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Efficacy of an essentifal oil of Cinnamomum zeylanicum against Psoroptes cuniculi
ARTICLE IN PRESS
Phytomedicine 14 (2007) 227–231 www.elsevier.de/phymed
SHORT COMMUNICATION
Efficacy of an essentifal oil of Cinnamomum zeylanicum against Psoroptes cuniculi G. Fichia, G. Flaminib, L.J. Zarallia, S. Perruccia, a
Dipartimento di Patologia Animale, Profilassi ed Igiene degli Alimenti–Faculty of Veterinary Medicine of Pisa University, Italy Dipartimento di Chimica Bioorganica e Biofarmacia–Faculty of Pharmacy of Pisa University, Italy
b
Abstract The aim of the present study was to investigate the in vitro and in vivo acaricidal effects of an essential oil of Cinnamomum zeylanicun leaves on Psoroptes cuniculi, a mange mite. In vitro, 2.5 ml of the essential oil diluted at different concentrations, from 10% to 0.03%, in paraffin oil were added to Petri dishes containing all motile stages of P. cuniculi. Mites mortality observed in these dishes was compared with that observed in untreated and treated (AcaCerulen Rs) control plates. In vivo, one group of six P. cuniculi infected rabbits was topically treated two times at seven days interval with two ml of the essential oil at the concentration of 2.5% in paraffin oil and compared with untreated and treated (AcaCerulen Rs) control groups of six rabbits each. After 24 h of contact, all concentrations of essential oil between 0.10 and 10% showed a good in vitro acaricidal efficacy if compared with the untreated controls (po0:01), but only the concentrations between 0.16 and 10% turned out as active as the drug. In vivo, the treatment with the essential oil cured all infested rabbits and no statistical differences were observed with the treated control group. r 2006 Elsevier GmbH. All rights reserved. Keywords: Psoroptes cuniculi; Cinnamomum zeylanicum; Essential oil; In vitro and in vivo acaricidal activity
Introduction Psoroptes cuniculi is a worldwide obligate ectoparasite mainly of rabbits, goats, horses, and sheep. It 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 crusts and scabs, that in the untreated animals can completely fill the external ear canal and the internal surface of the pinna (Bates, 1999). Therapy and control of both human scabies and animal mange are based mainly on the use of effective drugs and chemicals. At present, however, the use of drugs to Corresponding author. Tel.: +39 50 22 16949; fax: +39 50 22 16949. E-mail address: [email protected] (S. Perrucci).
0944-7113/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2006.01.004
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). Concern over toxicity (O’Brien, 1999) of many acaricides limits their use and reduces the number of safe effective products available. These problems have led to research efforts to discover new effective compounds. The identification of novel active plant derived natural compounds could increase the number of available chemotherapeutic agents, thereby reducing the frequency of resistance phenomena of pathogen arthropods and providing alternative drugs with greater acceptance, especially in terms of environmental safety (Beesley, 1973; Alawa et al., 2003). The human folk medicine reports the activity of the essential oil of Cinnamomum zeylanicum against bacteria, viruses and parasites and suggested its use for the
ARTICLE IN PRESS 228
G. Fichi et al. / Phytomedicine 14 (2007) 227–231
treatment of intestinal worms and skin parasites (Baltz, 2001). Modern studies showed that C. zeylanicum and its extracts have really anti-microbial and antiparasitic activities. The anti-bacterial activity of this drug was demonstrated against Helicobacter pylori (Tabak et al., 1999) and Listeria monocytogenes (Yust and Fung, 2002), while some studies reported the activity of essential oil on dermatophytes (Lima et al., 1993) and fungi involved in respiratory tract mycoses (Singh et al., 1995). The anti-helmintic activity of C. zeylanicum essential oil was demonstrated in vitro on Ascaris lumbricoides (Raj, 1975). In a recent study (Yang et al., 2005), C. zeylanicum oil and its main constituents showed ovicidal and adulticidal activities against the human head louse, Pediculus humanus capitis. The essential oil of another plant of the same genus, C. osmoploeum Kaneh, also showed a good insecticidal activity on mosquito larvae and termites (Chang and Cheng, 2002; Cheng et al., 2004). Thus this study was performed in order to evaluate the acaricidal properties of an essential oil of C. zeylanicum leaves on Psoroptes cuniculi.
