
Compounda
IC50 (µM)
HCT-8
H
H
3
H
Bel-7402 BGC-823 A549
A2780
(
0
3
δ = 3.95), H-9 (δ = 1.73) and H -17 (δ = 1.21), H -20 (δ =
5
> 10
8.68
> 10
3.20
3.67
3.26
3.29
3.47
3.49
9.72
6.48
4.04
3.10
3.11
2.94
1.51
2.49
0.29
H
H
3
H
3
H
.99) and H-8 (δ = 1.86), and H -20 (δ = 0.99) and H -21 (δ =
6
H
3
H
3
H
7
5.46
.78), unambiguously indicated that 3α-H, 5α-H, 7α-H, 8β-H, 9α-
Camptothecinb
12.51
H, and 18α-H were attached to rings, respectively. Also the NOE
correlation between H-14 (δ = 2.83) and H-15 (δ = 5.76) was in
H
H
HCT-8 = human colon cancer cell line; Bel-7402 = human hepatoma cell line;
favor of the E configuration for the double bound at C-13/C-15.
Thus, compound 4 was concluded to be methyl 3β-acetoxy-
BGC-823 = human gastric carcinoma cell line; A549 = human lung epithelial cell line;
A2780 = human epithelial carcinoma cell line. “ > 10” = inactive. a Compounds 3, 4,
and 8 were inactive (IC50 > 10 µM) for all cells. b Positive control
erythrosuamate.
The molecular formula of 5 was established as C23H34O . Its H
1
7
and 13C NMR data were very similar to those of 3, except the pres-
ence of an ethoxy group and lack of a methoxy group at C-16. The
is inferred that the length of the alkoxyl chain connected to C-16
in cassaine-type diterpenoids might influence the cytotoxicity.
This is the first detailed report on the leaves of Erythrophleum for-
dii with respect to its terpenoid and their glycosides. We found
two saponins containing oleanlic acid aglycon (1 and 2), which,
though common in other plants, have not been reported so far
from Erythrophleum genus. However, the ethyl esterified prod-
ucts, such as 5–7, might be artefacts formed during the extraction
process. Compounds 3–8 were evaluated for their cytotoxicity
against a small panel of cell lines; only 5–7 demonstrated moder-
ated cytotoxic activity.
correlations from oxymethylene protons H-22 (δ = 4.13) to car-
H
bonyl carbon C-16 (δ = 166.8) and methyl carbon C-23 (δ = 14.2)
C
C
in its HMBC spectrum confirmed the above deduction. Thus, the
structure of 5 was established as ethyl 3β-hydroxy-erythrosua-
mate.
Compound 6 had the molecular formula of C25H36O , as deduced
8
+
from the quasi-molecular ion peak at m/z = 465.2505 [M + H] by
the HR-ESIMS. The spectroscopic data (l Tables 2 and 3) of 6 re-
"
sembled those of 4 except for the replacement of the 16-methoxy
group with an ethoxy function. Thus, the structure of compound
6
was assigned as ethyl 3β-acetoxy-erythrosuamate.
Compound 7 was found by HR-ESIMS to possess the molecular
1
formula C23H34O , the same as 5. Detailed analysis of the H and
Acknowledgements
!
7
1
3
C NMR spectrum of 7 in comparison with those of 5 showed a
difference in the replacement of the 6-keto-7β-hydroxy in 5 by
the 6α-hydroxy-7-keto in 7. In particular, the HMBC correlations
of H-5 (δ = 1.36) with C-6 (δ = 75.4) and H-6 (δ = 4.60) with C-7
The research presented in this paper was supported by the Na-
tional Found for Distinguished Young Scholars (No. 30625040),
the National Science and Technology Project of China
(No.2009ZX09311-004) and the National Natural Science Foun-
dation of China (No. 90713039). Special thanks are due to Profes-
sor Songji Wei (Guangxi College of Chinese Traditional Medicine)
for collecting and identifying the plant materials. We would like
to thank the Department of Medicinal Analysis, Institute of Mate-
ria Medica, Chinese Academy of Medical Sciences and Peking
Union Medical College, for the measurements of IR, NMR, ESIMS,
and HRESIMS spectra, and the Department of Pharmacology of
our institute for cytotoxicity tests.
H
C
H
(
δ = 209.9) as well as the NOE interaction between H-6 and H -
C 3
20 confirmed the occurrence of 6α-hydroxy-7-keto. Accordingly,
compound 7 was identified to be ethyl 3β,6α-dihydroxy-cassa-
mate.
The molecular formula of compound 8 was determined to be
C27H40O11 by HR-ESIMS. Its spectroscopic properties indicated
the presence of a sugar unit and a diterpenoid unit analogous to
1
erythrofordin C [10]. In the H NMR spectrum, one anomeric pro-
1
3
ton [δ = 5.52 (1H, d, J = 8.0 Hz)] was observed, and in the
C
H
NMR spectrum, 6 signals were assigned to an β-glucopyranose.
The absolute configuration of the glucose was determined to be
in the D-series by the GC method described in the experiment
section [12,13]. Comparison of the C NMR data of the aglycon
of 8 with those of erythrofordin C indicated that they shared the
same skeleton, being the absence of hydroxy at C-3 the most no-
table difference, suggested by the disappearance of the great
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