Composition and Acaricidal Activity of Laurus novocanariensis and Laurus nobilis Essential Oils Against Psoroptes cuniculi

Abstract The major components of Laurus nobilis and L. novocanariensis leaf oils were identified and their acaricidal activity against Psoroptes cuniculi evaluated. Monoterpenes were predominant in L. nobilis oil (91.8%), while sesquiterpenes were only 1.4%. The main components of this oil were 1,8-cineole (39.2%), α-terpinyl acetate (11.3%), sabinene (10.6%) and linalool (7.4%). The acaricidal activity of L. nobilis oil, at a concentration of 10%, led to a mortality rate of 73%; at 5% the average activity was significantly reduced to 51%, while dilutions of 2.5%, 1.25% and 0.625% were ineffective. Laurus novocanariensis oil, compared to L. nobilis, was richer in sesquiterpenes; the main constituents were α-pinene (10.4%), 1,8-cineole (9.6%) and β-selinene (7.2%). After 24 h of contact, the oil of L. novocanariensis killed all the mites when used at 10% and 5% concentrations. At lower concentrations the mortality significantly decreased; a dilution of 0.625% was ineffective.


Introduction
The biological activity of essential oils against several organisms has been confi rmed in many reports, mainly associated with primary components mono-and sesquiterpenoids (1,2).
The composition of the leaf oils of L. azorica and L. nobilis has been reported (11)(12)(13). Prior to 2002, Laurus ssp.endemic of the macaronesian archipelagos of Azores, Madeira and Canaries-were represented as a single species, Laurus azorica. Recently, laurels from Madeira and the Canaries have been reconsidered as a separated taxon, classifi ed as Laurus novocanariensis (14).
The aim of the present study is the identifi cation of the major components of L. nobilis and L. novocanariensis leaf oils and the evaluation of their acaricidal activity against Psoroptes cuniculi.

Experimental
Leaves of Laurus nobilis were collected in June 2003 at Alberaccio (Asciano-Pisa, Italy) on wild trees. A voucher was deposited in the Herbarium of Pisa's botanical garden. Leaves of L. novocanariensis were collected in May 2001, at Ponta do Pargo on Madeira Island (Portugal) from a single wild tree. A voucher was deposited in the Herbarium of Madeira's botanical gardens.
The leaves were dried at room temperature away from direct sunlight before distillation. For each species, the oil was obtained from 100 g samples of fi nely cut dry leaves hydrodistillated for 2 h in a Clevenger-type apparatus.

Chemical Analysis
The oils were analyzed by gas chromatography with FID GC/MS detection. The GC analyses were accomplished with an HP-5890 Series II instrument equipped with HP-WAX and HP-5 capillary columns (30 m x 0.25 mm, 0.25 μm fi lm thickness) operated with the following temperature program: 60°C for 10 min, ramp of 5°C/min up to 220°C; injector and detector temperatures 250°C; carrier gas nitrogen (2 mL/min); detector dual FID; split ratio 1:30; injection of 0.5 μL. The identifi cation of the components was performed, for both the columns, by comparison of their retention times with those of pure authentic samples, and by means of their linear retention indices (LRI) relative to the series of n-hydrocarbons. The relative proportions of the oil constituents were expressed as percentages obtained by FID peak-area normalization, all relative response factors being taken as one.
GC/EIMS analyses were performed on a Varian CP-3800 gas chromatograph equipped with a DB-5 capillary column (30 m x 0.25 mm, coating thickness 0.25 μm) and a Varian Saturn 2000 ion trap mass detector. Analytical conditions: injector and transfer line temperatures 220°C and 240°C, respectively; oven temperature programmed from 60°-240°C at 3°C/min; the carrier gas was helium at 1 mL/min; injection of 0.2 μL (10% hexane solution); split ratio 1:30. Identifi cation of the constituents was performed as before by computer matching against commercial (NIST 98 and ADAMS) and homemade libraries of mass spectra built up from pure substances and components of known oils and MS literature data (15)(16)(17)(18)(19)(20). Moreover, the molecular weights of all the identifi ed substances were confi rmed by GC/CIMS using MeOH as CI ionizing gas.

Biological Analysis
Disks of fi lter paper were imbibed with 50 μL of each of the oils, diluted to several concentrations. The disks were put in concave glass capsules having the same dimensions of disks. Adult mites, collected from the ears of a massively infested rabbit, were put in groups of 30 over the disks. The capsules with the disks were then covered with parafi lm, fi nely punched to allow the circulation of air without allowing the mites to escape. (a) percentages obtained by FID peak-area normalization, all relative response factors being taken as one (HP-5 column); linear retention indices (HP-5 column); t = trace (< 0.1%) The mobility control of mites was performed after 24 h of contact with the substances. To verify mortality, immobile mites were collected one by one and placed on disks inside capsules without tested substances. Control was performed after another 24 h; the persistence of the immobility of the mites after their stimulation with a needle indicated their death.
The oils of L. nobilis and L. novocanariensis were used at 10%, 5%, 2.5%, 1.25% and 0.625%. Dilutions were obtained with paraffi n oil as the solvent. Paraffi n was also used as control to ensure its innocuousness for mites.

Statistical Analysis
Data were subjected to ANOVA in which oil type (Laurus novus and Laurus novocanariensis) and different concentration levels (10, 5, 2.5, 1.25, 0.65, control) were considered as fi xed effects and percentage mortality as the independent variable. Statistical analysis was undertaken using the statistical package JMP (SAS Institute, 2002).
The oil of L. novocanariensis, at concentrations of 10% and 5% after 24 h of contact with mites, killed all the mites, showing an acaricidal activity of 100%; at 2.5%, the mortality of the mites signifi cantly decreased to 22%; at 1.25% and at 0.65% concentrations, the mortality was further reduced, respectively, to 8% and 0%-largely ineffective. From these data, it can be established that the minimum concentration leading to death of the mite is 5%.
The acaricidal activity of L. nobilis oil, at a concentration of 10%, led to a mortality of 73% (average of three independent assays), while at 5% the average activity was signifi cantly reduced to 51%. Dilutions of 2.5%, 1.25% and 0.625% were found to be signifi cantly ineffective.