Chemical constituents from the roots of Periploca sepium with insecticidal activity

Article abstract of DOI:10.1080/10286020.2012.691880

Five compounds were isolated from the root powder of Periploca sepium. By mainly HR-ESI-MS, ¹H, ¹³C, and 2D NMR spectral data, they were characterized as periplocoside X (1), oligasaccharide A (2), periplocoside A (3), periplocoside E (4), and periplocoside N (5), respectively. Compounds 1-5 were found to possess insecticidal activities against the red imported fire ant. Among the compounds, periplocoside X showed significant activity with LD₅₀ values of 748.99, 116.62, 2169.58, and 3079.33mg/l against soldiers, workers, males, and alate females of red imported fire ant, respectively.

Full text of DOI:10.1080/10286020.2012.691880

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Chemical constituents from the roots
of Periploca sepium with insecticidal
activity
Yan Li a , Xin-Nian Zeng a , Wen-Zhong Wang a , Cai-Hong Luo a
Qin Yan a & Meng Tian a
,
a The Ministry of Education Key Laboratory of Pesticide
and Chemical Biology, South China Agricultural University ,
Guangzhou , 510642 , China
Published online: 14 Jun 2012.
To cite this article: Yan Li , Xin-Nian Zeng , Wen-Zhong Wang , Cai-Hong Luo , Qin Yan & Meng Tian
(2012) Chemical constituents from the roots of Periploca sepium with insecticidal activity, Journal
of Asian Natural Products Research, 14:8, 811-816, DOI: 10.1080/10286020.2012.691880
To link to this article: http://dx.doi.org/10.1080/10286020.2012.691880
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Journal of Asian Natural Products Research
Vol. 14, No. 8, August 2012, 811–816
Chemical constituents from the roots of Periploca sepium
with insecticidal activity
Yan Li, Xin-Nian Zeng*, Wen-Zhong Wang, Cai-Hong Luo, Qin Yan and Meng Tian
The Ministry of Education Key Laboratory of Pesticide and Chemical Biology, South China
Agricultural University, Guangzhou 510642, China
(Received 12 June 2011; final version received 3 May 2012)
Five compounds were isolated from the root powder of Periploca sepium. By mainly
HR-ESI-MS, 1H, 13C, and 2D NMR spectral data, they were characterized as
periplocoside X (1), oligasaccharide A (2), periplocoside A (3), periplocoside E (4),
and periplocoside N (5), respectively. Compounds 1–5 were found to possess
insecticidal activities against the red imported fire ant. Among the compounds,
periplocoside X showed significant activity with LD50 values of 748.99, 116.62,
2169.58, and 3079.33mg/l against soldiers, workers, males, and alate females of red
imported fire ant, respectively.
Keywords: Periploca sepium; the red imported fire ant; insecticidal activity;
periplocoside X
1. Introduction
As a result, five compounds were
isolated by bioassay guide including one
new steriod, periplocoside X (1), and the
red imported fire ant as a model insect. The
structure of 1 was elucidated on the basis of
spectroscopic analyses including HR-ESI-
MS, 1H and 13C NMR, HSQC, HMBC, and
TOCSY.
The crude drug ‘xiangjiapi’, which is root
barks of Periploca sepium Bunge (Ascle-
piadaceae), has been used as a traditional
Chineseherbalmedicinefor the treatmentof
rheumatoid arthritis and wound. It is widely
distributed in northeastern area and south-
western area of China [1]. Previously
isolated compounds included cardiac gly-
cosides, pregnane glycosides, oligosacchar-
ides, triterpenoids, and so on [2–9]. It has
been reported that the extract of P. sepium
possesses insecticidal activities against
Mythimna separate [10], Plutella xylostella,
Schizaphis graminum, Pieris rapae, and
Musca domestica [11]. In order to determine
the bioactive principles and to explore this
plant deeply, the methanol extract of
P. sepium has been studied.
2. Results and discussion
Compound 1 was obtained as white
25
amorphous powder with ½aꢀD 72.6
(c ¼ 0.1, CH3OH) and mp 255–2578C. Its
molecular formula C79H126O30, with 17
degrees of unsaturation, was deduced from
the pseudo-molecular ions at m/z 1578.8450
[M þ Na]þ and 1556.8632 [M þ H]þ using
HR-ESI-MS. Compound 1 showed positive
*Corresponding author. Email: zengxn@scau.edu.cn
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2012.691880
http://www.tandfonline.com

