New pregnane glycosides from Brucea javanica and their antifeedant activity

Article abstract of DOI:10.1002/cbdv.201000035

Three new pregnane glycosides, 3-O-β-D-glucopyranosyl-(1→2)- α-L-arabinopyranosyl-(20R)-pregn-5-ene-3β,20-diol (1), 3-O-α-L-arabinopyranosyl-(20R)-pregn-5-ene-3β,20-diol-20-O-β-D- glucopyranoside (2), 3-O-α-L-arabinopyranosyl-(20R)-pregn-5-ene-3β, 20-diol-20-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside (3) were isolated along with four known compounds, 4-7, from the leaves and stems of Brucea javanica. Their structures were determined by detailed analyses of 1D- and 2D-NMR spectroscopic data. All of the compounds isolated from Brucea javanica were tested for the antifeedant activities against the larva of Pieris rapae. Compounds 1, 3, and 5 showed significant antifeedant activities after 72 h incubation.

Full text of DOI:10.1002/cbdv.201000035

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
New Pregnane Glycosides from Brucea javanica and Their Antifeedant
Activity
by Yue-Yuan Chena), Qiao-Dan Panb), Dian-Peng Li*a), Jin-Lei Liua), Yong-Xin Wena),
Yong-Lin Huanga), and Feng-Lai Lua)
a) Department of Phytochemistry, Guangxi Institute of Botany, Guangxi Zhuangzu Autonomous Region
and Chinese Academy of Sciences, Guilin 541006, P. R. China
(phone: þ86-773-3550076; fax: þ86-773-3550067; e-mail: phytoldp@hotmail.com)
b) Department of Pharmacy, Youjiang Medical College for Nationalities, Baise 533000, P. R. China
Three new pregnane glycosides, 3-O-b-d-glucopyranosyl-(1!2)-a-l-arabinopyranosyl-(20R)-
pregn-5-ene-3b,20-diol (1), 3-O-a-l-arabinopyranosyl-(20R)-pregn-5-ene-3b,20-diol-20-O-b-d-glucopyr-
anoside (2), 3-O-a-l-arabinopyranosyl-(20R)-pregn-5-ene-3b,20-diol-20-O-b-d-glucopyranosyl-(1!2)-
b-d-glucopyranoside (3) were isolated along with four known compounds, 4–7, from the leaves and stems
of Brucea javanica. Their structures were determined by detailed analyses of 1D- and 2D-NMR
spectroscopic data. All of the compounds isolated from Brucea javanica were tested for the antifeedant
activities against the larva of Pieris rapae. Compounds 1, 3, and 5 showed significant antifeedant activities
after 72 h incubation.
Introduction. – Brucea javanica (L.) Merr. (Simaroubaceae) grows in the tropical
and the subtropics areas of Asia, Indonesia, and Australia, and is mainly distributed in
Guangdong and Guangxi provinces of China [1]. Its seeds have been used as antitumor
agent and as insecticide against amoeba. Its leaves have been used as folk medicine in
poultice against boils, ringworm, scurf, and centipede bites [2]. Quassinoids had been
isolated as major constituents from the seeds and roots [3–9]; a pregnane glycoside had
been reported in the leaves [10]. In the course of a screening, the crude extract from the
leaves and stems of Brucea javanica showed antifeedant activity. Fractionation of the
active extract afforded three new pregnane glycosides: 3-O-b-d-glucopyranosyl-(1!
2)-a-l-arabinopyranosyl-(20R)-pregn-5-ene-3b,20-diol (1), 3-O-a-l-arabinopyranosyl-
(20R)-pregn-5-ene-3b,20-diol-20-O-b-d-glucopyranoside (2), 3-O-a-l-arabinopyrano-
syl-(20R)-pregn-5-ene-3b,20-diol-20-O-b-d-glucopyranosyl-(1!2)-b-d-glucopyrano-
side (3), together with four known compounds, 4–7 (Fig. 1). Here, we report the
structure elucidation of 1–3 on the basis of spectroscopic analysis. In addition, the
antifeedant activities against the larva of Pieris rapae are also described.
