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Industrial Crops and Products 65 (2015) 170–174

Contents lists available at ScienceDirect

Industrial Crops and Products

jo u r n al homep age: www.elsev ier .com/ locate / indcrop

nsecticidal alkanes from Bauhinia scandens var. horsfieldii againstlutella xylostella L. (Lepidoptera: Plutellidae)

araporn Poonsri a, Wanchai Pluempanupat b,∗, Pawarun Chitchirachan b,asakorn Bullangpoti a,∗, Opender Koul a,c

Biopesticide Toxicology Specialty Research Unit, Department of Zoology, Faculty of Science, Kasetsart University, Phahonyothin Rd. Bangkok 10900,hailandDepartment of Chemistry and Centre of Excellence for Innovation in Chemistry, Faculty of Science Kasetsart University, Bangkok 10900, ThailandInsect Biopesticide Research Centre, 30 Parkash Nagar, Jalandhar- 144003, India

r t i c l e i n f o

rticle history:eceived 20 June 2014eceived in revised form0 November 2014ccepted 23 November 2014

eywords:

a b s t r a c t

The insecticidal activity of various extracts prepared from the dried stem of Bauhinia scandens var. hors-fieldii was determined against Plutella xytostella second instars via topical application. The best activitywas observed in case of dichloromethane extracts with an LD50 of 2.76 and 2.15 �g/larva after 24 and48 h, respectively. Further processing of this extract provided a mixture of long chain alkanes as activematerial exhibiting a LD50 of 2.81 �g/larva, 48 h post-treatment. Further screening of this mixture showedthat heptacosane and hexacosane were the major compounds and were toxic to P. xylostella larvae with

lutella xylostellaauhinia scandensotanical insecticidesetoxification enzymes

respective LD50 of 1.58 and 2.58 �g/larva after 48 h of treatment. This suggested that both compoundshave additive effect in the alkane mixture. Combined evaluation of heptacosane and hexacosane provedthat they were additive in activity when the two compounds were mixed in a natural ratio in which theyoccur in the alkane mixture. The impact of these compounds on detoxification enzymes was determinedto show the possibility of resistance development against these compounds.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

The diamondback moth, Plutella xylostella (Lepidoptera: Plutel-idae), is a serious insect pest of cruciferous crops throughout the

orld (Sarfaraz et al., 2006). Outbreaks of P. xylostella in Southeastsia often caused more than 90% crop loss (Verkerk and Wright,994). It is a serious pest of cauliflower, cabbage, broccoli, mus-ard, radish, and turnip (Eusebion and Rejsus, 1996; Capinera, 2001;

ohan and Gujar, 2003). The level of infestation varies accordingo plant type, locality, and the level of natural enemies. If no control

easures are undertaken, this insect can cause up to 100% crop lossShelton et al., 1993).

Chemical control of P. xylostella has become less effective dueo the development of resistance to almost all groups of insecti-ides, including organochlorines, organophosphates, carbamates,

yrethroids, insect growth regulators, abamectins, pyrazoles,xadiazines, neonicotinoids, spinosad, indoxacarb, and Bacillushuringiensis (Moham and Gujar, 2003; Abdel-Razek et al., 2006;

∗ Corresponding authors. Tel.: +66 2 5625555.E-mail addresses: fsciwcp@ku.ac.th (W. Pluempanupat), fscivkb@ku.ac.th

V. Bullangpoti).

ttp://dx.doi.org/10.1016/j.indcrop.2014.11.040926-6690/© 2014 Elsevier B.V. All rights reserved.