812
Y. Li et al.
reaction to the Liebermann–Burchard and
Molisch reactions, suggesting that 1 pos-
sessed steroidal glycoside. Its 13C NMR
spectroscopic data established that the
aglycone of 1 was identical to that of the
known natural product periplocoside A [4].
Furthermore, the characteristic carbon
signals at dC 97.8, 186.1, 148.2, 119.4,
68.8, 23.0, and 54.8, together with the
corresponding protonsignals atdH 5.03(1H,
s), 5.93 (1H, s), 4.18 (1H, m), 1.28 (3H, d,
J ¼ 5.7 Hz), and 3.78 (3H, OMe), soundly
confirmed the presence of additional similar
structure fragment 4,6-dideoxy-3-methoxy-
2-hexosuloside-3-ene in compound 1 [4].
The 13C NMR spectrum showed six
anomeric carbons at dC 103.6, 98.7, 98.8,
100.0, 100.3, and 101.9, and unusual signals
at dC 114.2 and 86.7 for sugar moiety. Also,
the 1H NMR spectrum showed the
corresponding anomeric protons at dH 4.73
(d, J ¼ 7.7 Hz), 5.25 (br d, J ¼ 9.9 Hz), 5.48
(br d, J ¼ 9.3 Hz), 4.86 (br d, J ¼ 9.4 Hz),
5.03 (br d, J ¼ 9.0 Hz), and 5.15 (br d,
J ¼ 9.9 Hz), and the typical signals at dH
5.38/4.97 (d, J ¼ 7.3 Hz) for 3,7-dideoxy
heptulose (Table 1) [4]. The HMQC
correlation between the carbon at dC 86.7
and each gem-proton at dH 4.97, 5.38 (d,
J ¼ 7.3 Hz),theHMBCcorrelationbetween
the gem-proton mentioned above and the
carbonsignalatdC 114.2,andthecorrelation
between the proton at dH 3.78 (m) and the
carbon signal at dC 114.2 were observed,
supporting the existence of heptulose, and
the heptulose was confirmed to be 3,7-
dideoxy-4-O-methyl-D-gluco-2-heptulo-
pyranose, based on the almost identical
carbon chemical shifts at dC 86.7, 114.2,
36.6,77.8,82.3,69.2,18.3,and56.5[12].By
analysisofthe1D, 2DNMRspectraandacid
hydrolysis-paper chromatography (PC) and
with the aid of reported NMR spectral data
[4], the sugar chain of1 was deduced tobeof
seven glycosyls, i.e. fourb-cymaropyranose
units, one 2-O-acetyl-b-digitalopyranose
unit, one b-canaropyranose unit, and one
3,7-dideoxyheptulose unit. The large coup-
ling constants (7.3–9.9 Hz) of the anomeric
protons indicated the b configuration for
each sugar unit. The HMBC correlations of
H-1digta/C-4cym4, H-1cym4/C-4cym3,
H-1cym3/C-4cym2, H-1cym2/C-4cym1,
H-1cym1/C-5hep, and H-1hep/C-3can
were observed, and thus the sequence of
the sugar chain in 1 was established as 2-O-
acetyl-b-digitalopyranosyl(1 ! 4)-O-b-
cymaropyranosyl(1 ! 4)-O-b-cymaropyr-
anosyl(1 ! 4)-O-b-cymaropyranosyl(1 !
4)-O-b-cymaropyranosyl(1 ! 5)-O-b-3,7-
dideoxy-4-methoxy-2-heptulopyranosyl
(2 ! 4)-dioxy-(1 ! 3)-O-b-canaropyrano-
syl. The location of sugar chain was
determined at C-20 based on the 1H13C
long-range correlation between the anome-
ric proton of canarose at dH 5.03 (br d,
J ¼ 9.0 Hz) and C-20at dC 83.1. Comparing
with the model compound 3, one more
cymarose unit was found in compound 1,
and this was the only difference found
between the two compounds. Thus, the
structureof1was elucidated as pregn-5-ene-
3b,17a,20(S)-triol-3-O-(4,6-dideoxy-3-
methoxy-2-hexosuloside-3-ene)-20-O-2-O-
acetyl-b-D-digitalopyranosyl(1 ! 4)-O-b-
cymaropyranosyl(1 ! 4)-O-b-cymaropyr-
anosyl(1 ! 4)-O-b-cymaropyranosyl(1 !
4)-O-b-cymaropyranosyl(1 ! 5)-O-3,7-
dideoxy-4-methoxy-2-heptulopyranosyl
(2 ! 4)-dioxy-(1 ! 3)-O-b-canaropyrano-
side, and named as periplocoside X
(Figure 1).
Compounds 2–5 were, respectively,
identified as oligasaccharide A [5], peri-
plocoside A [4], periplocoside E [5], and
periplocoside N [13] by comparison of
their spectral data with those reported in
the literature.
Compounds 1–5 were evaluated for
insecticidal activities against the different
castes such as the soldiers, workers, males,
andalatefemalesoftheredimportedfireant.
As shown in Table 2, all compounds were
found to have insecticidal activities. Among
these compounds, 1 possessed the strongest
activity. After 24h, LC50 values were found
to be 748.99, 116.62, 2169.58, and
3079.33 mg/l against the soldiers, workers,