Results and Discussion. – The structures of the known compounds, (20R)-3-O-a-l-
arabinopyranosylpregn-5-ene-3b,20-diol (4) [10], luteolin (5) [11], luteolin 7-O-b-d-
glucopyranoside (6) [11], and apigenin 7-O-b-neospheroside (7) [12], were deduced by
direct comparison of their spectroscopic data with those reported in the literature.
Compound 1, isolated as white needles, showed a quasi-molecular-ion peak at m/z
635.6 ([MþNa]þ ) in FAB-MS (positive-ion mode) and was assigned a molecular
ꢀ 2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
461
Fig. 1. Structures of 1–7
formula of C32H52O11, which was confirmed by HR-FAB-MS (positive-ion mode;
635.3459 ([MþNa]þ ; calc. 635.3407)) and also deduced by analysis of the 13C-NMR
spectrum combined with the DEPT data (Table 1). The IR spectrum revealed the
presence of OH groups (3400 cmꢀ1; br.), Me and CH2 groups (2934 and 2869 cmꢀ1
,
resp.; br.), and C¼O groups (1648 cmꢀ1). The 1H-NMR spectrum of 1 displayed signals
for two Me groups at d(H) 0.74 and 1.00, one Me doublet signal at d(H) 1.44 (d, J¼6.1),
suggesting a pregnane skeleton. Furthermore, two anomeric H-atom signals were
observed at d(H) 5.10 (d, J¼6.1) and 5.15 (d, J¼7.9 ) . The 13C-NMR spectrum
combined with DEPT data (Table 1) of 1 exhibited 32 C-signals, including those
corresponding to three Me (d(C) 12.7, 19.5, and 24.8), ten CH2, and 16 CH groups, and
three quaternary C-atoms (d(C) 141.1, 37.0, and 41.7). On acidic hydrolysis, 1 afforded a
sugar mixture of d-glucose and l-arabinose, which were identified by GC analysis. The
coupling constants of the anomeric H-atoms (J¼7.9 and 6.1) indicated b-glucosidic and
a-arabinosidic linkages, respectively. Signals of sugar units (Table 1) were assigned by
HMQC, pyranosyl configuration of the arabinosyl moiety was identified by comparison
1H- and 13C-NMR spectroscopic data with those in the literature [10]. Further
comparison of the NMR data with those of 4 showed that the two structures were
essentially analogous except that 1 displayed additional signals arising from a b-
glucopyranosyl moiety (anomeric C-atom signal at d(C) 106.1, anomeric H-atom signal
at d(H) 5.15 (d, J¼7.9)), and a glycosylation shift was observed for the signal of
arabinosyl C(2’) (þ 9.0 ppm), suggesting that the attachment of the additional sugar
was at C(2’) of the arabinosyl residue. This was confirmed by long-range correlations
between HꢀC(1’’) at d(H) 5.15 (d, J¼7.9) and C(2’) at d(C) 81.6 in the HMBC
spectrum of 1. Thus, the structure of 1 was established as 3-O-b-d-glucopyranosyl-(1!
2)-a-l-arabinopyranosyl-(20R)-pregn-5-ene-3b,20-diol.