Charleston et al., 2006; Zhao et al., 2006; Qian et al., 2008; Sheltonet al., 2008). The indiscriminate use of synthetic insecticides hasresulted in many ecological problems that include toxic residuesin soil and environment, harm to mammals and the pest resur-gence due to destruction of natural enemies (Shelton et al., 1993;Talekar and Shelton, 1993; Liang et al., 2003; Xu et al., 2004; Khanet al., 2005). These drawbacks emphasize the need for alternativesand one of the strategies would be the use of natural plant prod-ucts. Botanical insecticides, being comparatively safer for naturalenemies and environment than synthetic insecticides, provide agood option for the study of plant based products (Schmutterer,1995; Haseeb et al., 2004; Xu et al., 2004). There are many plantallelochemicals that are effective against many insect pests asinsecticides, antifeedants, growth inhibitors, and growth regula-tors, repellents, etc. (Dev and Koul, 1997; Koul, 2005, 2012; Koulet al., 2008) and specifically against P. xylostella as well (Morallo-Rejesus, 1986; Ling et al., 2008).

B. scandens var. horsfieldii (Miq.) K. & S.S. Larsen (Fabaceae)is a common tropical plant in Thailand used for mosquito con-

trol in folklore. From our earlier preliminary studies, B. scandensstem extract was observed to be active against P. xylostella (Poonsriet al., 2011) but no active ingredient was identified. Accordinglythe objective of the present work was to determine the active

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long chain alkanes with hexacosane and heptacosane as major com-pounds (Table 3). The mixtures of alkanes were toxic to P. xylostellalarvae with an LD50 of 3.45 and 2.81 �g/larva �g/larva after 24 and48 h post-treatment, respectively. Hexacosane and heptacosane

Table 1Yield and characteristics of B. scandens extracts obtained after solvent extraction.

Solvents used Characteristics Yield (%)

W. Poonsri et al. / Industrial Cro

ngredient/s from this plant species quantitatively and also to studyhe impact on detoxifying enzymes of the insect.

. Material and methods

.1. Insect rearing

P. xylostella insects were obtained from a kale farm in Non-haburi province, Thailand and were raised in an insect-rearingoom at the Faculty of Science, Kasetsart University. P. xylostellaarvae were reared on kale leaves that had never been exposed toesticides in a cage (29 × 19 × 3 cm) at 26 ± 1 ◦C, 75% RH and a pho-operiod of 16:8 h (L:D). The kale leaves were changed every twoays. Pupae were collected and kept in a net cage until the mothsmerged. Moths were fed with a 10% sugar solution. Cabbage leavesere used for oviposition. Second instars were randomly used for

he toxicity assay.

.2. Plant material, extraction, and structure elucidation

Dried B. scandens stem (1.5 kg) was obtained in October 2010rom Chachengsao Province, Thailand. The plant was identified bylant taxonomist of the Forest Herbarium, Department of Nationalark, Wildlife and Plant Conservation, Bangkok Thailand andoucher specimen retained under the voucher number BK066794.he extracts were prepared by soaking plant material at room tem-erature for seven days in hexane, dichloromethane, ethyl acetate,nd methanol, sequentially. Each crude extract was dried using aotary evaporator (R-215, Buchi, Thailand) and stored at 4 ◦C untilurther use in the experiments.

Most active dichloromethane extract was processed furthero isolate active compounds. The crude dichloromethane extract1.05 g) was subjected to silica gel column chromatography (Kieselel 60 G) with hexane/ethyl acetate (10:1) to give fraction 1.his fraction was purified further by preparative TLC with hex-ne to obtain sub-fraction 1–1 as a colorless oil (46.5 mg), whichas subsequently subjected to structural analysis using 1H NMR

nd GC–MS. Sub-fraction 1–1 was identified as a mixture ofong chain alkanes. 1H NMR (400 MHz, CDCl3) ı 1.15–1.37 (brm),.76–0.95 (brm). GC–MS analysis data of sub-fraction 1–1 showedighest percentage of hexacosane and heptacosane and wereonfirmed by authentic commercial heptacosane and hexacosaneurchased from Sigma–Aldrich that were subsequently used foretailed investigations. All other chemicals were obtained fromigma/Merck, unless otherwise noted. We chose to use hepta-osane and hexacosane for detailed study for two reasons; (i) bothompounds accounted for about 60% of the total isolates (Penta-osane = 19% only) and (ii) heptacosane and hexacosane fractionsere far superior in activity than pentacosane fraction.