Journal of Asian Natural Products Research
813
Table 1. 1H (400 Hz, CDCl3) and 13C NMR (150 MHz, CDCl3) spectral data of compound 1
(J in Hz).
No.
dH
dC
No.
dH
dC
Hexo
1
2
3
4
Cym 1
1
5.03, s
97.8
186.1
148.2
119.4
68.8
5.25, br d (9.9)
2.38/1.85, m
4.18, br s
3.20, dd (9.0, 2.5)
3.80, dq (9.3, 5.8)
1.28, d (5.7)
3.47, s
98.7
2
3
4
5
37.5
77.3
82.3
69.2
18.3
58.9
5.93, s
4.18, m
1.28, d (5.7)
3.78, m
5
6
3-OMe
23.0
54.8
6
OMe
Aglycone
1
2
3
4
5
6
7
8
Cym 2
1
0.96/1.82, m
1.84/1.40, m
3.78, m
37.2
30.0
78.9
39.0
140.5
122.4
31.5
31.5
50.2
36.6
19.4
37.8
46.0
51.4
24.0
30.0
85.3
19.4
14.5
83.1
17.2
5.48, br d (9.3)
2.75/1.00, m
3.61, br s
4.18, dd (5.6, 5.0)
3.95, dq (9.4, 6.2)
1.28, d (5.7)
3.47, s
98.8
37.6
77.8
83.1
69.1
18.4
58.7
2
3
4
5
2.41/1.99, m
5.48, br s
1.63, m
1.51, m
0.96, m
6
OMe
Cym 3
1
9
4.86, br d (9.4)
2.13/1.99, m
3.78, br s
4.18, dd (5.6, 5.0)
3.95, dq (9.4, 6.2)
1.28, d (5.7)
3.47, s
100.0
37.8
78.1
83.2
69.0
18.5
58.9
10
11
12
13
14
15
16
17
18
19
20
21
Can
1
2
3
4
5
1.50, m
1.28/1.85, m
2.13, m
1.20, m
0.96/1.84, m
6
OMe
Cym 4
1
5.15, br d (9.9)
2.38/1.85, m
4.18, br s
4.18, dd (5.6, 5.0)
3.95, dq (9.4, 6.2)
1.28, d (5.7)
3.47, s
101.9
39.0
78.9
83.9
69.2
21.0
58.9
0.96, s
0.76, s
3.39, m
2
3
4
5
1.33, d (6.3)
6
OMe
Digta
1
2
3
4
5
6
5.03, br d (9.0)
2.34/1.84, m
3.78, m
1.28, d, (5.7)
3.61, dq (8.8, 7.8)
1.28, d, (5.7)
100.3
36.9
78.1
79.7
69.2
18.2
2
3
4
5
4.73, d (7.7)
5.84, dd (9.2, 7.8)
3.61, dd (8.8, 4.2)
4.12, d (5.3)
3.78, m
103.6
70.5
80.0
67.7
70.1
17.2
169.8/21.2
58.0
6
Heptu
1
2
3
4
5.38/4.97 d (7.3)
86.7
114.2
36.6
77.8
82.3
69.2
18.3
56.5
1.33, d (6.3)
2.10, s
3.47, s
OAc
OMe
2.38/1.85, m
3.78, m
3.47, t (13.2)
3.55, dq (9.6, 6.2)
1.28, d (5.7)
3.40, s
5
6
7
OMe
males, and alate females of the red imported
fire ant, respectively. As expected, the
worker was the most sensitive caste and
the alate female was highly tolerant toward
all compounds. Thus, the bioassay indicated
that the order of sensibility to the tested
compounds was workers . soldiers .
males . alate females.

