Triterpenoid saponins with antifeedant activities from stem bark of Catunaregam spinosa (Rubiaceae) against Plutella xylostella (Plutellidae)

Article abstract of DOI:10.1016/j.carres.2011.07.022

Seven triterpenoid saponins, including four new compounds, catunarosides A-D (1-4), and three known compounds, swartziatrioside (5), aralia-saponin V (6), araliasaponin IV (7) were isolated from the stem bark of Catunaregam spinosa, a Chinese mangrove associate. Their structures were elucidated on the basis of their spectral data and hydrolysis experiments. The antifeedant activities of compounds 1-7 against Plutella xylostella were also evaluated.

Full text of DOI:10.1016/j.carres.2011.07.022

Carbohydrate Research 346 (2011) 2200–2205
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Triterpenoid saponins with antifeedant activities from stem bark of
Catunaregam spinosa (Rubiaceae) against Plutella xylostella (Plutellidae)
Guangchun Gao a,b, , Zhongxian Lu b, Shuhong Tao c, Si Zhang a, , Fazuo Wang a
⇑
⇑
a Guangdong Key Laboratory of Marine Material Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, PR China
b Institute of Plant Protection and Microbiology, Zhejiang Academy of Agriculture Science, Hangzhou 310021, PR China
c Guangdong Pharmaceutical University, University City, Guangzhou 510006, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 May 2011
Received in revised form 21 July 2011
Accepted 25 July 2011
Available online 4 August 2011
Seven triterpenoid saponins, including four new compounds, catunarosides A–D (1–4), and three known
compounds, swartziatrioside (5), aralia-saponin V (6), araliasaponin IV (7) were isolated from the stem
bark of Catunaregam spinosa, a Chinese mangrove associate. Their structures were elucidated on the basis
of their spectral data and hydrolysis experiments. The antifeedant activities of compounds 1–7 against
Plutella xylostella were also evaluated.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Triterpenoid saponins
Catunaregam spinosa
Catunarosides A–D
Plutella xylostella
Antifeedant activity
1. Introduction
R3
Catunaregam spinosa (Thunb.) Tirveng (Rubiaceae), known as
‘Madana and Mainphal’ in Indian and ‘Sacrifício de Cristo’ in Brazil,
is a folk medicine plant distributed in tropical and subtropical
regions.1,2 Its fruits are well known for their antispasmodic, anti-
dysenteric, antifertility and immunomodulatory properties.3,4 The
plant is used to cure emetic, asthma, jaundice, gonorrhea.5 Previ-
ous phytochemical investigations on this plant have led to the iso-
lation and identification of coumarin glucosides, iridoid glucosides,
triterpenoid saponins and lignans.1,3,6–10 Triterpenoid saponins
account for 9.5% of the EtOH extracts from fruits of C. spinosa.2 In
our phytochemical research on the stem bark of C. spinosa, four
new oleanane-type triterpenoid saponins, catunarosides A–D,
together with swartziatrioside, aralia-saponin V and araliasaponin
IV were isolated (Fig. 1), and their antifeedant activities againstPlu-
tella xylostella were also evaluated.
O
OH
OR2
OH
O
OH
O
O
O
OR1
HO
OH
OH
1
R1=β-D-Xyl
R2=H
R3=OH
R3=H
2 R1=α-L-Rha
3 R1=α-L-Rha
R2=H
R2=β-D-Glc
R2=β-D-Glc
R2=H
R3=OH
R3=OH
R3=H
4
R1=β-D-Xyl
5 R1=β-D-Xyl
6 R1=β-D-Glc
R2=β-D-Glc
R2=β-D-Glc
R3=H
7
R1=β-D-Xyl
R3=H
2. Results and discussion
Figure 1. Structures of compounds 1–7.
Catunaroside A (1) was obtained as an amorphous powder. It has
ten degrees of unsaturation as deduced from the molecular formula
C
47H76O18 determined by HRESIMS. The IR spectrum revealed
absorption bands at 1741 cmꢀ1 (C@O) and 1645 cmꢀ1 (C@C). There-
fore, the molecule has eight rings. The 13C NMR spectrum showed se-
ven methyl carbons at d 27.7, 16.4, 15.4, 17.4, 24.8, 28.8 and 24.9,
two olefinic carbons at d 123.1 and 144.9. The seven methyl groups
(d 1.28, 1.07, 0.84, 1.03, 1.68, 1.20 and 1.12) and an olefinic proton
⇑
Corresponding authors. Address: Institute of Plant Protection and Microbiology,
Zhejiang Academy of Agriculture Science, 198 Shiqiao Road, Hangzhou 310021, PR
China. Tel./fax: +86 571 86404077 (G.G.).
