Experimental Parasitology 115 (2007) 168–172 www.elsevier.com/locate/yexpr
EYcacy of an essential oil of Eugenia caryophyllata against Psoroptes cuniculi G. Fichi a, G. Flamini b, F. Giovanelli a, D. Otranto c, S. Perrucci a,¤ a
Dipartimento di Patologia Animale, ProWlassi ed Igiene degli Alimenti, Faculty of Veterinary Medicine, University of Pisa, Viale delle Piagge 2, 56124 Pisa, Italy b Dipartimento di Chimica Bioorganica e Biofarmacia, Faculty of Pharmacy, University of Pisa, Italy c Dipartimento di Sanità, Patologia, Farmaco-Tossicologia e Benessere degli Animali, Faculty of Veterinary Medicine, University of Bari, Italy Received 24 March 2006; received in revised form 3 July 2006; accepted 27 July 2006 Available online 14 September 2006
Abstract The acaricidal activity of Eugenia caryophyllata essential oil was evaluated in vitro and in vivo on Psoroptes cuniculi, a mange mite. In vitro, diVerent concentrations of the oil were tested and the observed mites mortality was compared with that observed in untreated and treated (Acacerulen R®) controls. In vivo, six P. cuniculi infected rabbits were topically treated with the oil diluted at 2.5% and compared with untreated and treated control groups of six rabbits each. In vitro, up to the concentration of 0.10% the oil gave highly signiWcant (P < 0.01) percentages of mite mortality respect to the untreated controls, but only up to 0.16% it showed the same eYcacy of Acacerulen R®. In vivo, the treatment with the essential oil cured all infested rabbits and no statistical diVerences were observed respect to the treated control group. The untreated rabbits remained infested. © 2006 Elsevier Inc. All right reserved. Index Descriptors and Abbreviations: Psoroptes cuniculi; Acari; Psoroptidae; Eugenia caryophyllata; Essential oil; In vitro and in vivo acaricidal activity
1. Introduction Therapy and control of both human scabies and animal mange are based mainly on the use of eVective drugs and chemicals. At present, however, the use of drugs to control these parasitic arthropods presents several problems including drug-resistance (Synge et al., 1995; Clark et al., 1996) and environmental damage (Halley et al., 1993; O’Brien, 1999). Ivermectin is increasingly being used to treat human scabies but often treatment failures, recrudescence and reinfection can occur (Currie et al., 2004); an outbreak of lindane-resistant scabies was also documented (Purvis and Tyring, 1991). Concern over toxicity (O’Brien, 1999) of many acaricides limits their use and reduces the number of safe eVective products available. These problems have lead to research eVorts to discover
*
Corresponding author. Fax: +39 050 2216941. E-mail address:
[email protected] (S. Perrucci).
0014-4894/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2006.07.005
new eVective compounds. The identiWcation of novel active plant derived natural compounds could increase the number of available chemotherapeutic agents, thereby reducing the frequency of resistance phenomena and providing alternative drugs with greater acceptance, especially in terms of environmental safety (Alawa et al., 2003). The biological activity of Eugenia caryophyllata (Myrtaceae) (synonym Syzygium aromaticum L., Eugenia aromatica L., Caryophyllus aromaticus L.) was investigated on several parasites. The antibacterial activity of diVerent extracts of E. caryophyllata was demonstrated against pathogenic bacteria (Burt and Reinders, 2003; Feres et al., 2005; Larhsini et al., 2001), and its antiviral eYcacy is also well documented on Herpex simplex (Kurokawa et al., 1995, 1998), and on Hepatitis C virus (Hussein et al., 2000). The fungicidal activity of the essential oil of E. caryophyllata was demonstrated on several food-borne fungal species (Ranasinghe et al., 2002; Velluti et al., 2004; Lopez et al., 2005), on fungi isolated from onychomicosis
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(Gayoso et al., 2005) and on the yeast model Saccharomyces cerevisiae (Chami et al., 2005). On arthropods, the inhibition of adult emergence by E. caryophyllata extracts was demonstrated on Culex pipiens larvae (El Hag et al., 1999). The essential oil of this plant showed eYcacious repellency on the mosquitoes Ades aegypti, Culex quinquefasciatus and Anopheles dirus (Trongtokit et al., 2005), insecticidal activity on Pediculus capitis (Yang et al., 2003), and acaricidal activity on Dermatophagoides farinae and D. pteronyssinus (Kim et al., 2003). Thus, this study was performed in order to evaluate the acaricidal properties of an essential oil of clove (E. caryophyllata) in vitro and in vivo on the mange mite Psoroptes cuniculi. The genus Psoroptes comprise obligate ectoparasites of animals, even if human episodes of Psoroptes ovis infestation have been reported (Mazyad et al., 2001). P. cuniculi lives in the external auricular meatus where it is thought to feed mainly on serous exudate, skin secretions, and blood (Bates, 1999). In rabbits the mite causes intense pruritus with the formation of scabs, that in the untreated animals can completely Wll the external ear canal and the internal surface of the pinna (Bates, 1999). 