Exp Appl Acarol (2008) 45:219228DOI 10.1007/s10493-008-9182-6

Repellent eVect of sweet basil compounds on Ixodes ricinus ticks

Simone Del Fabbro Francesco Nazzi

Received: 19 May 2008 / Accepted: 17 July 2008 / Published online: 1 August 2008 Springer Science+Business Media B.V. 2008

Abstract Diseases transmitted by ticks are causing increasing concern in Europe and allaround the world. Repellents are an eVective measure for reducing the risk of tick bite;products based on natural compounds represent an interesting alternative to common syn-thetic repellents. In this study the repellency of sweet basil (Ocimum basilicum L.) wastested against the tick Ixodes ricinus L., by using a laboratory bioassay. A bioassay-assistedfractionation allowed the identiWcation of a compound involved in the biological activity.Eugenol appeared to be as repellent as DEET at two tested doses. Linalool, which wasidentiWed in the active fraction too, failed to give any response. Repellency of eugenol wasproved also in the presence of human skin odour using a convenient and practical bioassay.

Keywords Basil Eugenol Ixodes ricinus Linalool Ocimum basilicum Tick repellents

Introduction

Diseases transmitted by ticks (e.g., Lyme borreliosis, tick borne encephalitis) are emergingdiseases in most European countries (Randolph 2006; Vorou et al. 2007). Along with vac-cines, repellents represent a fundamental resource for preventing tick transmitted diseases.Repellent compounds in diVerent commercial formulations are available for tick biteprevention; however, some arthropods have shown diVerential responses to these products(see for example Klun et al. 2004) and concern exists about possible adverse eVects ofsome of these compounds for human health (Abdel-Rahman et al. 2001). For these reasonsthe development of novel repellents could be of great value.

Plants produce secondary metabolites that are eVective against arthropod pests (DAles-sandro and Turlings 2006; Isman 2000) as well as blood feeding arthropods, such as mos-quitoes (Prajapati et al. 2005); repellents based on natural compounds from plants are

S. Del Fabbro F. Nazzi (&)Dipartimento di Biologia e Protezione delle Piante, Universit di Udine, via delle Scienze 208, 33100 Udine, Italye-mail: francesco.nazzi@uniud.it1 C

220 Exp Appl Acarol (2008) 45:219228

widely used to prevent arthropod bites and tend to be more appreciated than synthetic com-pounds by the consumers. However some authors (see for example Thorsell et al. 2006)suggested that plant essential oils or plant extracts may not be suitable for the applicationon humans due to their possible toxicity; for this reason the identiWcation of the active com-pounds responsible for the biological activity may be advisable in that their repelling andpotential toxic eVect can be better assessed.

The repellent eVect of some plant materials on the sheep tick (Ixodes ricinus L.) havebeen demonstrated by several authors (Gardulf et al. 2004; Jaenson et al. 2005, 2006;Mehlhorn et al. 2005; Thorsell et al. 2006; Trigg and Hill 1996; Tunn et al. 2006). Ocimumbasilicum L. (sweet basil) is used in traditional cookery as well as a natural remedy for selfmedication worldwide. Moreover sweet basil is regarded as an antimicrobial agent(Suppakul et al. 2003) and as a repellent for mosquitoes (Bindra et al. 2000; Erler et al.2006; Gbolade et al. 2000; Murugan et al. 2007; Prajapati et al. 2005; Ruberto et al. 1991;Seyoum et al. 2002). A little known paper reports the bioactivity of O. menthaefoliumHochst. O. gratissimum L. against the tick I. persulcatus (Dobrotvorskii et al. 1989);Mwangi et al. (1995) demonstrated that O. suave Willd. can be considered as repellent aswell an acaricide to Rhipicephalus appendiculatus Neumann ticks.

Preliminary experiments, carried out with a simple lab bioassay developed for thispurpose, suggested a biological activity of sweet basil on I. ricinus nymphs (Nazzi et al. inpress); the aim of this research was to conWrm previous observation about the repellency ofO. basilicum on I. ricinus and identify the compounds responsible for this eVect.

Materials and methods

Ticks used in this study

The ticks used in this study belonged to the species I. ricinus and were sampled bydragging, with a 1.0 1.0 m white Xeece blanket. The samplings were carried out in themountain area of Friuli Venezia Giulia (North-eastern Italy) in Spring, Summer andAutumn 2007.

Nymphs were used for this study as this is the most important developmental stageunder the epidemiological point of view, being both abundant in the environment andactive in human biting. Nymphs were kept inside sealed polypropylene tubes with a wetstrip of Wlter paper, and were maintained at room temperature in darkness until they wereused in the assays. Only nymphs that appeared to be active in the storing tubes were usedfor the bioassay. Each tick was used only once.

