Norsesquiterpenoid and sesquiterpenoid glycosides from Evodia austrosinensis

Article abstract of DOI:10.1055/s-2005-837762

Two new norsesquiterpenoid glycosides named austrosides A and B, together with four known glycosides, corchoionoside B, citroside A, icariside B ₁ and dihydrophaseic acid 4′-O-β-D-glucopyranoside were isolated from Evodia austrosinensis. Their structures were elucidated by 1D and 2D NMR data. Austroside A showed antibacterial activity against the Staphylococcus aureus antibiotic resistant strain.

Full text of DOI:10.1055/s-2005-837762

Norsesquiterpenoid and
Sesquiterpenoid Glycosides from
Evodia austrosinensis
Junsong Wang, Yuemao Shen, Hongping He, Wenyi Kang,
Xiaojiang Hao
Abstract
Fig. 1 Structures of compounds 1±6.
Two new norsesquiterpenoid glycosides named austrosides A
and B, together with four known glycosides, corchoionoside B, ci-
troside A, icariside B1 and dihydrophaseic acid 4¢-O-b-D-gluco-
pyranoside were isolated from Evodia austrosinensis.Their struc-
tures were elucidated by 1D and 2D NMR data.Austroside A
showed antibacterial activity against the Staphylococcus aureus
antibiotic resistant strain.
instead.In the HMBC, the 1H-13C long-range correlations bet-
ween the ketone carbon at d = 215.9 (s) and the protons at
d = 2.86 (H-2a), 1.84 (H-2b), 2.44 (H-4a) and 2.16 (H-4b) sug-
gested that the ketone group was located at C-3.The sugar link-
age was determined on the basis of HMBC experiments as well.A
cross peak of 1H-13C long-range coupling was observed between
the proton signal at d = 4.34 (Glc H-1) and the oxymethylene
carbon signal at d = 73.9 (C-10), indicating that the glucosyl
unit was linked with C-10 via oxygen.The relative stereochemis-
try of 1 was determined by ROESY experiments as shown in
Fig. 2.
Evodia austrosinensis fruit is traditionally used as Wu-Chu-Yu
[1]. The leaves of E. austrosinensis clear away heat, expel cold
and dampness as well as toxic materials which, in traditional
Chinese medicine, include viruses, fungi, and bacteria.Indeed,
contemporary research has shown that Evodia officinalis, used
in Indian medicine, showed antimicrobial activity and promising
preliminary results in the use against multi-drug resistant
strains of some human pathogens [2].Crude extracts of the
leaves and stem wood of E. elleryana have shown a wide range
of antibacterial activities as well [3].
Austroside B (2) was obtained as a colorless amorphous powder.
The UV spectra showed a maximal absorption at 231 nm (log e =
4.26). The IR spectrum indicated the presence of hydroxy groups
(3424 cm±1), an allenic structure (1938 cm±1), and a conjugated
carbonyl group (1668 cm±1).The proton and carbon signals in
the 1H-NMR and 13C-NMR spectra of 2 and 4 were nearly super-
These indications and the fact that no phytochemical work has
been done on E. austrosinensis, led to this study and the isolation
of two new glycosides which were named austrosides A (1) and B
(2) and four known ones (3±6; Fig.1) from the leaves of this
plant.The antibacterial activity of austroside A against antibiotic
resistant strains by utilizing the DD (disc diffusion) method [4]
was also examined.
