American Journal of Plant Sciences, 2012, 3, 1568-1572
http://dx.doi.org/10.4236/ajps.2012.311189 Published Online November 2012 (http://www.SciRP.org/journal/ajps)
Insecticidal Toxicity of Spilanthol from Spilanthes acmella
Murr. against Plutella xylostella L.
Anuradha Sharma1, Vishal Kumar2, Rameshwar Singh Rattan1, Neeraj Kumar2, Bikram Singh2*
1Entomology and Pesticide Residue Laboratory, Hill Area Tea Sciences (HATS) Division, Palampur, India; 2Natural Plant Product
Division, Institute of Himalayan Bioresource Technology (CSIR), Palampur, India.
Email: firstname.lastname@example.org, *email@example.com
Received August 12th, 2012; revised September 16th, 2012; accepted October 15th, 2012
The present study explored the Spilanthes acmella Murr. for insecticidal principle, a plant of high value. The seed ex-
tract showed insecticidal activity against Plutella xylostella. Further, bioassay guided isolation of bioactive compounds
resulted in insecticidal active molecule, which was identified with the help of ESI-MS and NMR. Highest activity of 95 -
100 percent was observed at low dose of 2 g/l with spilanthol, while 60 - 70 and 80 - 90 percent mortality at 5 g/l in
crude seed extracts prepared in methanol and hexane after 48 hours exposure, respectively. LC50 of 1.49, 5.14, 5.04,
11.75 g/l was observed with spilanthol, crude seed extract of methanol, hexane, deltamethrin, respectively. The findings
indicate the potential of S. acmella with potent insecticidal toxicity for the management of P. xylostella and other in-
sects of agricultural importance.
Keywords: Spilanthes acmella; Insecticidal Activity; Plutella xylostella; Spilanthol; Alkamides; Extracts; Toxicity
Spilanthes acmella Murr. is an annual herb belonging to
the compositae family distinguishable from other species
by its yellow flower head; the leaves and flower have
pungent taste accompanied by tingling and numbness on
the tongue. The flower heads and leaves have been used
for the treatment of toothache and skin diseases . Sev-
eral bioactive constituents including spilanthol have been
isolated from this species [2,3]. Phytochemical investiga-
tions of the genus Spilanthes, Acmella ciliata was found
to contain 20 amides [4,5]. The extracts or its constitu-
ents S. acmella showed toxic activity against insects [6-
8], bacteria [9-11], fungi [12-15] and nematodes .
The diamondback moth (DBM), Plutella xylostella (L.)
(Lepidoptera: Plutellidae), is one of the most destructive
insect pests of crucifers worldwide. Larvae of diamond-
back moth, P. xylostella feed on the foliage of the cruci-
ferous plants from the seedling stage to harvest, and
greatly reduce the yield and quality of produce. P. xylos-
tella has only become a significant pest relatively re-
cently, with major problems observed in the 1970s ap-
parently caused at least in part by the evolution of insec-
ticide resistance . It is an oligophagous species that
feeds on plant species of the family Brassicaceae ,
which include economically important crops such as
cabbage, cauliﬂower, broccoli, canola and Brussels sp-
routs. As such it is a worldwide pest, costing over the
past US$1 billion to control annually. Pesticides have
dominated attempts to control P. xylostella for more than
40 years [19-23]. It was the first crop insect reported to
be resistant to DDT and now in many crucifer-producing
regions. It has shown significant resistance to almost
every insecticide applied in field including biopesticides
such as crystal toxins from B. thuringiensis and spino-
syns from Saccharopolyspora spinosa under field condi-
tions [24,25]. Insecticide resistance associated crop fail-
ure has been reported in many parts of the World [26,27].
A large number of insecticides with different modes of
action are available for the control of susceptible P. xy-
lostella but resistance has been observed to all but the
newest modes of action in one or more regions. Largely,
because of the negative impact of pesticides and the in-
creasing difficulty encountered in controlling diamond-
back moth populations, much effort has been devoted to
find alternative control measures for this pest. Botanical
insecticides can inﬂuence the behavior and development
of the herbivorous insects that search for or use the plant
for their reproduction. The present investigations were
focused on exploring the insecticidal activity of S. acme-
lla extract and bioassay-guided isolation of its con-
Copyright © 2012 SciRes. AJPS
Insecticidal Toxicity of Spilanthol from Spilanthes acmella Murr. against Plutella xylostella L. 1569
stituents and structural analysis.
2. Materials and Methods
2.1. Plant Material and Extraction
The mature flower heads/leaves from the S. acmella
Murr. were collected for preparation of extract. The air-
dried flower heads of S. acmella (500 g) were ground to
fine powder and extracted with hexane (20 ml/g of dry
weight of the material, 20 hr), followed by filtration. The
extract was fractionated by dry column chromatography
over silica-gel to separate twelve fractions (A-L). The
combined fractions D-F eluted with 30% ethyl acetate in
hexane were rechromatographed on a silica-gel column.