Extraction and fractionation

Sweet basil (O. basilicum) plants used in this study were obtained from an organic farmand can be regarded as residue free. Extraction of sweet basil plants was carried out byimmersion of 100 g of leaves and stems into the solvent (680 ml of acetone) for 1 h.Extracts were kept at 20C until use. To isolate the active components of the extract,100 mg equivalents of the basil extract were injected into a Varian ProStar 230 high pres-sure liquid chromatograph (HPLC) equipped with a Supelcosil LC-SI column(25 cm 4.6 mm, 5 m) and a Varian ProStar 704 fraction collector. Solvent was a mix-ture of hexane and diethyl ether (80% and 20%, respectively).1 C

Exp Appl Acarol (2008) 45:219228 221

Four fractions were collected according to the elution time: from 0 to 5 min = fractionA = Fr A (5 ml); from 5 to 10 min = fraction B = Fr B (5 ml); from 10 to 15 min = fractionC = Fr C (5 ml); from 15 to 30 min = fraction D = Fr D (15 ml). The desired concentrationsof the basil extract and the fractions used in the bioassays were obtained by evaporating thesolvent under a gentle stream of nitrogen.

GC-MS analysis

The identiWcation of the volatile compounds present in the active fraction was carried outusing a Varian 3400 gas-chromatograph coupled to a Varian Saturn 2000 mass spectrome-ter (GC-MS). The CP-SIL 8 column (30 m 0.25 mm, Wlm thickness: 0.25 m) was main-tained at 40C for 1 min then programmed to 250C at 10C/min. The carrier gas washelium (Xow: 1 ml/min). Injection volume was 1 l in splitless mode. MS ionization energywas set to 70 eV. Tentative identiWcation of the compounds was based on the spectrum andchromatographic behaviour of unknown substances and was conWrmed by injection ofauthentic standards.

Pure compounds

Eugenol (99% purity), ()-Linalool (9597% purity) and DEET (97% purity) were obtainedfrom Sigma-Aldrich. The DEET based commercial product OFF! (scudo roll-on; JohnsonWax) was used for the in vivo bioassay.

Bioassays

In vitro bioassays

A circular glass arena, obtained placing upside down a 6 cm diameter Petri dish, was usedto observe the behaviour of ticks under the eVect of diVerent stimuli. Two concentric circleswere drawn on the inner surface of the Petri dish, having 1 cm radius (start line or line A) and2 cm radius (Wnish line or line B). The treatment was applied with a pipette outside theWnish line on the outer surface of the Petri dish in 100 l volume of solvent. The arena wasplaced on a wet piece of Wlter paper inside a larger Petri dish (Fig. 1a). A single nymph wasplaced with a Wne paint brush in the centre of the arena and the time spent to go from theline A over the line B was recorded. If the start line was not crossed before 45 s the nymphwas discarded; if after 3.5 min the tick did not cross the Wnish line, 210 s were recorded asthe Wnish time.

If no stimuli were applied or the stimulus was not active the nymph simply went straightfrom the centre of the arena to the edge of it (in Fig. 1b, the track obtained from the videoof a single tick assayed against the solvent alone is reported as an example). If a repellentsubstance was used, the tick walked up to the Wnish line, then turned again and again everytime the Wnish line was approached (an example obtained using 100 g of eugenol as astimulus is reported in Fig. 1c).

In vivo bioassays

In order to test if the repellent eVect of a compound was signiWcant even in presence ofhuman skin attractants, an in vivo bioassay was developed. A circle of Wlter paper (6 cmdiameter) was held on the palm of a persons hand. A central point, a start line (A) and a1 C

222 Exp Appl Acarol (2008) 45:219228

Wnish line (B) (1 and 2 cm radius, respectively) were drawn on the lower surface of the paperand the treatment applied outside line B on the upper surface as in the in vitro bioassay(Fig. 2). A single I. ricinus nymph was transferred onto the centre of the paper and timerecorded as usual. The same persons hand was used for all bioassays; after a set of bioas-says with the same stimulus was carried out, the hand was washed with common bath soap.