814
Y. Li et al.
O
OH
O
O
O
O
O
O
O
O
O
21
O
O
O
12
O
O
O
19
20
Can
OCH3
Heptu
OH
OAc
H3CO
OCH3
Cym1
6'
OCH3
Cym2
OCH3
Cym3
17
16
15
OCH3
Cym3
11 H 13
18
Digita
14
9
1
4
5'
2'
O
4'
3'
8
2
10
H
6
H
3
5
1'
7
H3CO
O
O
1
Hexo
O
OH
O
O
O
O
O
O
O
O
21
O
O
O
O
O
19
20
Can
OAc
OCH3
H3CO
OH
OCH3
Cym1
OCH3
Cym2
6'
12
H
OCH3
Cym3
Heptu
17
16
15
13
14
11
18
Digita
9
1
5'
2'
O
4'
3'
2
3
8
10
H
6
H
1'
5
7
4
H3CO
O
O
Hexo
3
Figure 1. Structures of compounds 1 and 3 from P. sepium.
3. Experimental
uncorrected (CBIO Biosciene & Technol-
ogies Co. Ltd, Beijing, China). IR spectra
were recorded on a Nicolet NEXUS-470
spectrometer (Madison, WI, USA). All
3.1 General experimental procedures
Meltingpoints were measuredwithanX-4A
micro melting point apparatus and are
Table 2. Toxicology of five compounds extracted from root of P. sepium against soldiers, workers,
males, and alate females of the red imported fire ant.
95%
Confidential
limit interval
Tested
castes
Regression equation,
y ¼ ax þ b
Coefficient LC50
correlation, r (mg/l)
Compounds
Soldiers
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
y ¼ 1.9665x–0.6527
y ¼ 2.0522x–1.0168
y ¼ 2.0450x–0.9527
y ¼ 2.5277x–2.4619
y ¼ 2.4412x–2.1237
y ¼ 1.2780x þ 2.3587
y ¼ 1.7075x þ 0.5477
y ¼ 1.4098x þ 1.7308
y ¼ 1.6754x þ 0.4741
y ¼ 1.8729x–0.1076
y ¼ 2.2261x–2.4270
y ¼ 2.0766x–2.3455
y ¼ 3.0581x–5.2358
y ¼ 2.3072x–3.0011
y ¼ 2.5555x–3.9970
y ¼ 2.4529x–3.5570
y ¼ 1.5651x–0.5873
y ¼ 2.5105x–3.8641
y ¼ 2.6539x–4.3929
y ¼ 2.0623x–2.3690
0.9546
0.9246
0.9350
0.9834
0.9942
0.9666
0.9853
0.9493
0.9407
0.9422
0.9965
0.9971
0.9917
0.9986
0.9939
0.9927
0.9939
0.9984
0.9954
0.9898
748.99 610.95–918.23
854.97 698.45–1046.56
814.41 665.84–996.14
895.41 751.17–1067.34
828.22 696.30–985.15
Workers
Males
116.62
65.04–209.12
405.01 308.28–532.08
208.42 137.16–316.71
502.69 390.74–646.71
533.41 422.74–673.06
2169.58 1818.78–2588.05
3446.27 2817.78–4214.96
2224.11 1932.50–2559.72
2937.09 2460.07–3506.60
3316.37 2783.54–3951.19
3079.33 2594.37–3654.94
3713.72 2868.79–4807.50
3394.65 2841.05–4056.11
3461.66 2906.53–4122.83
3742.08 3021.94–4633.83
Alate females

