Antifeedant Constituents from Leucaena leucocephala

Article history: Received on: 18/07/2016 Revised on: 04/10/2016 Accepted on: 30/10/2016 Available online: 28/12/2016 Hexane and methanolic extracts of Leuceana leucocephala Lam. leaves were tested against Spodoptera litura L. a polyphagus pest of cotton rice, tomato, ground nut, castor and legume and found to have antifeedant potential in the concentration of 2.5 μg/cm 2 . The bioassay-guided fractionation yielded the three compounds quercetin 3O-rhamnoside (1), quercetin (2) and D-onanitol, (3) with antifeedant activity.


INTRODUCTION
Botanical insecticides have long been used by men since ancient times in insect pest management and crop protection and are attractive alternatives to synthetic chemical insecticides for pest management because of little threat to the environment and human health.The mechanism of action of botanical pesticides may differ greatly and are often not yet well understood.They have an advantage that they combine a wide range of toxic potencies hence reducing the chance of pest to develop resistance (Nelson and William, 2004).In addition to that, residues are hardly expected on the products or in the environment since botanical pesticides are generally considered to be non persistent under field condition as that are readily degraded by light, oxygen and microorganism to less toxic products (Isman and Akhtar, 2007).The deleterious effects of crude plant extracts on insects are manifest in several ways, including toxicity, (Hiremath, 1997) feeding inhibition (Wheeler and Isman, 2001).The search for plant derived chemicals that have potential use as crop-protectant (insecticide, antifeedant, and growth inhibitor) often begins with screening of plant extracts (Peta and Pathipati, 2008).Insecticides of plant origin have been in use for long time.By applying plant extracts to other susceptible plant species the defense of susceptible plant is improved and use of natural products in agro ecosystem is emerging as one of the prime means to protect crops (Rattan, 2010).The cutworm, Spodoptera litura Fabricius (Lepidoptera: Noctuidae), is a polyphagous pest that has near about 150 host species.The cutworm, Spodoptera litura is an economically important polyphagous pest found in major part of the world.In India, the larval stages causes severe damage to a number of crops including tobacco, caster, groundnut, tomato, cabbage, cauliflower, cotton and other various crucsiferous crops (Rao et al., 2001) Crop loss due to insect pests varies between 10% and 30% for major crops (Ferry et al., 2004;Isman et al., 2007).Traditionally synthetic pesticides are used to control S. litura and hence the pest developed resistance against the commonly using pesticides.For this purpose, medicinal plants were screened and are being reported to contain bio-pesticidal property (Selvaraj et al., 2005).S. litura is often used to evaluate antifeedants in plants.(Pung and Srimongkolchai, 2011).Number of plants exhibited significant antifeedant activity against this pest.Several Himalayan plants are documented as insecticidal agents against spodoptera littura and other forest pests (Negi et al., 2016;Negi et al., 2006) Extracts of Swertia corymbosa, Phyllanthus deblis, Syzygium lineare, Curculigo orchioides, Evolvulus alsinoides, and Zanthoxylum limonella showed significant antifeedant activity against spodoptera litura (Jeyasankar et al., 2010;Arivoli et. al., 2012).Leucaena leucocephala (Mimosaceae) Lam.(Shu babol) is an unarmed small deciduous tree to 5 m height, generally used for soil conservation, fuel and feed purposes in India.(Gaur, 1999) It was known to be a rich source of tannin, proteinous and nonproteinous amino acid and other phenolics (Azeemoddin et al., 1988;Hossain et al., 1998).The plant commonly used as foliage, as a source of β-carotene, vitamin K, β-carotene, green manure, fuel wood or as drought resistance ( Lalitha et al., 1993).It is also known to have great medicinal importance (Salem et al., 2011).The roots of Leucaena leucocephala contains tannins which are known to exhibited nitrification inhibition effect (Erickson et al., 2000).The seed oil could be used as a potential bio inhibitor for corrosion of mild steel and copper.(Meena et al., 2013).The phytochemical investigation of Leucaena leucocephala revealed the presence of coumarins, terpenes, sterols and flavonoids.The seed extract of Leucaena leucocephala have been reported as antidiabetic, anthelmintic and has a broad spectrum antibacterial activity (Irene et al., 1997;Ademola et al., 2005;Syamsudin et al., 2010).The significant antioxidant activity and antimicrobial activity was recorded from the extract and the compounds isolated from Leucaena leucocephala (Reda et al., 2015).
In present investigation, we tested the antifeedant level of plant extract of Leucaena leucocephala and isolated compounds of Leucaena leucophela against third instar larvae of S. litura.(Lapidoptera), a polyphagous pest of groundnut, tomato, cotton, rice, tobacco, castor and legumes.