To check for possible diVerences in bioactivity in case the repellent was applied onpaper or directly to skin, a second version of the in vivo test was carried out, in which thebioactivity of a DEET based commercial product was tested with the bioassay describedabove and with a modiWed version of the same in which the Wlter paper dish was smaller(radius = 2 cm) and the treatment was applied outside the paper dish directly to the skin(Fig. 3). In this case, 10 Wlter paper dishes were treated and 10 nymphs tested on them withthe standard bioassay (6 cm diameter treated Wlter paper dishes on a clean hand); then theproduct was applied to a persons hand and 10 nymphs were tested with the modiWed ver-sion of the bioassay (4 cm diameter clean Wlter paper dishes on a treated hand). Data wererecorded as usual. In this case the dispenser of the commercial formulation of DEET wasused; in this way 26 mg of the product (corresponding to 5.3 mg of DEET) were smearedon the arena in each assay.

Experimental protocol

One to three replicates were run for each stimulus. Every replicate consisted of 712 bioas-says with single ticks against the stimulus to be tested and 712 bioassays against the nega-

Fig. 1 (a) Glass arena used in the in vitro bioassays. Line A: start line, 1 cm radius; line B: Wnish line, 2 cmradius; the dashed area represents the treated area. (b) Track of a single tick (dashed line) on an arena treatedwith the solvent alone (negative control). (c) Track of a single tick (dashed line) on an arena treated with arepellent (100 g of eugenol)1 C

Exp Appl Acarol (2008) 45:219228 223

tive control (in this case the arenas were treated with 100 l of the solvent alone). In somecases 712 bioassays against a positive control were carried out too, using a solution ofDEET as a reference repellent.

Bioassays were all run at room temperature under daylight conditions. The temperaturewas monitored at the beginning and at the end of each experimental session and showed avariation between 0 and 1.2C. The minimum and maximum temperature recorded in thewhole period of the bioassays were 20 and 28C, respectively.

Experiments carried out

Several stimuli at diVerent doses were tested in this study with the in vitro bioassay:

sweet basil acetone extract (at the doses of 10 and 100 mg equivalents): two replica-tions for each dose.

Fig. 2 Paper arena for the in vivo bioassay (1st version) held on the palm of a persons hand. Line A (startline, 1 cm radius); line B (Wnish line, 2 cm radius); the dashed area represents the treatment applied on thepaper arena

Fig. 3 Paper arena for the in vivo bioassay (2nd version) held on the palm of a persons hand. Line A (startline, 1 cm radius); line B (Wnish line, 2 cm radius); the dashed area represents the treatment applied directlyto skin1 C

224 Exp Appl Acarol (2008) 45:219228

basil extract HPLC fractions (Fr A, Fr B, Fr C, Fr D at 10 mg equivalents): three repli-cations for each fraction.

Pure compounds from Fr B: eugenol (1, 10, 100 and 1000 g): three replications foreach dose; linalool (10, 100 and 1000 g): one replication; DEET (1, 10, 100 and1000 g): one replication.

With the in vivo bioassay, eugenol and DEET were tested once at the same dose(100 g).

With the second version of the in vivo test, the DEET based commercial product wastested once.

Statistical analysis of the results

In order to check for possible signiWcant diVerences between treatments and negativecontrols, treatments and positive controls, negative and positive controls, the times spentby ticks to move from line A to line B against each tested substance were compared usingthe non-parametric tests of MannWhitney and Friedman (for simple and repeated exper-iments, respectively). Due to some unbalanced replications, a generalization of theFriedman test, proposed by Mack and Skillings (1980) and Groggel and Skillings (1986)was used.

Results

The sweet basil extract was repellent in comparison to the solvent (acetone) at both doses(10 and 100 mg; P < 0.01). Out of the four HPLC fractions of the active extract, only thesecond one (Fr B) appeared to be repellent (P = 0.056) (Table 1). Several compounds wereidentiWed by GC-MS in the active fraction (Fr B), linalool and eugenol being the mostabundant ones, each representing roughly the 30% of the compounds detected in thisfraction.

Eugenol was repellent at the doses of 100 and 1000 g (P < 0.01) and there were no sta-tistical diVerences between the medians of eugenol and DEET at these doses. Unlike euge-nol, DEET was also active at the dose of 10 g (P < 0.01), whereas no bioactivity wasrecorded at 1 g for both compounds. Linalool was not repellent at any dose (Fig. 4).

In the in vivo bioassay eugenol was active (median of time before crossing line B inarenas treated with eugenol: 87.5 s; median of time before crossing line B in arenas treatedwith the solvent alone: 12.5 s; P < 0.01), but DEET was more active than eugenol (medianof time before crossing line B in arenas treated with DEET: 203.5 s; P < 0.05).

No statistical diVerence were noted in the bioactivity of the standard commercial prod-uct applied directly to the hand or to the Wlter paper.