Compound 2, obtained as white needles, was assigned the molecular formula
C32H52O11 based on the HR-FAB-MS (positive-ion mode; m/z 635.3459 ([MþNa]þ ;
calc. 635.3407)), which was confirmed by the 13C-NMR and DEPT spectrum (Table 1)
and was the same as that of 1. The IR spectrum indicated the presence of OH groups

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
a
b
1
Table 1. 13C- and H-NMR Data for 1–3 in (D5)Pyridine ) )
Position
1
2
3
d(C)
37.7 (t)
d(H)
d(C)
37.7 (t)
d(H)
d(C)
37.7 (t)
d(H)
1
2
1.03–1.05 (m),
1.75–1.77 (m)
1.80–1.83 (m),
2.12–2.14 (m)
1.03–1.06 (m),
1.75–1.76 (m)
1.72–1.75 (m),
2.13–2.15 (m)
1.06–1.08 (m),
1.74–1.76 (m)
1.72–1.74 (m),
2.13–2.15 (m)
30.3 (t)
30.4 (t)
30.4 (t)
3
4
78.3 (d) 3.93–3.95 (m)
78.1 (d) 3.86–3.89 (m)
78.1 (d) 3.94–3.96 (m)
39.2 (t)
2.56 (d, J¼11.6),
39.3 (t)
2.43 (d, J¼11.6),
39.3 (t)
2.43 (d, J¼12.0),
2.73 (d, J¼10.4)
2.66 (d, J¼13.4)
2.66 (d, J¼11.0)
5
6
7
141.1 (s)
141.1 (s)
141.0 (s)
121.9 (d) 5.37 (br. s)
32.2 (t) 1.52–1.54 (m),
1.80–1.83 (m)
121.8 (d) 5.35 (br. s)
32.2 (t) 1.40–1.44 (m),
1.75–1.76 (m)
121.9 (d) 5.35 (br. s)
32.2 (t) 1.35–1.38 (m),
1.80–1.82 (m)
8
9
10
11
12
31.8 (d) 1.40–1.43 (m)
50.5 (d) 0.88–0.89 (m)
37.0 (s)
31.9 (d) 1.34–1.35 (m)
50.4 (d) 0.85–0.87 (m)
37.0 (s)
31.9 (d) 1.32–1.35 (m)
50.4 (d) 0.87–0.88 (m)
37.0 (s)
21.1 (t)
39.1 (t)
1.47–1.48 (m)
1.10–1.12 (m),
1.89–1.91 (m)
21.1 (t)
39.2 (t)
1.36–1.40 (m)
1.06–1.08 (m),
1.83–1.85 (m)
21.1 (t)
39.2 (t)
1.30–1.32 (m)
1.11–1.14 (m),
1.79–1.80 (m)
13
14
15
41.7 (s)
56.9 (d) 0.96–0.97 (m)
24.6 (t)
26.6 (t)
41.6 (s)
56.7 (d) 0.87–0.90 (m)
24.5 (t)
27.2 (t)
41.7 (s)
56.8 (d) 1.01–1.03 (m)
24.5 (t)
27.0 (t)
1.10–1.12 (m),
1.60–1.61 (m)
1.86–1.89 (m),
2.12–2.14 (m)
1.03–1.06 (m),
1.44–1.48 (m)
1.83–1.85 (m),
2.34–2.35 (m)
1.10–1.11 (m),
1.64–1.66 (m)
1.97–1.99 (m),
2.57–2.59 (m)
16
17
18
19
20
21
Ara’
1’
2’
3’
4’
59.4 (d) 1.52–1.54 (m)
12.7 (q) 0.74 (s)
19.5 (q) 1.00 (s)
69.0 (d) 3.93–3.95 (m)
24.8 (q) 1.44 (d, J¼6.1)
58.4 (d) 1.55–1.59 (m)
12.5 (q) 0.68 (s)
19.4 (q) 0.93 (s)
81.3 (d) 3.86–3.89 (m)
23.3 (q) 1.55 (d, J¼6.1)
58.2 (d) 1.66–1.68 (m)
12.5 (q) 0.60 (s)
19.4 (q) 0.92 (s)
81.4 (d) 3.81–3.83 (m)
22.8 (q) 1.52 (d, J¼6.1)
100.9 (d) 5.10 (d, J¼6.1)
81.6 (d) 4.58 (d, J¼6.5)
73.0 (d) 4.42–4.44 (m)
68.1 (d) 4.40–4.42 (m)
103.1 (d) 4.84 (d, J¼6.7)
72.6 (d) 4.40–4.42 (m)
74.6 (d) 4.19–4.21 (m)
69.5 (d) 4.25–4.26 (m)