E-mail address: gaogc@scib.ac.cn (G. Gao).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.07.022

G. Gao et al. / Carbohydrate Research 346 (2011) 2200–2205
2201
HO
O
O
OH
OH
3'
OH
OH
OH
O
OH
1'
O
3
1'
OH
O
3
O
OH
O
1'''
O
O
O
O
1'''
3'
O
1''
HO
OH
OH
HO
1''
OH
OH
OH
O
O
OH
OH
CH3
OH
OH
HO
HO
HO
O
O
O
O
28
28
OH
OH
OH
OH
1''''
1''''
OH
O
O
OH
O
O
1'
1'
3
OH
3
O
OH
O
O
O
O
OH
OH
1'''
HO
O
O
3'
OH
OH
1'''
HO
3'
O
HO
1''
OH
OH
1''
OH
HO
OH
OH
O
O
OH
OH
CH3
OH
OH
HO
Figure 2. Key HMBC correlations for the sugar sequence of 1–4 (from H to C).
(d 5.55, br s) were also observed in the 1H NMR spectrum. The infor-
mation from the 1H NMR spectrum coupled with the 13C NMR spec-
trum indicated that 1 possessed a siaresinolic acidic aglycon.11 The
chemical shifts of C-3 (d 89.6) and C-28 (d 180.9) revealed that 1
was a monodesmosidic glycoside. In the 13C NMR spectrum, 27 of
47 resonances were assigned to the oligosaccharide moieties. The
1H NMR and 13C NMR spectra of 1 exhibited three sugar anomeric
protons at d 4.83 (d, J = 7.7 Hz), 5.37 (d, J = 7.9 Hz) and 5.60
(d, J = 7.7 Hz), and carbons at d 105.1, 104.8 and 104.6. The monosac-
contain one L-rhamnose and two D
-glucose residues. The 13C NMR
chemical shifts of C-3 (d 88.7) and C-28 (d 180.8) indicated that 2
was a monodesmosidic glycoside in which the sugar moiety was
connected at C-3.13 The sequence of the oligosaccharide chain,
as well as the glycoside sites, was determined by an HMBC exper-
iment. In the HMBC spectrum, the following correlations were
observed: H-10 (d 4.87) of the inner glucose and C-3 (d 88.7) of
aglycon part; H-100 (d 5.15) of the terminal glucose and C-30
(d 89.6) of inner glucose; H-1000 (d 6.51) of the terminal rhamnose
and C-20 (d 78.7) of inner glucose (Fig. 2). The b-anomeric config-
charides were identified as D-glucose and D-xylose by TLC and GC-
MS analysis, The glycan part signals on NMR spectrum of 1 were
identical with those of swartziatrioside (5),12 and the sequence of
the glycan part was deduced from the following HMBC correlations:
H-100 (d 5.37) of terminal glucose with C-30 (d 88.9) of inner glucose,
H-1000 (d 5.60) of terminal xylose with C-20 (d 79.0) inner glucose, H-10
(d 4.83) of inner glucose with C-3 (d 89.6) of the aglycon (Fig. 2). The
b-anomeric configurations of the glucose and xylose units were
urations of the glucose units and a-anomeric configuration of the
3
rhamnose unit were separately determined by their JH1,H2 cou-
1
pling constants (7.5 and 7.8 Hz) and the JC1,H1 coupling constant
(169 Hz). On the basis of this evidence, the structure of compound
2, named as catunaroside B, was elucidated to be 3-O-a-L-
rhamnopyranosyl-(1?2)-[b-D-glucopyranosyl-(1?3)]-b-D-gluco-
pyranosyl-oleanolic acid.
3
determined by the JH1,H2 coupling constants (7.7–7.9 Hz). On the
Compound 3 was shown to have the molecular formula
basis of all the foregoing evidence, compound 1 was elucidated as
C54H88O23 based on its HRESIMS spectrum. A comparison of the
3-O-b-
D
-xylopyranosyl-(1?2)-[b-
D
-glucopyranosyl-(1?3)]-b-
D
-
1H and 13C NMR spectra of 3 with those of 1 clearly revealed that
the aglycon of 3 was identical to that of 1. Acid hydrolysis of 3 gave
glucopyranosyl-siaresinolic acid.