Journal of Asian Natural Products Research
815
NMR experiments were carried out on a
BRUKER AVANCE 400 spectrometer,
utilizing tetramethylsilane as an internal
standard with 1H and 13C NMR data
observed at 400 MHz. HR-ESI-MS data
were obtained on a Bruker Apex IV FT-MS
spectrometer (Billerica, MA, USA). Tan-
dem mass spectrometry (ESI-MS) exper-
iment was carried out on an LCMS-8030
mass spectrometer in Shimadzu Co. Ltd,
Kyoto, Japan. Column chromatography
(CC) was carried out on silica gel (100–
200, 200–300 mesh; Qingdao Meigao
Chemical Ltd, Qingdao, China). Silica gel
GF254 (Qingdao Meigao Chemical Ltd) was
used for TLC. All chemicals used in the
study were of analytical grade. High-
performance liquid chromatography
(HPLC) was carried out on SHIMADZU
LC-6AD HPLC apparatus (600 Epump,
UN486 detection and WDL95) (Shimadzu
Co. Ltd). Chromatography was carried out
on a Hypersil ODS2 C18 column (10mm,
10 £ 250 mmDalianEliteAnalyticalInstru-
ments Co. Ltd, Dalian, China).
give fractions A-1–A-15. A-9 (8.19 g) was
subjected to silica gel (200–300 mesh)
column eluted with gradient ethyl acetate:
acetone: petroleum ether: chloroform from
1:2:10:10 to 1:2:4:4 to give 2 (0.3623 g).
A-10 (6.07 g) was subjected to silica gel
(200–300 mesh) column eluted with gra-
dient ethyl acetate: acetone: petroleum
ether: chloroform from 2:1:8:8 to 2:1:2:2
to give 1 (0.5234 g). A-11 (4.31 g) was
subjected to silica gel (200–300 mesh)
column eluted with gradient ethyl acetate:
acetone: petroleum ether: chloroform from
1:2:10:10 to 1:2:1:1 to give 3 (0.3506 g).
A-12 (10.42g) was subjected to silica gel
(200–300 mesh) column eluted with
gradient ethyl acetate: acetone: petroleum
ether: chloroform from 3:3:5:7 to 3:3:2:4 to
give 4 (0.9755 g). A-14 (8.65 g) was
subjected to silica gel (200–300 mesh)
column eluted with gradient ethyl acetate:
acetone: petroleum ether: chloroform from
4:3:5:5 to 4:3:1:1 to give subfractions A-14-
1–A-14-8. A-14-1 (1.594g) was isolated by
RP-HPLC (mobile phase MeOH: H2O 85:
15; flow rate 1 ml/min; 230 nm) to afford
compound 5 (251mg).
3.2 Plant material
The roots of P. sepium were collected in
August 2007, in Yanan, Shanxi Province of
China and labeled as A-1. The sample was
identified by Associate Professor Gao-ying
Liu. A voucher specimen (sample No.
198410132) has been deposited at the
Shanxi Institute of Pharmaceutical Industry.
3.3.1 Periplocoside X (1)
Whiteamorphouspowder;½aꢀ2D5 72.6(c0.1,
CH3OH); mp 255–2578C; IR (KBr) nmax
:
3497, 2935, 1750, 1639, 1453, 1373, 1316,
1238, 1057, 1003 cm21. 1H and 13C NMR
spectral data: see Table 1. HR-ESI-MS: m/z
1578.8450 [M þ Na]þ (calcd for
C79H126O30Na, 1578.8465).
3.3 Extraction and isolation
The air-dried root powder of P. sepium
(2.5kg) was supersonically extracted with
MeOH(6liters £ 3)atambienttemperature.
The extract was concentrated in vacuo to
giveacruderesidue(496g).Theresiduewas
suspended in water and extracted with
CHCl3 (2 liters £ 3) to give a chloroform
fraction (102g). Part of this fraction (98 g)
was subjected to chromatography on silica
gel (100–200 mesh) eluting successively
with gradient petroleum ether–EtOAc
(10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 0:10, v/v) to
3.4 Acid hydrolysis
Compound 1 (5 mg) was hydrolyzed with
5 ml of 0.025 M H2SO4 in 50% aqueous
MeOH, stayed overnight. The hydrolyzates
were dried with nitrogen and isolated by
means of CC on silica gel using CHCl3–
MeOH (100:0, 99:1, 98:2, 97:3, 96:4, 95:5
v/v) and EtOAc as eluting solvents to give
subfractions H-1–H-7. Also, based on the
1
data of 13C and H NMR of 1 and those
reported in the literature [12], D-canarose,

816
Y. Li et al.
D-cymarose, and D-digitalose were ident-
ified by direct comparison with authentic
samples on PC from H-3 to H-7.
Prof. Josep Coll from the Institute of Chemistry
and Environment in Barcelona, CSIC, Spain,
for his guidance and help in the compound
identification.
3.5 Bioassays
References
The red imported fire ants were reared in
South China Agricultural University in
Guangzhou, China, and fed according to
the method of Zhong [14]. The compounds
were dissolved in acetone and the concen-
tration of every compound was set as 5000,
3000, 1000, 500, and 100 mg/l. Then, 20 of
the red imported fire ants (soldiers, workers,
males, and alate females) were transferred
into a plastic beaker of 10cm diameter.
Total 1 ml of the solutions was sprayed to
0.1 g chippingofbiscuit aspoisonbaits. And
the control had only equal volume of
acetone sprayed on it. Then, the baits were
put into the plastic beaker after the solvent
was volatilized. Each treatment was done
with three replicates. The mortality was
assessed after 24h and the parameters of the
dose–mortality regression line were con-
ducted with the method of the probit
analysis.
[1] H.M. Wang, J. Chin. Med. Mater. 21,
43 (1999).
[2] J.P. Xu and K. Takeya, Phytochemistry
29, 344 (1990).
[3] S. Kawanishi, Chem. Pharm. Bull. 20,
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Acknowledgments
Thanks are due to the Scientific and Techno-
logical Plan of Guangdong Province for its
financial support to the present research
(2009B050400004). We are also grateful to
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