2. Materials and methods 2.1. Essential oil analysis The essential oil of E. caryophyllata cloves used in this study was a commercial sample by FLORA srl (Lorenzana, Pisa, Italy). GC/EIMS analyses were performed with a Varian CP3800 gas-chromatograph equipped with a DB-5 capillary column (30 m £ 0.25 mm; coating thickness 0.25 m) interfaced with a Varian Saturn 2000 ion trap mass detector. Analytical conditions were: injector and transfer line temperatures 220 and 240 °C, respectively; oven temperature programmed from 60 °C to 240 °C at 3 °C/min; carrier gas helium at 1 ml/min; injection volume, 0.2 l of a10% hexane solution; split ratio 1:30. The identiWcation of the components was performed, for both the columns, by comparison of their retention times with those of pure authentic samples and by mean of their linear retention indices (lri) relative to C8–C17 n-alkanes on HP-wax and HP-5 columns and by computer matching against commercial mass spectra libraries (NIST 98 and ADAMS) and a home-made library, built up from pure substances and components of reference oils, and MS literature data (Adams, 1995; Davies, 1990; Jennings and Shibamoto, 1980; Masada, 1976; Stenhagen et al., 1974; Swigar and Silverstein, 1981). Moreover, the molecular weights of all the identiWed substances were conWrmed by GC/CIMS, using MeOH as CI ionizing gas. 2.2. Acaricidal activity in vitro Psoroptes cuniculi mites were isolated from naturally infected rabbits. The scabs and the cerumen, collected from
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the infected ears, were observed by means of a stereoscopic microscope to isolate all motile stages, i.e. larvae, nymphs and adults of both sex. Mites were collected with a needle and placed in 6-cm Petri dishes (approximately 100 adult mites/each dish). The essential oil of E. caryophyllata was diluted from the concentration of 10% to 0.03% (10%, 5%, 2.5%, 1.25%, 0.62%, 0.31%, 0.16%, 0.10% 0.06%, 0.03%) in paraYn oil and 2.5 ml of each solution were directly added to Petri dishes. Six replications were made for each concentration. As untreated control, six Petri dishes containing only 2.5 ml of paraYn oil were used, while six Petri dishes containig 2,5 ml of a pyrethrum extract containing 25% of pyrethrins (Acacerulen R®, Teknofarma s.p.a., Torino), a drug used for topically treatment of psoroptic mange of the rabbit, represented the treated control. All plates containing each concentration of the oil and untreated and treated control plates were placed in separate humidity chambers in saturated humidity conditions at 22 °C (High Performance Incubator 2800, Galli, Milan, Italy). After 24 h, mites were placed in clean Petri dishes containig 2.5 ml of paraYn oil and after further 24 h all the motionless mites were stimulated with a needle; lack of reactions and persistent immobility indicated their death. The main component of the essential oil whose acaricidal activity on P. cuniculi was still unknown, was also tested at the same concentrations used for the essential oil and compared with untreated and treated control plates. Data were statistically elaborated with the exact test of Fisher (Glantz, 2003a) using Mc Graw Hill software (Glantz, 2003b). In addition, the EYcacy Dose 50 (ED50) was calculated (Previtera, 1976). 2.3. Acaricidal activity in vivo In order to assess the in vivo eYcacy of E. caryophyllata essential oil, 18 New Zealand rabbits were experimentally infected with P. cuniculi with the method reported by Perrucci et al. (2001). After two months, at a clinical and microscopical examination all rabbits were found infested in both ears and they were randomly divided in three groups of six animals. In order to evaluate the diVerent eYcacy of the essential oil in presence or in absence of scabs in the ear of the rabbits, from each animal and just before the treatment all the scabs were collected only from the right ear by means of dissecting forceps, while they were not collected from the left ear. One group of six rabbits was treated topically with two ml of a solution of essential oil at the concentration of 2.5% in a mixture composed by 98% of saline solution and 2% of paraYn oil. Another group of six rabbits was treated with two millilitre of a mixture composed by 98% of saline solution and 2% of paraYn oil (untreated control group), and the last group of six rabbits was treated with two millilitre of Acacerulen R® (treated control group). All rabbits were treated two times, at the beginning of the treatment (0 day) and seven days later.