103.2 (d) 4.84 (d, J¼6.7)
72.6 (d) 4.43–4.45 (m)
74.6 (d) 4.16–4.18 (m)
69.5 (d) 4.32–4.34 (m)
5’
65.0 (t)
3.76–3.79 (m),
4.27–4.29 (m)
66.8 (t)
3.79 (d, J¼10.4),
66.8 (t)
3.79 (d, J¼10.4),
4.30–4.32 (m)
4.33–4.35 (m)
Glc’’
1’’
2’’
3’’
4’’
106.0 (d) 5.15 (d, J¼7.9)
76.2 (d) 4.11 (t, J¼8.5)
78.2 (d) 4.21 (t, J¼8.9)
71.7 (d) 4.27–4.28 (m)
78.6 (d) 3.76–3.79 (m)
106.1 (d) 4.95 (d, J¼7.9)
75.8 (d) 3.88–3.91 (m)
78.7 (d) 4.21–4.22 (m)
71.9 (d) 4.19–4.20 (m)
78.2 (d) 3.86–3.88 (m)
103.8 (d) 4.95 (d, J¼7.3)
83.1 (d) 4.19–4.21 (m)
78.4 (d) 4.33–4.35 (m)
71.7 (d) 4.15–4.17 (m)
78.1 (d) 3.83–3.85 (m)
5’’
6’’
62.8 (t)
4.42–4.43 (m),
4.53–4.56 (m)
63.1 (t)
4.30–4.33 (m),
4.56 (d, J¼10.4)
63.0 (t)
4.28–4.30 (m),
4.48–4.50 (m)
Glc’’’
1’’’
2’’’
3’’’
4’’’
105.7 (d) 5.39 (d, J¼7.3)
76.9 (d) 4.13–4.15 (m)
78.0 (d) 4.35–4.37 (m)
71.9 (d) 4.26–4.27 (m)
78.4 (d) 3.85–3.89 (m)
5’’’
6’’’
62.8 (t)
4.30–4.31 (m),
4.50–4.52 (m)
a) Me4Si was used as an internal standard. b) Assignments were confirmed by HMQC, HMBC, and
1H,1H-COSY experiments; due to severe overlapping, only detectable J values are reported; d in ppm, J
in Hz.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
463
(3400 cmꢀ1; br.). On acidic hydrolysis, 2 afforded a sugar mixture of d-glucose and l-
1
arabinose, which were identified by GC analysis. The H- and 13C-NMR spectra
(Table 1) of the aglycone portion of 2 were almost identical with those of 4, except that
the signal due to C(20) at d(C) 69.0 was shifted to d(C) 81.3, and the signals due to the
C-atoms around C(20) were slightly changed: signal of C(16) was shifted downfield by
0.57 ppm, and those of C(17) and C(21) were shifted upfield by 0.97 and 1.49 ppm,
respectively. In addition, the NMR spectra of 2 displayed additional signals arising from
a b-glucopyranosyl moiety (anomeric C-atom signal at d(C) 106.1, anomeric H-atom
signal at d(H) 4.95 (d, J¼7.9)). These findings suggested that the additional
glucopyranosyl group was at C(20) of the aglycone, which was further confirmed by
HMBC spectrum showing long-range correlations between the HꢀC(1’’) (d(H) 4.95 (d,
J¼7.9)) and C(20) of the aglycone (d(C) 81.3), and between HꢀC(1’) (d(H) 4.84(d, J¼
6.7)) and C(3) of the aglycone (d(C) 78.1). Accordingly, the structure of 2 was
determined to be 3-O-a-l-arabinopyranosyl-(20R)-pregn-5-ene-3b,20-diol-20-O-b-d-
glucopyranoside.