Compound 2 was analyzed to have the molecular formula of
48H78O17 by its HRESIMS spectrum (m/z: 949.5341 [M+Na]+).
L-rhamnose and D-glucose, which were determined by comparing
C
the Rf values in TLC analysis and the retention times in GC–MS anal-
ysis with that of authentic samples. Four anomeric proton signals at
d 4.85 (d, J = 7.5 Hz), 5.13 (d, J = 7.9 Hz), 6.49 (br s) and 6.37 (d,
J = 8.5 Hz) in the 1H NMR spectrum were observed. The downfield
13C NMR chemical shift at d 88.8 and the upfield 13C NMR chemical
shift at d 177.3 suggested that 3 was a bisdesmosidic glycoside with
glycosidic linkages at C-3 through an ether bond and at C-28
through an ester bond.14 By comparison of their signals in 13C
NMR spectrum with those of 2, the glycan parts were the same, ex-
cept one more glucose residue, which was connected to C-28. The
sequence of the glycan parts and glycoside sites were also proven
by the following HMBC correlations: H-10 (d 4.85) of the inner
glucose and C-3 (d 88.8) of aglycon part; H-100 (d 5.13) of the glucose
and C-30 (d 89.5) of inner glucose; H-1000 (d 6.49) of the rhamnose and
C-20 (d 78.7) of inner glucose (Fig. 2); H-10000 (d 6.37) of the glucose
The signals of seven methyl carbons at d 28.0, 17.1, 15.6, 17.4,
26.2, 33.3, 23.7, two olefinic carbons d 122.5 and 144.8, and one
carbonyl carbon at d 180.8 were present in the 13C NMR spectrum.
In combination with the 1H NMR spectrum, the aglycon part of this
compound was determined as oleanolic acid, based on compari-
sons of the 1H, 13C and DEPT NMR signals of 2 with those from
3-O-[b-D-glucopyranosyl-(1?3)-b-D-galactopyranosyl]-olean-12-
en-3b-ol-28-oic acid.9 From the signals in the anomeric region of
the 13C NMR spectrum (resonances at d 104.9, 103.9 and 101.7),
compound 2 had three sugar residues. Acid hydrolysis suggested
that the monosaccharides of this compound were
L-rhamnose and
D
-glucose, which were identified by GCMS analysis of their chiral
derivatives. According to the characteristic proton signal at d 1.70
(3H, d, J = 6.0 Hz) in the 1H NMR spectrum, 2 could be deduced to

2202
G. Gao et al. / Carbohydrate Research 346 (2011) 2200–2205
and C-28 (d 177.3) of aglycon part (Fig. 2). The 1JC1,H1 coupling con-
stant of the rhamnose unit (170 Hz) confirmed that the anomeric
and activities of monodesmosidic saponins, and can be converted
into the biologically active monodesmosidic form by removal of
the sugar at the C-28 position.18 Our results show bidesmosidic
saponins 3, 4, 6 and 7 exhibit weaker antifeedant activity than
monodesmosidic saponins 1, 2 and 5. These findings suggest bides-
mosidic saponins in C. spinosa would be deglycosylated to
monodesmosidic saponins at C-28 position when the plant is at-
tacked by P. xylostella.
proton was equatorial (
configurations of the
coupling constants (7.5–8.5 Hz). On the basis of the above evidence,
the structure of compound 3 was elucidated as 3-O- -rhamnopyr-
anosyl-(1?2)-[b- -glucopyranosyl-(1?3)]-b- -glucopyranosyl-
siaresinolic acid 28-O-b- -glucopyranoside.
a-pyranoid anomeric form). The b-anomeric
3
D
-glucose were determined by their JH1,H2
a-L
D
D
D
Compound 4 has the molecular formula of C53H86O23 based on
its HRESIMS spectrum. The 1H and 13C NMR signals of 4 were
assigned after DEPT, HSQC and HMBC experiments. A comparison
of the 1H and 13C NMR spectra of 4 with those of 3 clearly revealed
that the aglycon part of 4 was identical to that of 3, and 4 was sug-
gested to be a 3,28-bisdesmoside with four monosaccharide units.