The column was eluted with hexane and gradually in-
creasing the polarity with ethyl acetate. Eluting with
hexane-ethyl acetate (9:1) yielded compound 1 (490 mg),
which was identified with the help of ESI-MS and NMR
analysis. The filtrates of the first extraction were com-
bined and evaporated in vacuo to give a crude hexane
extract (9.9 g). Similarly, the extraction was performed
using ethyl acetate and methanol to obtain the corre-
sponding ethyl acetate (2.5 g) and methanol (28.1 g) ex-
2.2. Screening Bioassay
Leaf dip method was used to evaluate the activity of
plant extract/fraction against test insect. The cabbage
leaves were cut into circular shape with 34 cm2 area (di-
ameter 6.5 cm). The leaves were allowed to dry at ambi-
ent condition. The circular leaf discs were treated for
about 30 seconds. The 2nd instar larvae were released on
circular leaf discs placed in Petri plate. The Petri plates
were sealed with Parafilm®. Bioassay was conducted on
laboratory reared 2nd instar larvae of P. xylostella. The
test was repeated 4 times (10 larvae/replicate) with three
replications. The mortality was observed after 24 and 48
hours. Each experiment was repeated with negative con-
trol. Toxicity effects were reported as LC50 and LC90
representing the concentration in g/l with 50 and 90 per-
cent larval mortality rate after 48 hours of exposure. All
experiment were conducted in a growth chamber set at
25˚C ± 2˚C, 16:8 h (dark:light) photoperiod and 65% RH.
The larval mortality data were corrected for control mor-
tality by the formula of Abbott . LC50, LC90 and 95%
confidence limits (upper and lower) were analyzed using
EPA Probit analysis (Version 1.5) software based on
Finney’s Probit Analysis [29,30].
NMR spectra were recorded on a Bruker Avance-300
spectrometer. Mass spectra were recorded on QTOF-
Micro of Waters Micromass. TLC was performed on
silica gel 60 F254 plates (60 - 120 mesh) for column chro-
matography (Sisco Research Laboratories Pvt. Ltd.).
3. Results and Discussion
The present study revealed the toxicity of botanical ex-
tract from the flower heads of Spilanthes acmella against
2nd instar larvae of P. xylostella. Compound 1 i.e. spi-
lanthol showed the 95 - 100 percent mortality as com-
pared to crude extracts of hexane (70 - 80) and methanol
(60% - 70%) after 48 hour exposure. The LC50 of spi-
lanthol, crude seed extracts in hexane and methanol was
1.49, 5.14, and 5.04 g/l, respectively (Table 1). Del-
tamethrin (Decis®) was used as reference standard in the
studies (LC50: 11.75). In preliminary studies, leaf extracts
in hexane showed only 2 - 3 percent mortality against
2nd instar of P. xylostella larvae after 48 hours of expo-
sure. During the experiment, it was observed that there is
a striking difference between the levels of mortality
caused by these extracts. i.e. 70 - 80 per cent larvae ob-
served as moribund with in one hour of exposure to spi-
lanthol at a concentration of 2 g/l. The insecticidal activ-
ity of plant extracts could be attributed to either the ma-
or compound of oil, or to the synergistic/or antagonistic
effects of all the components present in the mixture
[31,32]. Volatile compounds extracted from plants and
their constituents have been shown to be potent source of
botanical pesticide [33-35]. On the other hand, the
chemical composition could also vary depending on the
geographical area, the collection season, the parts of the
plant used for distillation (leaf, stem, flowers, roots), and
the presence of chemotypes or chemical components
present within the same species [36,37].
Twelve fractions (A-L) were collected and combined.
Fractions D-F were eluted with hexane:ethyl acetate (7:3).
The bioactive fractions (D-F) were purified by column
chromatography over silica-gel eluting with hexane and
gradually increasing the polarity by ethyl acetate. Eluting
with hexane-ethyl acetate (9:1) yielded compound 1 (490
mg), which was identified as spilanthol with the help of
ESI-MS and NMR analysis .
Spilanthol was found active against P. xylostella. This is
the first report of the compound with insecticidal activity
against P. xylostella from the ﬂower heads of S. acmella.
Previously, the extracts from Spilanthes acmella have
been mostly identified toxic against different mosquito
species (i.e. Anapheles, Culex and Aedes). Electrophysio-
logical studies showed immediate hyperexcitation fol-
lowed by complete inhibition of cercal nerve activity
. Maximum mortality occurred with flower head ex-
tracts (Table 1). The authors concluded that spilanthol
and alkamides are responsibe for toxic effects . Be- l
Copyright © 2012 SciRes. AJPS
Insecticidal Toxicity of Spilanthol from Spilanthes acmella Murr. against Plutella xylostella L.
Copyright © 2012 SciRes. AJPS
Table 1. Insecticidal toxicity of S. acmella seed extracts against P. xylostella.