Compound 3 was shown to have the molecular formula of C38H62O16 on the basis of
the HR-FAB-MS (positive-ion mode; 797.4078 ([MþNa]þ ; calc. 797.3936)), and the
13C-NMR and DEPT spectrum (Table 1). The 1H-NMR spectrum of 3 exhibited signals
for three anomeric H-atoms at d(H) 4.84 (d, J¼6.7), 4.95 (d, J¼7.3), and 5.39 (d, J¼
7.3), as well as signals for three Me groups at d(H) 0.60, 0.92, and 1.52 and one Me
doublet at d(H) 1.52 (d, J¼6.1), also suggesting a pregnane skeleton. Comparison of
the 13C-NMR spectrum of 3 with that of 4 suggested that the two structures were similar
except that the spectra of 3 displayed additional signals arising from two b-
glucopyranosyl moieties (anomeric C-atom signals at d(C) 103.8, 105.7, and anomeric
H-atom signals at d(H) 4.95 (d, J¼7.3), 5.39 (d, J¼7.3)). In addition the signal due to
C(20) was shifted downfield by 12.41 ppm, and the signals due to the C-atoms around
C(20) were somewhat displaced: signals due to C(16) was shifted downfield by
0.40 ppm, and those due to C(17) and C(21) were shifted upfield by 1.17 and 1.96 ppm,
respectively. On the other hand, the signal due to C(3) was unchanged, and the signals
of the a-l-arabinopyranosyl moiety (anomeric C-atom signal at d(C) 103.2 and
anomeric H-atom signal at d(H) 4.84 (d, J¼6.7)) remained almost unshifted,
suggesting that 3 is related to 4 with two additional sugar moieties at C(20) of the
aglycone. As compared to 2, the signal due to C(2’’) was shifted downfield by 7.28 ppm,
suggesting that the sugar chain at C(20) was b-glucopyranosyl-(1!2)-b-glucopyrano-
syl. The glycosylation sites and the interglycosidic linkage were confirmed by the
HMBC spectrum showing long-range correlations of d(H) 5.39 (HꢀC(1’’’)) with d(C)
83.1 (C(2’’)), of d(H) 4.95 (HꢀC(1’’)) with d(C) 83.1 (C(2’’)), of d(H) 4.95 (HꢀC(1’’))
with d(C) 81.4 (C(20)), and of d(H) 4.84 (HꢀC(1’)) with d(C) d78.1 (C(3)) (Fig. 2).
Hence, the structure of 3 was elucidated as 3-O-a-l-arabinopyranosyl-(20R)-pregn-5-
ene-3b,20-diol-20-O-b-d-glucopyranosyl-(1!2)-b-d-glucopyranoside.
Pregnanes have been already reported to possess antifeedant activities [13][14]. It
is assumed that their antifeedant activity is related to the effects on chemoreceptors for
glucose, sucrose, and inositol [15]. In this study, the antifeedant activities of compounds
1–7 were tested against the larva of Pieris rapae (Table 2). Treated and control leaves
were incubated 24, 48, and 72 h with the larvae. As a result, compounds 1, 3, and 5
showed significant antifeedant activities after 72 h incubation, as well as 1 and 5, after

464
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Fig. 2. Selected 1H,1H-COSY and HMBC correlations of compound 3
Table 2. Antifeedant Activity of 1–7 against the 3rd Larva of Pieris Rapae
Compound
AI%a)
24 h
48 h
72 h
1
2
3
4
5
6
7
94.4ꢂ3.3**b)
0.0ꢂ0.0
84.4ꢂ1.5**
70.1ꢂ0.6**
29.7ꢂ1.8
58.2ꢂ2.0*
6.6ꢂ0.6
ꢀ1.7 ꢂ0.7
50.6ꢂ2.3*
ꢀ10.9ꢂ3.2
53.3ꢂ2.9*
54.4ꢂ3.1*
18.1ꢂ4.2
61.1ꢂ3.4**
31.1ꢂ4.2*
72.2ꢂ5.6**
77.8ꢂ4.6**
66.7ꢂ2.5**
53.7ꢂ4.6*
41.9ꢂ6.8
29.5ꢂ1.8
a) Antifeedant index calculated as AI [%]¼[1ꢀ(T/C)]ꢁ100, data expressed as meansꢂSD of ten
replicates. Significant differences between consumption of treated and control leaves (Wilcoxon signed
rank test). b) *: P<0.05; **: P<0.01.
24 and 48 h. Compound 1 was the most active with an antifeedant index exceeding 70%
for all incubation times.