.3. Bioassays

Second instars of P. xylostella were used in all the experimentso determine the median lethal dose (LD50) by the topical method.ix concentrations of each extracts were prepared in acetone (ARrade) in the range of 0–15,000 ppm; all treatments were givenopically using 1 �l dose to the thorax region of second instars. Inach treatment 90 larvae were used in three replicates. In case ofontrols, only solvent was applied topically. The treated larvae and

ontrol larvae were then placed in Petri dishes (100 mm diame-er) and provided organic kale leaves for feeding. Mortality wasecorded after 24 and 48 h post-treatment. P. xylostella behavioralesponses such as paralysis or knockdown were recorded.

d Products 65 (2015) 170–174 171

2.4. Enzyme assays

2.4.1. Enzyme extraction methodSurvived 2-day-old treated second instars were used for enzyme

assay 24 h post-treatment. Larvae of same weight were used for theassay in order to avoid any influence of growth between treated andcontrol larvae. Larvae were homogenized in 0.5 mL buffer (100 mMPhosphate buffer, pH 7.2, and 1% Triton-X-100). The homogenatewas centrifuged at 10 000 × g for 15 min at 4 ◦C, and the supernatantwas used as an enzyme source.

2.4.2. Enzyme activity analysisCarboxylesterase (CE) activity was determined by modified

method of Bullangpoti et al., (2012). Enzyme solution (40 �l) wasmixed with p-nitrophenylacetate (pNPA) (40 �l; 10 mM in DMSO)and phosphate buffer (200 �l; 50 mM, pH 7.4). Enzyme activity wasmeasured at 410 nm and 37 ◦C for 90 s with the microplate readerin the kinetic mode. The activity of CE was determined by using theextinction coefficient of 176.4705 for pNPA.

The method for determining glutathione-s-transferase (GST)activity was that of Oppenoorth et al., (1979). The reaction solutioncontained 100 �l of enzyme solution, 200 �l of 50 mM potassiumphosphate buffer (pH 7.3), 10 �l of 10 mM glutathione reducedform, and 10 �l of 150 mM 1-chloro-2,4-dinitrobenzene (CDNB).Optical density was recorded at intervals of 30 s for 3 min at 37 ◦Cand 340 nm with a microplate reader. The GST activity was deter-mined from the extinction coefficient of 0.0096 for CDNB.

2.4.3. Protein determinationThe protein content of each fraction used as enzyme source

was determined by the Bradford method (Bio-Rad Laboratories,Hercules, CA) with bovine serum albumin as the standard beforemeasuring enzyme activities.

2.5. Statistical analysis

The LD50 values were determined by Probit analysis using theStatPlus Program (2008 version).

3. Results

3.1. Toxicity of B. scandens

Various extracts obtained after sequential solvent extractionsof B. scandens stem showed that maximum yield was in ethanol(5.0243%) and the least in dichloromethane (1.0161%) (Table 1).Overall, the toxicity of these extracts was dose and time depen-dent (P < 0.05) and dichloromethane extracts was most toxic(LD50 = 2.76 �g/larva, Table 2). Within five minutes after treatmentwith all crude extracts, P. xylostella larvae displayed symptomsincluding hyperactivity and knockdown that led to paralysis anddeath which was indicative of action at the neurotransmitter level.

Screening of the most active dichloromethane extract contained

Hexane light green viscous semisolid 3.5917Dichloromethane Dark green viscous semisolid 1.0161Ethyl acetate green viscous semisolid 1.1356Ethanol Dark green viscous semisolid 5.0243

172 W. Poonsri et al. / Industrial Crops and Products 65 (2015) 170–174

Table 2Toxicity of various B. scandens extracts against P. xylostella second instars via topical application after 24 and 48 h treatment.a

Time(hours)

LD50 (�g/larva) 95% Fiducial limit Slope(±SE) Intercept LD90 (�g/larva)

Lower Upper

Hexane 24 4.89 4.19 5.83 2.01(0.22) 2.42 21.2348 2.45 1.77 3.20 1.15(0.20) 1.11 32.56

Dichloromethane 24 2.76 2.49 3.05 3.56(0.29) 7.25 6.3248 2.15 1.88 2.42 2.94(0.27) 4.80 5.87

Ethyl acetate 24 7.25 6.33 8.55 2.74(0.28) 5.58 21.2648 4.11 3.42 5.08 1.5816(0.20) 0.72 13.61

Ethanol 24 15.71 14.25 17.18 2.23(0.46) 4.62 27.3748 4.62 1.91 11.17 2.524(0.73) 4.25 12.20

a The toxicity was determined with no mortality occur for the control treatment (acetone).