It has the same linkage sequence of sugar moiety and the same
ester-glycoside chain as araliasaponin IV,15 which was further con-
firmed by acid hydrolysis and the key HMBC correlations (Fig. 2).
From the above information, the structure of 4 was determined
3. Experimental
3.1. General
Column chromatography (CC) was performed on silica gel (200–
300 mesh, Qingdao Haiyang Chemical Plant), macroporous resin
(D101, Nankai University Chemical Plant), RP-18 silica gel (40–
60 mesh, Merck) or on Sephadex LH-20 (Pharmacia). IR spectra
were obtained using Bruker EQUINOX55 spectrometer. Optical
rotations were taken on an ATAGO Polax-2L polarimeter. NMR
spectra were recorded on a Bruker DRX-500, in pyridine-d5, either
at 500 (1H) or 125 (13C) MHz, using tetramethylsilane (TMS) as the
internal standard. ESI mass spectra were collected on MDS SCIEX
API 2000 LC/MS/MS instrument and Bruker BioTOF Q spectrometer
for HR-ESI mass spectra. Precoated Si gel plates were used for ana-
as 3-O-b-
D-xylopyranosyl-(1?2)-[b-
D
-glucopyranosyl-(1?3)]-b-
D-
glucopyranosyl-siaresinolic acid 28-O-b-
D-glucopyranoside.
Compounds 5–7, an amorphous powder, had similar IR spectra
with 1–4. Their 1H and 13C NMR signals were also assigned after
DEPT, HSQC and HMBC experiments. They were elucidated as
swartziatrioside (5),12 aralia-saponin V (6)16 and araliasaponin IV
(7)15 separately by HRESIMS, IR data, 1D and 2D NMR data. Com-
pounds 1–4 were new compounds, which are named as catunaro-
sides A–D.
The antifeedant activities of compounds 1–7 against second-
instar larvae of P. xylostella were tested. The results are shown
in Table 4. All the isolated compounds show potent antifeedant
activity against second-instar larvae of the diamondback moth.
lytical TLC. Preparative HPLC (Waters-600) equipped with
Waters-996 photodiode array detector was made by using an
ODS column (YMC-Pack ODS-5-A, 250 ꢁ 10 mm i.d., 5
a
lm; YMC).
3.2. Plants and insects
The stem bark of C. spinosa was collected in February 2006 from
Sanya, Hainan Province, PR China. The specimen was identified by
professor Si Zhang, South China Sea Institute of Oceanology, Chinese
Academy of Sciences. A voucher specimen has been deposited in the
South China Sea Institute of Oceanology, Chinese Academy of Sci-
ences (accession number: GKLMMM020). The P. xylostella tested
were obtained from established laboratory stock culture maintained
Compound 5, with the AFC50 value 106.73 lg/mL, is the most ac-
tive one of all compounds. Our findings support the earlier sug-
gestion that triterpenoid saponins are repellent or deterrent to
some insect herbivores.17
Bisdesmosidic saponins have two sugar chains, usually one at
the C-3 carbon, and one at C-28. They lack many of the properties
Table 1
1H NMR spectroscopic data (d) for the sugar moieties of compounds 1–7 (600 MHz in pyridine-d5)
Position
1
2
3
4
5
6
7
3-O
10
Glu
4.83 (d, 7.7)
4.19
4.25
4.06
3.85
4.56, 4.29
Xyl
5.60 (d, 7.7)
4.08
4.32
Glu
4.87 (d, 7.5)
4.04
4.22
4.28
4.10
4.48, 4.27
Rha
6.51 br s
4.62
4.82
4.31
4.77
1.70 (d, 6.0)
Glu
5.15 (d, 7.8)
3.90
4.04
4.19
4.12
4.56, 4.27
Glu
4.85 (d, 7.5)
4.03
4.21
4.26
4.11
4.48, 4.28
Rha
6.49 br s
4.60
4.82
4.29
4.75
1.69 (d, 6.0)
Glu
5.13 (d, 7.5)
3.88
4.01
4.18
4.10
4.55, 4.26
Glu
Glu
4.82 (d, 7.5)
4.23
4.22
4.04
3.82
4.54, 4.27
Xyl
5.60 (d, 7.5)
4.05
4.29
Glu
4.83 (d, 7.5)
4.19
4.24
4.04
3.84
4.54, 4.26