This work was supported by the National Natural Science Foundation of China (No. 30269002), and
we express our appreciation to Mr. Tsuyoshi Ikeda and Mr. Toshihiro Nohara of Kumamoto University
for recording the NMR spectra.
Experimental Part
General. Column chromatography (CC): silica gel (SiO2; 100–200 mesh and 200–300 mesh;
Qingdao Haiyang Chemical Company, Qingdao, P. R. China), macroporous absorption resin D101
(Tianjin Chemical Industry, Tianjin, P. R. China), and Chromatorex ODS (30–50 mm, Fuji Silysia
Chemical Ltd.). GC: Agilent 6890N gas chromatograph, cap. column (28 mꢁ0.32 mm i.d., HP-5), FID
detector, operated at 2608 (column temp. 1808); N2 as carrier gas (40 ml/min). M.p.: Yanaco-50949 micro
melting point apparatus; uncorrected. Optical rotations: P-1010 polarimeter (JASCO, Japan) at 258. IR
1
Spectra: 5-DX spectrometer (Nicolet), in KBr pellets. NMR Spectra: at 500 MHz for H and 125 MHz
for 13C; JNC-A500 NMR spectrometer; chemical shifts given on a d [ppm] scale with Me4Si as internal

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
465
standard; standard pulse sequences for DEPT and HMBC experiments. HR-FAB-MS: JEOL JMS-
DX303 HF spectrometer; in a glycerol matrix containing NaI.
Plant Material. Leaves and stems of Brucea javanica were obtained from Jingdao County, Fangcheng
City of Guangxi, P. R. China, in September 2004 and identified by Professor Yan Liu (Guangxi Institute
of Botany). A voucher specimen (BJ040912) of the plant is deposited with the Herbarium of Guangxi
Institute of Botany, P. R. China.
Extraction and Isolation. The air-dried cut leaves and stems of Brucea javanica (2.34 kg) were
extracted three times for 2 h with 95% EtOH (15.0 l) under reflux. The extract was concentrated under
reduced pressure to afford EtOH extract (205 g). The extract was suspended in H2O (0.6 l) and
successively partitioned with petroleum ether (PE; 0.5 lꢁ2), CHCl3 (0.5 lꢁ3), and BuOH (0.5 lꢁ3),
resp. The CHCl3 extracts were concentrated in vacuo to afford a residue (18 g) which was subjected to CC
(SiO2; CHCl3/MeOH from 20 :1 to 1:1 (v/v)), to afford five fractions. Fr. 2 (2.25 g) was subjected
repeatedly to CC (SiO2; CHCl3/MeOH 10 :1 (v/v)) to afford 5 (225.2 mg) and 4 (73.8 mg). Fr. 3 (1.85 g)
was repeatedly subjected to CC (SiO2; CHCl3/MeOH 5 :1 (v/v)), followed by further purification with
CC (ODS; 50–60% MeOH) to afford 2 (25.6 mg) and 1 (11.3 mg). The BuOH extract (75 g) was
dissolved in H2O and subjected to macroporous absorption resin D101 (EtOH/H2O 0 :100, 50 :50,
80 :20). The fraction (35.8 g) eluted with EtOH/H2O 50 :50 (v/v) was repeatedly subjected to CC (SiO2;
CHCl3/MeOH from 10 :1 to 1:1 (v/v)) to afford nine fractions. Further purification of Frs. 6 (1.5 g), 7
(2.4 g), and 8 (1.1 g) by CC (SiO2; CHCl3/MeOH 7:3 (v/v)) gave 6 (32.0 mg), 3 (195.8 mg), and 7
(25.0 mg), resp.
(3b,20R)-20-Hydroxypregn-5-en-3-yl 2-O-b-d-Glucopyranosyl-a-l-arabinopyranoside (1). White
needles. M.p. 290.5–292.08. [a]2D5 ¼ ꢀ13.4 (c¼ ꢀ0.10, MeOH). IR (KBr): 3415, 2934, 2869, 1455,
1379. 1H- and 13C-NMR: see Table 1. FAB-MS (pos.): 635.6 ([MþNa]þ ). HR-FAB-MS (pos.): 635.3459
([MþNa]þ , C32H52NaO1þ1 ; calc. 635.3407).