96
Table 1 1H- (400 MHz , J in Hz)and 13C- (100 MHz)NMR data of 1 and
2 in CD3OD
Position
1
2
13C
1H
13C
1H
1
2
44.2 s
52.2 t
36.9 s
48.0 t
Austroside A (1) was obtained as an amorphous powder.The mo-
lecular formula was determined to be C19H30O9 by 13C-NMR,
DEPT experiments and HR-FAB-MS analysis.The 1H-,13C- and
DEPT NMR spectra of 1 (Table 1) showed signals indicating three
methyl, three methylene, two methine, two carbonyl carbons
and three sp3 quaternary carbons, suggesting that 1 had a skele-
ton similar to that of corchoionoside B (3).The 1H- and 13C-NMR
spectra showed that 1 had one carbon-carbon double bond less
than 3 [5], but showed one more methylene and methine group
2.86 (d, 13.6),
1.84 (dd, 13.6, 2.0)
2.03 (d, 11.5)
3
4
215.9 s
45.7 t
72.6 d
46.7 t
4.34 (m)
2.44 (overlap),
2.16 (m)
2.31 (d, 12.5)
5
6
37.5 d
79.0 s
2.43 (overlap)72.5 s
119.8 s
7
152.4 d
127.9 d
198.3 s
73.9 t
7.10 (d, 16.0)211.1 s
6.64 (d, 16.0)101.3 d
8
5.89 (s)
202.2 s
27.0 q
9
10
4.77 (d, 17.2),
4.53 (d, 17.2)
2.26 (s)
Affiliation: The State Key Laboratory of Phytochemistry and Plant Resources in
West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunm-
ing, Yunnan, P.R.China
11
25.1 q
25.0 q
16.4 q
104.2 q
75.0 q
78.2 d
71.5 q
77.7 q
62.7 t
0.99 (s)32.5 q
1.11 (s)
1.40 (s)
12
0.95 (s)29.3 q
13
0.88 (d, 5.2)30.8 q
4.34 (d, 7.6)102.6 d
3.26±3.37 (m)75.0 d
3.26±3.37 (m)78.1 d
3.26±3.37 (m)71.6 d
3.26±3.37 (m)77.9 d
1.32 (s)
Correspondence: Xiaojiang Hao ´ The State Key Laboratory of Phytochemistry
and Plant Resources in West China ´ Kunming Institute of Botany ´ Chinese
Academy of Sciences ´ Heilongtan ´ Kunming ´ Yunnan 650204 ´ People's Repub-
lic of China ´ Phone: +86-871-522-3263 ´ Fax: +86-871-515-0227 ´ E-mail:
haoxj@mail.kib.ac.cn
Glc-1
Glc-2
Glc-3
Glc-4
Glc-5
Glc-6
4.41 (d, 7.6)
3.11±3.35 (m)
3.11±3.35 (m)
3.11±3.35 (m)
3.11±3.35 (m)
Received: April 14, 2004 ´ Accepted: August 20, 2004
3.86 (d, 12.0),
3.65 (d, 12.0)
62.7 d
3.85 (d, 11.5),
3.66 (d, 11.5)
Bibliography: Planta Med 2005; 71: 96±98 ´ ꢀ Georg Thieme Verlag KG Stutt-
gart ´ New York ´ DOI 10.1055/s-2005-837762 ´ ISSN 0032-0943

Fig. 2 ROESY correlations
of 1.
tions were performed on an HP Agilent 1100 series pumping sys-
tem equipped with diode array detector and with an XTerra C18-
PREP column (10 mm, 19”150 mm, Waters).TLC was conducted
on plates precoated with silica gel F254.Uncorrected melting
points were determined using an XRC-1 micromelting appara-
tus.
The aerial parts of Evodia austrosinensis were collected in Xish-
uangbanna prefecture, (Yunnan province) in March 2002.A vou-
cher specimen (No.BN200208) was deposited in the Herbarium
of Kunming Institute of Botany.