Xyl
5.59 (d, 7.4)
4.05
4.29
Glu
4.83 (d, 7.5)
4.34
4.24
4.01
3.80
4.48, 4.30
Glu
5.72 (d, 8.0)
4.06
4.25
4.14
3.87
4.44
Glu
5.37 (d, 7.5)
4.02
4.19
4.12
4.17
Glu
4.82 (d, 7.5)
4.2
4.23
4.00
3.81
4.50, 4.22
Xyl
5.56 (d, 7.5)
4.03
4.24
20
30
40
50
60
20-O
100
200
300
400
4.26
3.60, 4.30
4.23
3.60, 4.31
4.24
3.60, 4.31
4.17
3.55, 4.26
500
600
30-O
1000
2000
3000
4000
5000
6000
28-O
10000
20000
30000
40000
50000
60000
Glu
5.37 (d, 7.9)
4.07
4.24
4.18
Glu
5.37 (d, 7.5)
4.04
4.24
4.16
4.03
4.45, 4.25
Glu
6.38 (d, 8.0)
4.22
4.37
Glu
5.36 (d, 7.8)
4.05
4.23
4.15
Glu
5.34 (d, 8.0)
4.03
4.17
4.14
4.02
4.42, 4.20
Glu
6.29 (d, 7.5)
4.17
4.18
4.05
4.47, 4.25
4.04
4.45, 4.24
4.23
Glu
6.33 (d, 8.0)
4.18
4.22
4.31
4.31
4.37
6.37 (d, 8.5)
4.20
4.36
4.28
4.36
4.23
4.37
4.43
4.32
4.31
4.37
4.41

G. Gao et al. / Carbohydrate Research 346 (2011) 2200–2205
2203
Table 2
13C NMR spectroscopic data (d) for the aglycon moieties of compounds 1–4 (125 MHz in pyridine-d5)
Position
1
2
3
4
Position
1
2
3
4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
38.5
26.6
89.6
39.7
56.0
18.6
33.3
40.0
48.2
37.1
24.1
39.0
26.7
88.7
39.5
56.1
18.5
33.2
39.7
48.0
37.0
23.7
39.0
26.7
88.8
39.5
56.3
18.8
33.2
40.2
48.3
37.2
24.1
38.6
26.5
89.7
39.6
56.0
18.7
33.0
40.2
48.2
37.1
24.1
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
28.4
46.1
44.8
81.3
35.7
29.2
33.7
27.7
16.4
15.4
17.4
24.8
23.7
46.7
42.0
46.5
31.0
34.1
33.2
28.0
17.1
15.6
17.4
26.2
28.1
46.5
44.6
81.2
35.6
29.1
33.1
28.1
17.0
15.6
17.6
25.0
28.0
46.5
44.6
81.1
35.5
29.0
33.2
27.8
16.4
15.4
17.5
24.9
123.1
144.9
42.1
122.5
144.8
42.2
123.1
144.3
42.2
123.7
144.3
42.1
180.9
28.8
24.9
180.8
33.3
23.7
177.3
28.7
24.7
177.0
28.7
24.7
29.2
28.3
29.0
29.0
for >15 generations at 25 1 °C temperature, 75 10% RH and a
14:10 h (L: D) photoperiod. Cabbage plants (Brassica oleraceae) were
grown in greenhouse at Institute of Plant Protection and Microbiol-
ogy, Zhejiang Academy of Agriculture Science, PR China, without any
application of insecticides.
Table 3
13C NMR spectroscopic data (d) for the sugar moieties of compounds 1–7 (125 MHz in
pyridine-d5)
Position
1
2
3
4
5
6
7
3-O
10
Glu
Glu
Glu
Glu
Glu
Glu
Glu
105.1
79.0
88.9
70.2
77.8
62.3
Xyl
104.9
78.7
89.6
76.9
69.8
62.7
Rha
104.9
78.7
89.5
77.0
69.9
62.7
Rha
105.0
79.0
89.0
70.2
77.8
62.2
Xyl
105.0
79.0
88.9
70.1
77.8
62.3
Xyl
104.9
79.3
88.7
70.0
77.6
63.4
Glu
105.1
79.0
88.9
70.2
77.8
62.3
Xyl
20
3.3. Extraction and isolation
30
40
The n-BuOH extract of air-dried and powered plant material
(9.0 kg) was extracted by the same method as previously re-
ported.7 The n-BuOH soluble fraction (170 g) was passed through
a macroporous resin (D101) column eluted with H2O and EtOH–
H2O (3:7, 6:4, 95:5), whereas the EtOH–H2O (6:4, 35 g) eluted por-
tion was subjected to silica gel CC eluting with CHCl3–MeOH
(90:10?50:50) to give fractions A–F. Fraction D was separated
by ODS silica gel CC eluting with MeOH–H2O (50:50) and purified
by preparative HPLC using MeOH–H2O–H3PO4 (65:35:0.1) to give
compounds 6 (15 mg) and 7 (132 mg); Fraction F was separated
by silica gel CC eluting with CHCl3–MeOH–H2O (80:20:5), then
purified by preparative HPLC using MeOH–H2O–H3PO4
(55:45:0.1) to give compounds 3 (176 mg) and 4 (13 mg). The
EtOH–H2O (95:5, 10 g) eluting portion was subjected to silica gel
CC eluting with CHCl3–MeOH (98:2?50:50) to give fractions a–g.