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At 0, 7, 14 and 30 days after the beginning of the treatment, rabbit ears were examined with an otoscope in order to evaluate the presence of scabs. From each ear of each rabbit, the cerumen and/or scabs were collected and microscopically observed to evaluate the presence of mites. The degree of infestation was evaluated on the basis of the following scoring system: 0 D absence of scabs and/or mites; 0.5 D irritation in ear canal but no mites observed; 1 D small number of scabs in the ear canal, mites present; 2 D external ear canal Wlled with scabs, mites presents; 3 D scabs in ear canal and proximal 1/4 of pinna, mites present; 4 D 1/2 pinna Wlled with scabs, mites present; 5 D 3/4 of the pinna Wlled with scabs, mites present; 6 D all internal surface of the pinna full of scabs, mites present (Guillot and Wright, 1981). The experiment was carried out in accordance with the guidelines given by the Italian law on the use of animals in research. Data were statistically elaborated with Analysis of Variance and the test of Student–Newman–Keuls for multiple comparisons between the three groups (Glantz, 2003a) using Mc Graw Hill software (Glantz, 2003b). 3. Results 3.1. Essential oil and -caryophyllene Table 1 shows the chemical composition of the essential oil of E. caryophyllata. The most representative compounds of this oil resulted to be eugenol (59.3%) and -caryophyllene (24.9%). The acaricidal activity in vitro of eugenol on P. cuniculi, both by direct contact and by contact only with their vapour phase,was already reported in literature (Perrucci et al., 1995). In the present study, the active principle -caryophyllene did not show any acaricidal activity at all concentrations tested. The essential oil of E. caryophyllata showed in vitro a good acaricidal activity against P. cuniculi (Table 2). Respect to the untreated controls, the percentage of mites mortality resulted highly signiWcant (P < 0.01) up to the concentration of 0.10%, while it resulted only signiWcant (P < 0.05) and no signiWcant at 0.06% and 0.03%, respec-
Table 1 Percentage of the principal components of E. caryophyllata oil Compound
%
1,8-Cineole Linalool Eugenol -Copaene -Caryophyllene -Humulene -Cadinene Epizonarene -Muurolene Eugenyl acetate -Cadinene
0.1 0.2 59.3 1.2 24.9 2.8 0.5 0.1 0.1 4.2 3.6
Table 2 Percentage of mortality of P. cuniculi treated for 24 h with scalar concentrations (10–0.03%) of the essential oil of E. caryophyllata, with Acacerulen R® and untreated 5.00a,A 100.00C 77.88B 23.85b,A 21.15A 100.00C
Untreated control OE E.caryophyllata 0.16% OE E.caryophyllata 0.10% OE E.caryophyllata 0.06% OE E.caryophyllata 0.03% Acacerulen R®a
The percentage of mortality is presented as mean of percentage followed by diVerent small letters when there are signiWcantly diVerences P < 0.05 and diVerent capital letters when there are highly signiWcantly diVerences P < 0.01 (Exact Test of Fisher). a Drug containing 25% of pyrethrins as active components.