imposable, except for some signals of the structure near C-8.The
olefinic proton signal (H-8) and the methyl signal at d = 2.26 (H-
10) were shifted downfield by 0.1 ppm, and the carbon signal of
C-9 was shifted downfield by 1.3 ppm. This can be best interpret-
ed by the different relative stereochemistries of C-8 in 2 and 4
(see Fig.1).Acid hydrolysis of 2 and 4 gave 2a (m.p.150±152 8C,
ethyl acetate-hexane) and 4a (m.p. 134±136 8C, acetone-ben-
zene) as the aglycones, respectively.The downfield 1H-NMR shift
of H-8 of 4a versus 2a was shifted by 0.11 ppm in CDCl3.There-
fore, 2a and 4a [7], [8], as well as 2 and 4 are diastereomeric at
C-8.The sugar linkage was determined on the basis of HMBC ex-
periments.A cross peak of 1H-13C long-range coupling was ob-
served between the proton signal at d = 4.41 (Glc H-1) and the
carbon signal at d = 72.6 (C-3), indicating that the glucose was
linked to C-3.The presence of the allenic moiety was confirmed
further by HMBC correlations from the methyl protons at
d = 2.26 (H-10) to the carbon at d = 202.2 (C-9) and the proton
at d = 5.89 (H-8) to carbons at d = 211.1 (C-7) and 202.2 (C-9).
The NMR assignments of 4 and 5 for C-7 and C-9, C-2 and C-4 in
the literature [6], [7] should be reversed with the aid of HMBC
experiments.
Air-dried and powdered material (6.0 kg) was extracted three
times with 95% ethanol under reflux, the residue was suspended
in water and partitioned with petroleum ether, EtOAc, and n-
BuOH, respectively.The n-BuOH extract (60.0 g) was purified by
MPLC on silica gel (7”60 cm), with eluents of increasing polarity
[CHCl3-MeOH-H2O (90:10:1, 80:20:2, and 70:30:5) and
MeOH, 3000 mL each] to afford eight fractions (EA1±8).The
fraction EA5 (4.192 g) was chromatographed on silica gel (150 g)
eluted with EtOAc-CH3OH (96:4, 90:10, 1500 mL each) to give
subfractions EA5-1±EA5-5.EA5-2 (2177 mg) was chromato-
graphed on a reversed-phase silica gel (100 g) column and eluted
with CH3OH-H2O (3:7, 5:5, 1000 mL each) and MeOH to produce
5 fractions EA5-2-1±EA5-2-5.EA5-2-2 (369 mg) and EA5-2-3
(425 mg) were purified by preparative HPLC (MeOH-H2O, 1:9,
flow rate 10 mL/min) to give compounds 1 (17 mg, tR = 24.2
min) and 3 (20 mg, tR = 21.4 min), 4 (27 mg, tR = 27.8 min) and
5 (15 mg, tR = 37.3 min), respectively. Compound 2 (22 mg,
tR = 40.5 min) was obtained from fraction EA5-2-4 (120 mg)
after purification through preparative HPLC with MeOH-H2O
(1:9).EA5-4 (1793 mg) was chromatographed on a Sephadex
LH-20 column eluted with CH3OH (containing 1% Et3N) and col-
lecting 24 fractions of 10 mL volume each.Fractions 7±10 were
purified by preparative HPLC eluting with CH3OH-H2O (5:95,
containing 0.1% TFA) to obtain compound 6 (25 mg, tR = 37. 7
min).
Compounds 3±6 (see Fig.1) were determined to be corchoiono-
side B ([a]D25: 136.7, c 2.5, MeOH) (Lit. [5], [a]D28: 113. 7,c 0.4,
MeOH), icariside B1 ([a]D25: ±72. 6,c 1.3, MeOH) (Lit. [6], [a]D25
:
97
±73.5, c 1.0, MeOH), citroside A ([ a]D25: ±76. 2,c 1.0, MeOH) (Lit.
[7], [a]D23: ±95. 7,c 0.44, MeOH), and dihydrophaseic acid 4¢-O-
b-D-glucopyranoside ([a]D25: ±35. 0,c 0.5, MeOH) (Lit. [9], [10],
[a]D27: ±20, c 0.209, EtOH), respectively, on the basis of their
physical and spectral data.All these compounds were obtained
for the first time from the genus Evodia (Rutaceae).