(3b,20R)-3-(a-l-Arabinopyranosyloxy)pregn-5-en-20-yl b-d-Glucopyranoside (2). White needles.
1
M.p. 254.5–255.58. [a]2D5 ¼ ꢀ36.2 (c¼0.12, MeOH). IR (KBr): 3393, 2934, 2869, 1455, 1379. H- and
13C-NMR: see Table 1. FAB-MS (pos.): 635.6 ([MþNa]þ ). HR-FAB-MS (pos.): 635.3459 ([MþNa]þ
,
C32H52NaOþ11 ; calc. 635.3407).
(3b,20R)-3-(a-l-Arabinopyranosyloxy)pregn-5-en-20-yl 2-O-b-d-Glucopyranosyl-b-d-glucopyrano-
side (3). White needles. M.p. 236–2378. [a]2D5 ¼ ꢀ13.4 (c ¼ 0.1, MeOH). 1H- and 13C-NMR: see Table 1.
FAB-MS (pos.): 797.1 ([MþNa]þ ). HR-FAB-MS (pos.): 797.4078 ([MþNa]þ , C38H62NaO1þ6 ; calc.
797.3936).
Antifeedant Activity Tests. Antifeedant activity of compounds 1–7 (Fig. 1) was studied using leaf disc
no-choice method [16]. Fresh Brassica alboglabra leaf discs of 2 cm in diameter were punched using cork
borer and were dipped in 1 mg/ml test compound, respectively. Leaf discs treated with acetone and H2O
were used as negative control. Treated and control leaf discs were then placed in two separate Petri dishes
(1.5 cmꢁ7 cm) covered with a piece of wet filter paper, and one third larvae of Pieris rapae (starved for
4 h) were introduced into each Petri dish. Progressive consumption of treated and control leaf by the
larvae after 24, 48, 72 h was recorded using leaf-area meter. Leaf area, eaten by larvae in treatment, was
compared to that from the negative control. Tests were performed in ten replicates. The measurements
were always performed by the same operator. The antifeedant index (AI [%]) was calculated as [1ꢀ(T/
C)]ꢁ100, where T and C represent the consumption of treated and control leaf areas, resp.
Acid Hydrolysis and Sugar Analysis by TLC and GC. Each soln. of the compounds 1–3 (each 5 mg)
in a mixture of MeOH (2.0 ml) and 1m H2SO4 (2.0 ml) was refluxed for 2 h on a H2O bath. The
hydrolysate was allowed to cool, H2O (8.0 ml) was added, and the soln. was extracted with AcOEt (3ꢁ
8.0 ml). The aq. layer was neutralized with aq. Ba(OH)2 and concentrated in vacuo to give a residue. The
residues were analyzed by TLC (SiO2) by comparison with standard sugars. The solvent system was
CHCl3/MeOH/H2O 8 :5 :1 (v/v), and spots were visualized by spraying with 95% EtOH/H2SO4/
anisaldehyde 9 :0.5 :0.5 (v/v/v), then heated at 1208 for 10 min. For sugars of 1–3, the Rf values of glucose
and arabinose by TLC was 0.31, 0.55 resp. The results were further confirmed by GC analysis. The
residues were dissolved in anh. pyridine (100 ml), then 0.1m l-cysteine methyl ester hydrochloride (200 ml;
Sigma) was added, and the mixture was warmed at 608 for 1 h. The trimethysilylation reagent HMDS/
TMCS (hexamethyldisilazane/trimethylchlorosilane/pyridine 2 :1:10; Acros Organics, Belgium) was

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
added, and the mixture was warmed at 608 for 30 min. The thiazolidine derivatives were analyzed by GC
for sugar identification. The tR values of l-arabinose (5.26 min) and d-glucose (12.43 min) were
confirmed by comparison with authentic standards.
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Received November 23, 2009