Insecticidal Toxicity of Spilanthol from Spilanthes acmella Murr . against Plutella xylostella L .

The present study explored the Spilanthes acmella Murr. for insecticidal principle, a plant of high value. The seed extract 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 insects of agricultural importance.


Introduction
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 [1].Several bioactive constituents including spilanthol have been isolated from this species [2,3].Phytochemical investigations of the genus Spilanthes, Acmella ciliata was found to contain 20 amides [4,5].The extracts or its constituents S. acmella showed toxic activity against insects [6][7][8], bacteria [9][10][11], fungi [12][13][14][15] and nematodes [16].
The diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae), is one of the most destructive insect pests of crucifers worldwide.Larvae of diamondback moth, P. xylostella feed on the foliage of the cruciferous plants from the seedling stage to harvest, and greatly reduce the yield and quality of produce.P. xylostella has only become a significant pest relatively recently, with major problems observed in the 1970s apparently caused at least in part by the evolution of insecticide resistance [17].It is an oligophagous species that feeds on plant species of the family Brassicaceae [18], which include economically important crops such as cabbage, cauliflower, broccoli, canola and Brussels sprouts.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][20][21][22][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 spinosyns from Saccharopolyspora spinosa under field conditions [24,25].Insecticide resistance associated crop failure 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. xylostella 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 increasing difficulty encountered in controlling diamondback 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. acmella extract and bioassay-guided isolation of its con-stituents and structural analysis.

Plant Material and Extraction
The mature flower heads/leaves from the S. acmella Murr.were collected for preparation of extract.The airdried 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 increasing 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 combined 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 corresponding ethyl acetate (2.5 g) and methanol (28.1 g) extracts, respectively.

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 cm 2 area (diameter 6.5 cm).The leaves were allowed to dry at ambient 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 control.Toxicity effects were reported as LC 50 and LC 90 representing the concentration in g/l with 50 and 90 percent 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 mortality by the formula of Abbott [28].LC 50 , LC 90 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].

Experimental
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 F 254 plates (60 -120 mesh) for column chromatography (Sisco Research Laboratories Pvt.Ltd.).

Results and Discussion
The present study revealed the toxicity of botanical extract from the flower heads of Spilanthes acmella against 2nd instar larvae of P. xylostella.Compound 1 i.e. spilanthol showed the 95 -100 percent mortality as compared to crude extracts of hexane (70 -80) and methanol (60% -70%) after 48 hour exposure.The LC 50 of spilanthol, crude seed extracts in hexane and methanol was 1.49, 5.14, and 5.04 g/l, respectively (Table 1).Deltamethrin (Decis ® ) was used as reference standard in the studies (LC 50 : 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 exposure.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 observed as moribund with in one hour of exposure to spilanthol at a concentration of 2 g/l.The insecticidal activity of plant extracts could be attributed to either the maor 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][34][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 [38].
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-degradable, little or no resistance to target pest and non toxic to environment, will better contribute to the sustainable agricultural production of the world.

Conclusion
The present study reports the successful isolation of a diverse group of bioactive metabolites.The major insecticidal 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 chemoprotective 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 potential insecticide.In addition, further experiments are in progress to elucidate the understanding of spectrum of action, specificity to target insect pests and structure activity relationship of active molecules.This has shown that the flower head extract of S. acmella possesses remarkable 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.

Acknowledgements
Authors are grateful to the Dr. P. S. Ahuja, Director, IHBT, Palampur, for his encouragement to carry out work and