Fraction d was separated by ODS silica gel CC eluted with
MeOH–H2O (50:50?80:20), then fractions 29–30 and fractions
41–45 were purified by preparative HPLC, respectively, using
50
60
20-O
100
104.6
76.2
79.3
71.4
67.2
101.7
72.5
72.5
73.9
69.8
18.6
Glu
101.7
72.5
72.5
73.9
69.9
18.6
Glu
104.6
76.2
79.3
71.4
67.2
104.6
76.2
79.3
71.4
67.2
103.8
75.4
78.6
72.6
77.8
62.2
Glu
104.7
76.3
79.4
71.5
67.2
200
300
400
500
600
30-O
1000
2000
3000
4000
5000
6000
28-O
10000
20000
30000
40000
50000
60000
Glu
Glu
Glu
Glu
104.8
75.4
78.6
71.6
78.6
62.6
103.9
77.8
75.2
78.4
71.5
62.3
103.9
77.8
75.2
78.4
71.5
62.4
Glu
104.7
75.4
78.6
71.6
78.6
62.6
Glu
104.7
75.4
78.6
71.6
78.6
62.6
104.7
76.4
78.6
71.6
78.6
62.6
Glu
104.8
75.4
78.6
71.6
78.6
62.7
Glu
95.9
74.2
78.9
71.1
79.3
62.2
95.9
74.2
78.9
71.1
79.3
62.3
95.7
74.2
78.9
71.1
79.3
62.3
95.8
74.2
78.9
71.2
79.3
62.4
MeOH–H2O–H3PO4
(65:35:0.1)
and
MeOH–H2O–H3PO4
(62:38:0.1) to give compounds 1 (96 mg), 5 (13 mg), and 2 (6 mg).
3.3.1. Catunaroside A (1)
Table 4
Antifeedant activities of compounds 1–7 against P. xylostella larva
Colorless amorphous powder, ½a D20
ꢂ
+10.4 (c 0.8, MeOH); IR (KBr)
m
max cmꢀ1: 3400, 2920, 1741, 1645, 1038; 1H NMR (500 MHz, pyr-
Compound no.
Regression equation
r
AFC50 (lg/ml)
idine-d5): d 3.25(1H, dd, J = 4.5, 11.6 Hz, H-3), 5.55 (1H, br s, H-12),
3.63 (1H, br s, H-18), 3.60 (1H, br s, H-19), 1.28 (1H, s, H-23), 1.07
(1H, s, H-24), 0.84 (1H, s, H-25), 1.03 (1H, s, H-26), 1.68 (1H, s, H-
27), 1.20 (1H, s, H-29), 1.12 (1H, s, H-30); 1H NMR data of the sugar
part, see Table 1; 13C NMR (125 MHz, pyridine-d5) data, see Table 2
and Table 3; ESIMS (positive mode) m/z: 951 [M+Na]+, 967 [M+K]+;
HRESIMS (positive mode) m/z: 951.4938 ([M+Na]+, C47H76O18Na+;
calcd 951.4930).
1
2
3
4
5
6
7
Y = 4.673+0.137x
Y = 4.250+0.297x
Y = 4.300+0.235x
Y = 4.673+0.137x
Y = 4.299+0.346x
Y = 4.286+0.191x
Y = 4.250+0.297x
0.986
0.999
0.992
0.952
0.981
0.992
0.996
243.23
369.63
946.13
608.42
106.73
5467.82
335.54
Table 1; 13C NMR (125 MHz, pyridine-d5) data, see Table 2 and Ta-
ble 3; ESIMS (positive mode) m/z: 949 [M+Na]+, 965 [M+K]+; HRE-
SIMS (positive mode) m/z: 949.5349 ([M+Na]+, C48H78O17Na+; calcd
949.5341).