General Experimental Procedure
Melting points is uncorrected and was taken in open capillary.NMR spectra were recorded at 400 MHz for 1 H and 100 MHz for 13 C on Bruker AVANCE 400 spectrometer in DMSO-d 6 , CDCl 3 with TMS as internal standard.Proton detected heteronuclear correlation were measured using HMQC (optimized for 1 J HC =145 Hz) and HMBC (optimized for J HCCC =7 Hz).The IR spectra were recorded on a Perkin Elmer Infrared 15 spectrometer using KBr pellets.MS data were obtained on a JEOL SX-102 spectrometer.Silica gel (60-120 mesh Merck) for column chromatography and silica gel G (Merck) for TLC were used.

Plant Material
Leaves of Leucaena leucocephala were collected at Chauras campus of HNB Garhwal University, Srinagar Garhwal, Uttarakhand, India.A voucher specimen (GUH 16280) is deposited in the Herbarium of Chemistry Department HNB Garhwal University, Srinagar Garhwal, Uttarakhand, India.

Pest
Field collected Spodoptera litura L. larvae were cultivated in the laboratory at 25+2 o C and third instar larvae from laboratory culture were used for antifeedant assay.

Extraction and Isolation
Shed dried powder leaves part (2.4kg) was exhaustively extracted at 60 0 C with 90 % EtOH.The extract was evaporated in vacuo to give the crude residue A-1.It was partitioned with hexane and methanol, and gave hexane-soluble fraction, A-2 and methanol-soluble fraction, A-3.A-3 was charged to gross chromatography on silica gel using CHCl 3 -MeOH as eluent with increasing proportion of methanol, which afforded Fr 01 and Fr 02.Fr 01 was re-chromatographed on CC using CHCl 3 -MeOH (9:1) afforded 1 (45mg) and 2 (18mg), while Fr 02 gave 3 (108mg) further purified by MeOH.

Antieedant Activity
Crude extract and isolated compounds were tested against third instar larvae of Spodoptera litura L. (Lepidoptera).The dual choice leaf disc method was performed (Kannan, et al., 2013).Field collected Ricinius communis leaves were cut in to circular discs (180 cm 2 ) with the medium vain as marker between two equal halves.Hexane and methanolic extracts and isolated compounds were dissolved in solution, which was sprayed on half of circular leaf disc with 2.5-μg/cm 2 concentrations.Other half of the leaf treated with solvent.
Azadirachtin A, a potent insect antifeedant and growth regulatory compound, was kept as active control (Govindachari, et al., 1995;Govindachari, et al., 1996).After drying, each leaf disc was placed in a Petri dish (15 cm dia).Five freshly moulted insect third instar larvae of S. litura were placed in the center of leaf and left to feed for 36 hr.For each extracts and compounds, five replicate were maintained.After 24 hrs the leaves were removed and unfed area in the treated and control halves were measured using ΔT area measurement meter.Percent feeding index (PFI) was calculated as:

CONCLUSION
Flavonol glycoside Quercitin 3-O-rhamnoside along with Quercitin and D-onanitol were isolated from the dried leaves Leucaena leucocephala leaves.Hexane and methanolic extracts and isolated compounds were test for antifeedant activity against spodoptera litura by duel choice leaf disc method and result was evaluated in terms of Percentage feeding index.A comparison of antifeedant activity against S. Litura indicated that the methanolic extract and the active compound Quercitin 3-O-rhamnoside expressed significant antifeedant potential.These results could be useful in eco-friendly formulations for insect control.

Table 1 :
1 H and 13 C data and HMBC correlation of 1

H/C atom 13 C δ J in Hz 1 H δ J in Hz HMBC
Compounds 2 and 3 were identified as quercetin and Donanitol by comparison of co-TLC with authentic samples and NMR data with those of data reported in literature(Lowry et al.,  1984; Dorman et al., 1970)(Fig1).For antifeedant activity the crude extracts were tested on dual choice leaf disc method to know the Percentage Feeding Index (PFI).

Table 2 :
Antifeedant activity of extracts and isolated compounds.