Austroside A (1): A white powder; [a]D25: ±40.8 (c 1.3, MeOH); UV
(MeOH): lmax (log e) = 230 nm (4.22); IR (KBr): nmax = 3429,
2931, 2880, 1698, 1630, 1286, 1076, 1038 cm±1; 1H- and 13C-NMR
data, see Table 1; HR-FAB-MS: found: 425.1782, calcd. for
Among these compounds, only austrosid A (1) showed inhibitory
activity against the penicillin resistant strain of Staphylococcus
aureus (CMEC 26003) (190 mg/disc, zone diameter: 9 mm).Peni-
cillin G as a positive control showed an inhibitory zone diameter
of 13 mm at 600 mg/disc and 0 mm at 190 mg/disc.Compound 3
did not inhibit the growth of this strain, showing that the intro-
duction of a double bond at C4-C5 of 1 depresses its biological
activity, which was consistent with the results of diterpenes re-
ported by Metteo et al.[11].Compound 3 is less flexible than
compound 1, which may lead to a loss of the pharmacophoric
conformation.
C
19H30O9Na: 425.1787; EI-MS: m/z (rel.int). = 402 (3, M +), 240
(50, [M±Glc + H]+), 222 (47, [M±OGlc + H]+).
Austroside B (2): A white powder; [a]D25: ±33. 8 (c 0.8, MeOH); UV
(MeOH): lmax (log e) = 231 nm (4.26); IR (KBr): nmax = 3424,
2928, 1938, 1668, 1243, 1159, 1075, 1034 cm±1; 1H- and 13C-NMR
data, see Table 1; HR-FAB-MS: found: 409.1831; calcd. for
C19H30O8Na: 409.1838; FAB-MS: m/z = 385 [M±1].
Acid hydrolysis of 2, 4, 5: A solution of austroside B (ca.5 mg) in
50% AcOH (2 drops) was heated at 708C for 2 h.The reaction
mixture was passed through a porous resin D101 column, which
was washed with water.The methanol eluate was purified by
HPLC (XTERA RP-18, 5 mm, 7.8”150 mm; H2O-CH3CN (80:20) to
give the aglycone as colorless needles (ethyl acetate-hexane),
m.p. 150±152 8C.From 4 and 5 (5 mg), the aglycones were ob-
tained in the same manner as colorless needles (acetone-ben-
zene), m.p.134±136 8C.
Material and Methods
The NMR spectra were recorded on a Bruker AM-400 spectrome-
ter.FAB-MS was recorded on a VG AutoSpec-3000 spectrometer.
Infrared (IR) spectra were obtained with a Bio-Rad FTS-135 infra-
red spectrometer with KBr pellets.UV spectra were obtained on a
Shimadzu double-beam 210A spectrophotometer.HPLC separa-
Letter¼ Planta Med 2005; 71: 96±98

3 Khanu MR, Kihara M, Omoloso AD.Antimicrobial activity of Evodia
elleryana.Fitoterapia 2000; 71: 72±4
Antimicrobial activity: Staphylococcus aureus (CMEC 26003) was
spread with an aseptic spreader on the surface of the agar medi-
um dish (containing beef extract).The compounds were respec-
tively added to each paper disk (diameter 0.5 cm) under sterile
conditions.After 1 h, the dried paper disks were placed onto the
medium dish and were cultured at 378C for 24 h.The antibacter-
ial activity was calculated by the diameter of the inhibitory zone.
4 Gnanou JC, Sanders P.Antibiotic resistance in bacteria of animal ori-
gin: methods in use to monitor resistance in EU countries.Interna-
tional Journal of Antimicrobial Agents 2000; 15: 311±22
5 Yoshikawa M, Shimada H, Saka M, Yoshizumi S, Yamahara J, Matsuda
H.Absolute stereostructures of corchoionosides A, B, and C, histamine
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6 Miyase T, Ueno A, Takizawa N, Kobayashi H, Karasawa H.Studies on
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Letter¼ Planta Med 2005; 71: 96±98