3.3.2. Catunaroside B (2)
Colorless amorphous powder, ½a D20
ꢂ
+32.4 (c 0.8, MeOH); IR (KBr)
m
max cmꢀ1: 3410, 2925, 1739, 1640, 1030; 1H NMR (500 MHz, pyr-
idine-d5): d 3.40(1H, dd, J = 3.9, 12.0 Hz, H-3), 5.47 (1H, br s, H-12),
3.39 (1H, dd, J = 10.1, 3.8 Hz, H-18), 1.30 (1H, s, H-23), 1.17 (1H, s,
H-24), 0.82 (1H, s, H-25), 0.98 (1H, s, H-26), 1.26 (1H, s, H-27), 0.95
(1H, s, H-29), 1.01 (1H, s, H-30); 1H NMR data of the sugar part, see
3.3.3. Catunaroside C (3)
Colorless amorphous powder, ½a D20
ꢂ
+37.5 (c 0.4, MeOH); IR (KBr)
mmax cmꢀ1: 3408, 2926, 1743, 1644, 1035; 1H NMR (500 MHz,

2204
G. Gao et al. / Carbohydrate Research 346 (2011) 2200–2205
pyridine-d5): d 3.38 (1H, dd, J = 3.8, 11.5 Hz, H-3), 5.50 (1H, br s, H-
12), 3.52 (1H, br s, H-18), 3.57 (1H, br s, H-19), 1.25 (1H, s, H-23),
1.18 (1H, s, H-24), 0.88 (1H, s, H-25), 1.13 (1H, s, H-26), 1.62 (1H, s,
H-27), 1.14 (1H, s, H-29), 0.98 (1H, s, H-30); 1H NMR data of the su-
gar part, see Table 1; 13C NMR (125 MHz, pyridine-d5) data, see Ta-
ble 2 and Table 3; ESIMS (positive mode) m/z: 1127 [M+Na]+;
HRESIMS (positive mode) m/z: 1127.5562 ([M+Na]+, C54H88O23Na+;
calcd 1127.5565).
0.25 lm). Column temperature: 100–180 °C, with a rate of 10 °C/
min, then 180–240 °C, with a rate of 3 °C /min, keeping at 240 °C
for 5 min, and the carrier gas was He (1.2 mL/min), split ratio 1/
50, injection temperature: 250 °C. Injection volume: 1
absolute configurations of the monosaccharides were confirmed
to be -rhamnose, -xylose, and -glucose, by comparison of the
retention times of monosaccharide derivatives with those of stan-
dard samples: -rhamnose (9.69 min), -xylose (12.45 min) and
lL. The
L
D
D
L
D
D-
glucose (13.61 min), respectively.
3.3.4. Catunaroside D (4)
Colorless amorphous powder, ½a D20
ꢂ
+68.5 (c 0.15, MeOH); IR
3.5. Antifeedant assay
(KBr) mmax cmꢀ1
:
3408, 2929, 1741, 1645, 1038; 1H NMR
(500 MHz, pyridine-d5): d 3.25 (1H, dd, J = 3.5, 11.5 Hz, H-3), 5.50
(1H, br s, H-12), 3.52 (1H, br s, H-18), 3.58 (1H, br s, H-19), 1.28
(1H, s, H-23), 1.09 (1H, s, H-24), 0.87 (1H, s, H-25), 1.13 (1H, s,
H-26), 1.65 (1H, s, H-27), 1.14 (1H, s, H-29), 0.98 (1H, s, H-30);
1H NMR data of the sugar part, see Table 1. 13C NMR (125 MHz,
pyridine-d5) data, see Table 2 and Table 3. ESIMS (positive mode)
m/z: 1114 [M+Na]+, 1127 [M+K]+; HRESIMS (positive mode) m/z:
1113.5471 ([M+Na]+, C53H86O23Na+; calcd 1113.5463).