Discussion

The repellency of an acetone extract of O. basilicum against the tick I. ricinus, observed inthis study, conWrms previous Wndings about the biological activity of this plant species onthe sheep tick (Nazzi et al. in press), integrating the data about the biological eVects ofO. basilicum and related species against other arthropods, including other tick species(Dobrotvorskii et al. 1989; Mwangi et al. 1995).1 C

Exp Appl Acarol (2008) 45:219228 225

A bioassay-assisted fractionation led to the isolation of one active fraction (Fr B); thisallowed a subsequent easier identiWcation of the pure compounds responsible for the bioac-tivity. Out of the compounds identiWed in the active fraction, linalool showed no eVectsagainst I. ricinus nymphs, although its repellent properties against other arthropods havebeen known for a long time (Chapman et al. 1981; Hwang et al. 1985; Inazuka 1983).Instead eugenol seems to be as repellent as the reference substance DEET, except at thelower doses tested. The repellent activity of eugenol against insects has already been dem-onstrated (see for a review Isman 2000); data about eugenol repellency on I. ricinus ticksare available as well (Thorsell et al. 2006; Tunn et al. 2006), although details about thesize of the samples and the statistical signiWcance of the observed eVects are lacking in thecited studies.

Many published studies deal with the biological activity of essential oils or plantextracts on I. ricinus ticks (Gardulf et al. 2004; Jaenson et al. 2005; Jaenson et al. 2006;

Table 1 Bioactivity in the in vitro bioassay of the sweet basil extract and the fractions of the extract vs thenegative control

Stimulus Dose Replicationnumber

Treatmentmedian (s)

Negative controlmedian (s)

P

Basil acetone extract 10 mg equivalents 1 14.5 8.0

226 Exp Appl Acarol (2008) 45:219228

Mehlhorn et al. 2005; Thorsell et al. 2006; Trigg and Hill 1996; Tunn et al. 2006). In thiscase an attempt to identify the single components responsible for the bioactivity of sweetbasil was carried out. A similar approach was pursued because polymorphism, as well asother factors, determines a high variability in sweet basil chemical composition (Hasegawaet al. 1997; for a discussion on factors aVecting variability in sweet basil chemical compo-sition, see Suppakul et al. 2003). Moreover such an approach is advisable in view of devel-oping possible repellents for human protection because diVerent components of essentialoils and plant extracts may have diVerent eVects on human health. Thus the use of a fewcompounds with deWned concentrations and well-known characteristics seems to be morereliable and also safer than the use of whole plant extracts.

The diVerence in the repellency of the two substances tested in this study (eugenol andlinalool) underlines once again the opportunity of an approach based on the identiWcationof the compounds responsible for the biological activity rather than using whole plantextracts or essential oils, that could contain substances which do not contribute to the repel-lency but may have some side eVects on human health. Other authors (Dautel 2004; Sch-reck 1977) pointed out that whatever the identity of a tick repellent this should be testedagainst host-associated attractant stimuli before it is regarded as active. Therefore a funda-mental aspect in the development of repellents is the use of adequate tests to evaluate theeYcacy of the compounds. DiVerent kinds of bioassays have been developed for this goal(Dautel 2004). In this study a serious attempt to accomplish this target was made, in thatin vivo laboratory bioassays were used to conWrm previous in vitro lab tests, that areeasily standardizable and simpler to carry out.

In any case more studies would be needed before implementing the identiWed compoundin a protective strategy for humans against ticks (Katz et al. 2008). In particular, withrespect to the mode of application, eugenol may not be very suitable as a skin-repellent butrather for a distribution on clothing, because of possible skin sensitisation eVects (Schnuchet al. 2007). As regards the formulation, the weaker eYciency at the lower dose tested com-pared to the DEET is not a matter of concern, since the DEET in the commercial productsis actually used at much higher concentrations than those tested in this study. Finally, inorder for eugenol to be used as an eVective tick repellent, further in-depth studies shouldexamine its eYcacy in Weld control assays and the persistence of its repellent eVect, as itwas done for other substances by some authors (Garboui et al. 2006).

Acknowledgements We are grateful to Dr. Iris Bernardinelli who assisted in the collection of ticks, Prof.Corrado Lagazio for his support in the statistical analysis, the Azienda Agricola Achille Ermacora (Paviadi Udine, UD, Italy) for providing the basil plants used in this study.

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Repellent eVect of sweet basil compounds on Ixodes ricinus ticksAbstractIntroductionMaterials and methodsTicks used in this studyExtraction and fractionationGC-MS analysisPure compoundsBioassaysIn vitro bioassaysIn vivo bioassays

Experimental protocolExperiments carried outStatistical analysis of the results

ResultsDiscussionReferences

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