(Fiducial limits 95% confidence)(Fiducial limits 95% confidence)
Compound code LC50 (µg/l)
Hexane 5.14 4.33 5.66 8.02 7.34 9.29 9.48
Methanol 5.04 3.77 6.41 9.64 8.40 13.08 8.78
Compound 1 (Ethyl acetate) 1.49 1.40 1.57 1.99 1.85 2.27 7.85
Deltamethrin 11.75 9.10 14.78 64.61 43.78 122.38 12.59
Figure 1. Structure of (2E,6Z,8E)-N-isobutyl-2,6,8-deca-
trienamide. Compound 1: (2E,6Z,8E)-N-isobutyl-2,6,8-deca-
trienamide (spilanthol): Colourless oil. 1H NMR (300 MHz,
CDCl3): δ 6.76 - 6.83 (m, 1H), 6.22 - 6.30 (m, 1H), 6.14 (m,
1H), 5.83 - 5.88 (m, 1H), 5.91 - 5.97 (m, 1H), 5.67 (br s, 1H),
5.22 - 5.24 (m, 1H), 3.11 (d, 2H, J = 4.2 Hz), 2.24 - 2.26 (m,
4H), 1.74 (m, 4H), 0.90 (d, 6H, J = 6.3 Hz) ppm; 13C NMR
(75 MHz, CDCl3): δ 166.5, 143.7, 130.2, 129.8, 128.0, 127.0,
124.6, 47.2, 32.5, 28.9, 26.7, 20.5, 18.6 ppm; ESI-MS m/z
sides, non-volatile sesquiterpenoids and saponins were
also reported [40,41]. Aqueous extracts of S. acmella was
toxic against Phyllotreta nemorum (70%) . On the
other hand ethanolic extract of flower heads of S.
acmella has shown a potent ovicidal, insecticidal and
pupaecidal activity at dose of 7.5 ppm concentration with
100% of Anapheles, Culex and Aedes mosquito . The
hexane extract of dried flower buds of S. acmella (3
N-isobutylamides: spilanthol, undeca-2E,7Z,9E-trienoic
acid isobutylamide and undeca-2E-en-8,10-diynoic acid
isobutylamide) was found active against Aedes aegypti
larvae and Helicoverpa zea neonates at concentrations of
12.5 and 25.0 µg/ml, respectively . Ethanolic extracts
of S. acmella (whole plants) was screened against early
4th instar larvae of Culex quinquefasciatus; LC50: 61.43
ppm . However, in another study, the extracts of S.
acmella showed no effect against C. quinquefasciatus
The active component from extracts of S. acmella was
identified as N-isobutyl-2,6,8-decatrienamide (spilanthol)
and was shown to be toxic against adults of P. americana.
It was the most potent compound when compared with 3
conventional insecticides (carbaryl, lindane and biores-
methrin) with potency found to be 1.3, 3.8 and 2.6 times
more toxic, respectively. Lemos et al.  characterized
eighteen compounds (GC/MS) in the essential oil of S.
acmella plants collected in Brazil. The major constituents
identified were beta-caryophyllene (30.2%), gamma-
cadinene (13.3%) and thymol (18.3%). While, Baruah
and Leclercq [47,48] identified twenty compounds from
essential oil of S. acmella by GC-MS, and the main con-
stituents were limonene (23.6%), beta-caryophyllene
(20.9%), and germacrene D (10.8%). Only a few reports
have been published on the constituents of S. acmella [40,
The development of natural insecticides will help to
decrease the negative effects associated with chemical
insecticides (such as environmental and health hazards).
Bio-insecticides that will be effective, selective, bio-de-
gradable, little or no resistance to target pest and non
toxic to environment, will better contribute to the sus-
tainable agricultural production of the world.
The present study reports the successful isolation of a
diverse group of bioactive metabolites. The major insec-
ticidal component identified was (2E,6Z,8E)-N-isobutyl-
2,6,8-decatrienamide from S. acmella. In this and other
studies, these compounds possessed marked insecticidal,
fungicidal, and antimicrobial activities including chemo-
protective effects on human health. Promisingly, (2E,6Z,
8E)-N-isobutyl-2,6,8-decatrienamide (spilanthol) and crude
extracts of ethyl acetate and hexane exhibited insecticidal
activity. The data support the use of S. acmella as a po-
tential insecticide. In addition, further experiments are in
progress to elucidate the understanding of spectrum of
action, specificity to target insect pests and structure ac-
tivity relationship of active molecules. This has shown
that the flower head extract of S. acmella possesses re-
markable insecticidal toxicity against P. xylostella. Thus,
there is possibility of developing as a source of alternate
insecticidal agent for sustainable management of insect
pests of economic importance and mosquito control. This
will have the important benefit of helping to reduce the
present excessive use of synthetic insecticides, which has
been causing concern for sometime now.
Authors are grateful to the Dr. P. S. Ahuja, Director, IHBT,
Palampur, for his encouragement to carry out work and
Insecticidal Toxicity of Spilanthol from Spilanthes acmella Murr. against Plutella xylostella L. 1571
gratefully acknowledge CSIR for financial grant.
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