A conventionalnon-choice leaf disc method experiment was con-
ducted to evaluate the antifeedant activities.20 Each sample was dis-
solved in water containing 0.05% Tween 80 (Polysorbate 80) to
obtain serial concentrations of 0.1, 1, 10, 100 and 1000 ppm. Water
containing 0.05% Tween 80 was used as a control separately. Leaf
disks (1.5 cm diameter) cut from cabbage leaves with a cork borer,
were dipped for 10 s in the sample and control solutions and dried
in the air for 3 h at room temperature. Four leaf disks were trans-
ferred to Petri dishes (9 cm diameter and 2 cm depth) with a moist
filter paper on the bottom. Five second instar larvae of P. xylostella
were starved for 2 h and then placed alongside the leaf disks at the
center of the Petri dish. Each treatment was replicated six times
and conducted at 25 1 °C temperature, 75 10% RH and a photope-
riod of 14:10 h (L:D). The amount of leaf consumed within 48 h was
recorded. The area of each disk consumed during this period was
determined by placing leaf remains on the graph paper and then
counting the squares within the consumed area. Then the antifee-
dant index (%) was calculated as (C ꢀ T)/C ꢁ 100 where C is the con-
sumed leaf area of negative control disks and T is the consumed leaf
area of treated and positive control disks. Leaf area consumed in
treatment was corrected from the control. The antifeedant concen-
tration of 50% (AFC50) values generated by linear regression was
determined by probit analysis (SPSS V13.0).
3.3.5. Swartziatrioside (5)
Colorless amorphous powder, ½a D20
ꢂ
+35.0 (c 0.15, MeOH); IR
(KBr) mmax cmꢀ1
:
3403, 2929, 1740, 1645, 1038; 1H NMR
(500 MHz, pyridine-d5): d 3.28 (1H, dd, J = 4.0, 11.0 Hz, H-3), 5.47
(1H, br s, H-12), 3.29 (1H, dd, J = 13.0, 4.0 Hz, H-18), 1.28 (1H, s,
H-23), 1.07 (1H, s, H-24), 0.81 (1H, s, H-25), 0.98 (1H, s, H-26),
1.30 (1H, s, H-27), 0.96 (1H, s, H-29), 1.01 (1H, s, H-30); 1H and
13C NMR data of the sugar part, see Table 1 and Table 3; ESIMS (po-
sitive mode) m/z: 935 [M+Na]+, 951 [M+K]+.
3.3.6. Aralia-saponin V (6)
Colorless amorphous powder, ½a D20
ꢀ15.0 (c, 0.2, MeOH); IR
ꢂ
(KBr) mmax cmꢀ1
:
3409, 2924, 1738, 1641, 1038; 1H NMR
(500 MHz, pyridine-d5): d 3.27 (1H, dd, J = 4.0, 11.5 Hz, H-3), 5.42
(1H, br s, H-12), 3.19 (1H, dd, J = 12.5, 3.8 Hz, H-18), 1.25 (1H, s,
H-23), 1.08 (1H, s, H-24), 0.81 (1H, s, H-25), 1.08 (1H, s, H-26),
1.25 (1H, s, H-27), 0.91 (1H, s, H-29), 0.88 (1H, s, H-30), 4.83 (1H,
d, J = 7.5 Hz, H-10); 1H and 13C NMR data of the sugar part, see
Table 1 and Table 3; ESIMS (positive mode) m/z: 1127 [M+Na]+.
Acknowledgments
This study was financially supported by the National Basic Re-
search Program of China (No. 2010CB833801) and the Scientific
Research Special Funds Project of the Ministry of Finance
(KSCX2-YW-Z-1018).
3.3.7. Araliasaponin IV (7)
Colorless amorphous powder, ½a D20
ꢂ
+80.0 (c = 0.5, MeOH); IR
(KBr) mmax cmꢀ1
:
3411, 2928, 1743, 1640, 1032; 1H NMR
Supplementary data
(500 MHz, pyridine-d5): d 3.26(1H, dd, J = 4.0, 11.0 Hz, H-3), 5.42
(1H, br s, H-12), 3.19 (1H, dd, J = 13.0, 4.0 Hz, H-18), 1.27 (1H, s,
H-23), 1.07 (1H, s, H-24), 0.84 (1H, s, H-25), 1.08 (1H, s, H-26),
1.27 (1H, s, H-27), 0.92 (1H, s, H-29), 0.88 (1H, s, H-30); 1H and
13C NMR data of the sugar part, see Table 1 and Table 3; ESIMS
(positive mode) m/z: 1098 [M+Na]+.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2011.07.022.
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