Table 3GC–MS analysis data of sub-fraction 1–1 of dichloromethane extract.

Compound Results

Retention time % Area % Match

Tetradecane 24.13 0.33 94Hexadecane 39.74 0.87 98Octadecane 46.78 1.26 99Nonadecane 49.28 0.28 95Eicosane 51.49 1.39 98Heneicosane 53.49 0.91 98Docasane 55.37 2.51 99Tricosane 57.15 3.52 98Tetracosane 59.01 8.28 99Pentacosane 61.35 19.01 98Hexacosane 64.28 25.88 99

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Fig. 1. Combined toxicity of hexacosane and heptacosane after 24 and48 h topical treatment at LD25 (A1 and B1) and LD50 (A2 and B2) dosage

((Kraikrathok et al., 2013), family Fabaceae is one of the major

TT

T

Heptacosane 68.11 34.67 99

eing the major compounds were responsible for the activity oflkane mixture and LD50 values were 3.96 and 2.43 �g/larva, 24 host-treatment, respectively. The toxicity increased significantly8 h post-treatment and LD50 were potentially better (Table 4) withecrease in slope in probits. Heptacosane was most active alkaneith an LD50 of 1.58 �g/larva after 48 h of treatment. Combination

f hexacosane and heptacosane accounts substantially to the totalctivity of the alkane fraction and it was observed that when twoompounds were combined at the LD25 or LD50 values in 1:1 orn 1:1.35 (natural ratio in the fraction) ratio, they were additive inctivity against P. xylostella larvae (Fig. 1).

.2. Effect on detoxification enzymes

All crude extracts were studied to determine the effectn detoxification enzymes in vivo. While ethanol and hexanextracts induced glutathione-s-transfernase activity in treated lar-

ae (Table 5), dicholoromethane extract and active hexacosane andeptacosane from this extract inhibited glutathione-s-transferasend carboxylesterase (Table 5).

able 4oxicity of alkane mixture, hexacosane and hepatcosane to P. xylostella larvae after 24 an

Time(hours)

LD50 (�g/larva) 95% Fid

Lower

Alkane mixture 24 3.45 3.10

48 2.81 2.42

Hexacosane 24 3.96 2.59

48 2.58 1.48

Heptacosane 24 2.43 1.25

48 1.58 0.89

he toxicity was determined with no mortality recorded for the control treatment (aceto

levels. A1 = heptacosane:hexacosane (1:1); B1 = heptacosane:hexacosane(1:1.35, the ratio in natural mixture); A2 = heptacosane:hexacosane (1:1);B2 = heptacosane:hexacosane (1:1.35, the ratio in natural mixture).

4. Discussion

The new botanical insecticides have several ecological advan-tages related to their inherent nature compared to the organicallysynthesized insecticides. The plant kingdom is the most efficientproducer of chemical compounds synthesizing many productshaving wide array of functions that are used in defense againstinsects in general (Croteau et al., 2000). In case of diamond-back moth, P. xylostella, there are few reviews available thatshow the potential of botanical insecticides against this insect(Grainge et al., 1984; Morallo-Rejesus, 1986; Rattan and Sharma,2011). Though, there are variety of plant families that have poten-tial as insecticidal plants, like piperaceae against P. xylostella

tropical plant families possessing insecticidal properties. Ourstudy is based on B. scandens which is least exploited speciesbut abundantly available in Thailand and our earlier preliminary

d 48 h post-treatment.

ucial limit Slope(±SE)

Intercept LD90 (�g/larva)

Upper

3.98 3.28(0.74) 6.29 6.253.18 2.89(0.59) 4.72 5.856.82 3.59(1.22) 7.93 7.445.67 1.95(0.58) 1.66 6.946.72 1.62(0.54) 0.49 8.152.72 2.19(0.59) 2.02 5.47

ne).