tively. Respect to the treated controls, the oil showed the same eYcacy of Acacerulen® with the exception of the concentrations of 0.10%, 0.06% and 0.03% (P < 0.01). The concentration of 0.074% resulted the ED50 of used E. caryophyllata essential oil. 3.2. Acaricidal activity in vivo The dose of E. caryophyllata (2 ml) at the concentration of 2.5% was selected in order to have a quantity of eugenol (31.5 mg) less than 100 mg/24 h, the toxically dose for contact in the rabbits (http://ntp.niehs.nih.gov). Table 3 shows the main results of the trial. At the day 0 no diVerence appeared between the three groups, both for the parasitological (presence of mites) and clinical score (presence of scabs/crusts). Treatment with the essential oil gave a clinical cure in all rabbits in each ear. From day 7 after the beginning of the treatment, E. caryophyllata treated group and treated control group resulted negative for the presence of P. cuniculi mites and/or eggs. No signiWcant diVerences regard to the degree of infestation and lesion scores resulted between the treated control group and the essential oil group and no side eVects was observed
Table 3 Lesion scores in right ears (R) and in left ears (L) (after collection of all the crusts) of rabbits infested by Psoroptes cuniculi and treated with 2.5% of Eugenia caryophyllata essential oil, with Acacerulen R® and untreated at 0, 7, 14, and 30 days after the treatment Day
Ear
E. caryophyllata
AcaCerulen R®b
Untreated control
0 7 14 30 0 7 14 30
L L L L R R R R
3.00 0.00a 0.00a 0.00a 3.33a 0.11a 0.00a 0.00a
2.83 0.00a 0.00a 0.00a 3.17a 0.06a 0.00a 0.00a
3.33 3.00b 2.83b 2.67b 3.33a 2.83b 2.83b 3.00b
R: right ear L: left ear (after collection of all the scabs). The lesion score is presented as mean value followed by diVerent small letters when there are signiWcantly diVerence P < 0.05 (test of Student–Newman–Keuls for multiple comparisons). b Drug containing 25% of pyrethrins as active components.
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in this last group. The untreated control group remained infested until the end of the trial and, in the ears of three rabbits where the crusts were not collected, the clinical conditions get worse. From day 7, in this latter group the degree of infestation was signiWcantly higher respect to E. caryophyllata treated group and treated control group for both ears. 4. Discussion The chemical composition of the essential oil of clove E. caryophyllata investigated in this study shows that eugenol represents the main component of the oil. The neuroinsecticidial activity of eugenol was demonstrated on Periplaneta americana by Enan (2001). A study about the structure/activity relationship of some monoterpenes as acaricides against P. cuniculi (Perrucci et al., 1995) showed a high in vitro activity of eugenol on this mite species both by direct contact and by contact only with their vapour phase. The presence of this active principle could be the reason of the good acaricidal activity against P. cuniculi showed both in vitro and in vivo by the essential oil of E. caryophyllata. The in vitro eYcacy of this oil resulted so good as Acacerulen R®, the reference drug used in the present study, at concentrations (from 10% to 0.16%) much lower respect to that of pyrethrins (25%) contained in Acacerulen R®. The in vivo trial conWrmed the good acaricidal activity of this essential oil and showed the same eYcacy both in presence and in absence of scabs in the ears of infected rabbits. The method of application of topical treatments for P. cuniculi mange mites, as Acacerulen R ®, often suggests the collecting of the scabs; however, in this study both essential oil and Acacerulen R® showed that the presence of scabs do not reduce their therapeutic eYcacy. The acaricidal activity previously reported for this oil on D. farinae and D. pteronyssinus (Kim et al., 2003) is conWrmed also on P. cuniculi. Results obtained in vivo in the present study indicate that E. caryophyllata oil could represent a possible alternative for the topical treatment of psoroptic mange in rabbits and encourage further studies to evaluate its eYcacy also on other mange mites responsible for otoacariasis, such as Otodectes cynotis, or skin diseases, such as Sarcoptes scabiei, in other animal species and in humans (Arlian, 1996; Wrenn, 1996). The antibacterial (Burt and Reinders, 2003; Larhsini et al., 2001) and fungicidal activity (Gayoso et al., 2005) reported for E. caryophyllata oil could be useful for the treatment or prevention of secondary infections that are often responsible of compliance in scabies and mange (McCarthy et al., 2003; Van den Broek and Huntley, 2003), while the local anaesthetic activity of -caryophyllene (Ghelardini et al., 2001), found in high percentage (24.9%) in this oil, could reduce pruritus, a characteristic symptom of these diseases (McCarthy et al., 2003; Van den Broek and Huntley, 2003).
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