W. Poonsri et al. / Industrial Crops and Products 65 (2015) 170–174 173

Table 5Detoxification enzymes activities of survived P. xylostella larvae after 24 h exposure to B. scandens extracts, mixture of long chain alkanes, hexacosane, and heptacosane atLD50 dose.

Treatment Dose(�g/larva)

Carboxylesterase(nM p-nitrophenol/min/mg protein)a

(C/T)c

Activity Glutathione-s-transferase(glutathione conjugatedproduct/min/mg protein) 1

(C/T)c

Activity

Controlb – 0.040 ± 0.0040f – 1.14 ± 0.45d –Hexane crude extract 4.89 0.013 ± 0.0025b(3.08) Inhibition 1.64 ± 0.73f(0.699) InductionDichloromethane crude extract 2.76 0.017 ± 0.0013d(2.35) Inhibition 1.04 ± 0.68d(1.095) No effectEthylaceate crude extract 7.25 0.017 ± 0.0012d(2.35) Inhibition 1.11 ± 0.36d(1.03) No effectEthanol crude extract 15.71 0.020 ± 0.0049e(2.00) Inhibition 1.53 ± 0.67e(0.75) InductionMixture of long chain alkanes 3.45 0.015 ± 0.0013c(2.67) Inhibition 0.422 ± 0.12c(2.70) InhibitionHexacosane 3.96 0.014 ± 0.0030c(2.85) Inhibition 0.381 ± 0.09b(2.99) InhibitionHeptacosane 2.43 0.011 ± 0.0021a(3.63) Inhibition 0.322 ± 0.05a(3.54) Inhibition

a Values with the same letter in a column are not significantly different at P < 0.05 according to Duncan’s New Multiple’s Range Test. The toxicity was determined with nom

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ortality recorded for the control treatment (acetone).b Control = 20% acetone in distilled water.c C/T ratio = The ratio of the enzyme activity of control group over the treated gro

tudies did point to its potential as an insecticidal plant againstiamondback moth (Poonsri et al., 2011). However, there arether species like Bauhinia variegata rich in Lupeol, �-Sitosterol,aempferol, and Quercetin (Jash et al., 2014) and others that con-

ain terpenoids, flavonoids, tannins, saponins, reducing sugars,teroids, and cardiac glycosides with pharmacological activi-ies like anticancer, antioxidant, hypolipidemic, antimicrobial,nti-inflammatory, nephroprotective, hepatoprotective, antiulcer,mmunomodulating, molluscicidal, and wound healing effects (Al-nafi, 2013; Hazra and Chatterjee, 2008). However, no reports ofnsecticidal effect, specifically against P. xylostella are yet known.

In present study B. scandens dichloromethane extract was toxico second instars of P. xylostella via topical application. Apparentlyichloromethane crude extracts seem to be very potential fraction

n many other studies where various plants were used and wereffective against Spodoptera exigua (Khumrungsee et al., 2010),podoptera frugiperda (Bullangpoti et al., 2012) and Aedes aegyptiPluempanupat et al., 2013). Dichloromethane extract of B. scan-ens contained long chain alkanes that were equally active as theain extract.

Hexacosane and hepatcosane were identified as the most activeompounds from alkane fraction and accounted substantially forhe total activity of the extract. It was obvious that combination ofeptacosane and hexacosane (1:1.35; natural concentration in thelkane mixture) showed increased activity and was approximatelybsolute (100%) when concentration combination was at individualD50 levels. This implies that these alkanes provide additive effect in

mixture in the main extract of B. scandens, suggesting significancef multi-component defense of the plant allelochemicals. This wasbvious from other data as well when combinations were used in:1 or 1:1.35 ratio at LD25 dose, where the mortality was in theange of 46.84–52.80% (Fig. 1), i.e., about twice of 25% mortality.

The acute toxicity of B. scandens or the active alka-es present do compare with the efficacy of several otherlant materials active against diamond back moths, like Aca-

ypha fruticosa Forssk (Lingathurai et al., 2011), pongam oilPavela, 2012) but were less toxic to well known neem,zadirachta indica (LC50 = 0.54 ppm; Robert et al., 1993), Annonaquamosa seed extract (EC50 = 0.10 ppm; Leatemia and Murray,004), Syzygium aromaticum flower extracts (LC50 = 1.09 ppm;antakom et al., 2007), Piper sarmentosum Roxburgh leaf extractLC50 = 4.34 ppm; Varasutpisal, 2008), Mammea americana seedxtract (LC50 = 5.90 ppm; Issakul et al., 2011), and Wedelia trilobataLD = 0.443 �g/larva; Junhirun et al., 2012). Some alkanes have

50een reported as effective against P. xylostella in mixtures with sin-

grin that reduce rate of insect movement (Spencer et al., 1999).hese studies also show that sinigrin + alkanes led to increased

oviposition compared to that in response to sinigrin treatmentsalone.

B. scandens extract or isolated compound treatments to P.xylostella displayed paralysis and death which was indicative ofaction at the neurotransmitter level. Phytochemicals are known toaffect one or the other neuron-transmitters. However, this cannotbe generalized for the B. scandens compounds in the present studyuntil detailed investigation to understand the mechanism of actionis established.

It is well known that detoxification enzymes of resistant insectpests, including cytochrome P450 monooxygenases, glutathione-s-transferase, and carboxyl/cholinesterases show elevated activitiescompared to susceptible ones in order to metabolize otherwisedeleterious plant secondary metabolites (Simon and Hsu 1993;Ramsey et al., 2010; Li et al., 2007). This implies that n-alkanesalso could induce the resistance as P. xylostella is known to rapidlyadapt to toxins. Enzyme based experiments in the current studyrevealed that carboxylesterase activity of P. xylostella was inhibitedby all B. scandens crude and purified extract; however, glutathione-s-transferase activity was inhibited by alkanes and not by thehexane and ethanol crude extracts. Carboxylesterases normallyplay an important role as detoxification enzymes in allelochemicalmetabolism and tolerance, although the roles have been validatedonly at the biochemical level in a few cases (Li et al., 2007).There are also many studies, which describe carboxylesterases andglutathione-s-transferases responsible for inducing resistance inP. xylostella and other insect pests. Sonoda and Tsumuki (2005)showed that glutathione-s-transferase gene involved in chlorflu-azuron resistance of P. xylostella L. and Doichuanngam et al., (1989)described non-specific esterases play an important role in insecti-cide resistance to malathion in P. xylostella. The organophosphates’resistance in the B-biotype of Bemisia tabaci is associated withover expression of carboxylesterase (Alon et al., 2008). Therefore,inhibition of carboxylesterase in the present case could proba-bly be attributed, although theoretically, to the interference withhydrolytic process of neurotransmitter acetylcholine or juvenilehormone, which are among the specific functions served by car-boxylesterase (Taylor and Radic, 1994; Riddiford et al., 2003). This,however, needs to be established in future studies with otherdetoxication enzymes such as Cytochrome P450 and some neuronenzyme targets. Present results, however, demonstrate the poten-tial of B. scandens dichloromethane extracts or alkanes present init to control P. xylostella. They also inhibit carboxylesterase andglutathione-s-transferase which suggest meager potential for resis-

tance development. The inhibition of these enzymes by alkanesfrom B. scandens may thus constitute a useful alternative approachfor pest management.

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cknowledgements

This research was supported by ScRF and Teacher Assistantunding from Faculty of Science, Kasetsart University and theunding from Graduated School, Kasetsart University and KURDI,asetsart University. W.P. is grateful to Center of Excellence for

nnovation in Chemistry (PERCH-CIC), Office of the Higher Educa-ion Commission, Ministry of Education for the financial support.

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