Materials and methods Essential oil The essential oil of Cinnamomum zeylanicum leaves used in this study was a commercial sample by FLORA srl (Lorenzana, Pisa, Italy). GC/EIMS analyses were performed with a Varian CP-3800 gas-chromatograph equipped with a DB-5 capillary column (30 m 0.25 mm; coating thickness 0.25 mm) interfaced with a Varian Saturn 2000 ion trap mass detector. Analytical conditions were: injector and transfer line temperatures 220 and 240 1C respectively; oven temperature programmed from 60 1C to 240 1C at 3 1C/min; carrier gas helium at 1 ml/min; injection volume, 0.2 ml of a10% hexane solution; split ratio 1:30. The identification 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; Massada, 1976; Stenhagen et al., 1974; Swigar and Silverstein, 1981). Moreover, the molecular weights of all the identified substances were confirmed by GC/CIMS, using MeOH as CI ionizing gas.
Acaricidal activity in vitro P. cuniculi mites were isolated from naturally infected rabbits. The scabs and the cerumen, collected from 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. The essential oil of leaves of C. zeylanicum was diluted from the concentration of 10% to 0.03% (10%, 5%, 2.5%, 1.25%, 0.62, 0.31%, 0.16%, 0.010% 0.06%, 0.03%) in paraffin oil and 2.5 ml of each solution were added to Petri dishes. Six replications were made for each concentration. As untreated control, six Petri dishes containing only 2.5 ml of paraffin oil were used, while six Petri dishes containig 2.5 ml of a pyrethrum extract containing 25% of pyretrhins (AcaCerulen Rs, Teknofarma s.p.a., Torino), a drug used for topically treatment of psoroptic mange of the rabbit, represented the treated control. Plates were placed in a humidity chamber in saturated humidity conditions at 22 1C (High Performance Incubator 2800, Galli, Milan, Italy). After 24 h, mites were placed in clean Petri dishes containing 2.5 ml of paraffin oil and after further 24 h all the motionless mites were stimulated with a needle; lack of reactions and persistent immobility indicated their death (Perrucci et al., 2001). Data were statistically elaborated with the exact test of Fisher between the three groups (Glantz, 2003a) using Mc Graw Hill software (Glantz, 2003b). In addition, the Efficacy Dose 50 (ED50) was calculated (Previtera, 1976).
Acaricidal activity in vivo In order to assess the in vivo efficacy of C. zeylanicum essential oil, 18 New Zealand rabbits were experimentally infected with P. cuniculi with the method reported by Perrucci and collaborators (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 different efficacy 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 paraffin oil. Another group of six rabbits was treated with two ml of a mixture composed by 98% of saline solution and 2% of paraffin oil (untreated control group), and the last group of six
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rabbits was treated with two ml of Acacerulens (treated control group). All rabbits were treated two times, at the beginning of the treatment (0 day) and seven days later. 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 ¼ absence of scabs and/or mites; 0.5 ¼ irritation in ear canal but no mites observed; 1 ¼ small number of scabs in the ear canal, mites present; 2 ¼ external ear canal filled with scabs, mites present; 3 ¼ scabs in ear canal and proximal 14 of pinna, mites present; 4 ¼ 12 pinna filled with scabs, mites present; 5 ¼ 34 of the pinna filled with scabs, mites present; 6 ¼ all internal surface of the pinna full of scabs, mites present (Guillot and Wright, 1981). 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).
Table 1. Principal components of Cinnamomum zeylanicum essential oil (leaves)
Results
Treatment
Dead mites (%)
C. zeylanicum EO 0.03% C. zeylanicum EO 0.06% C. zeylanicum EO 0.10% C. zeylanicum EO 0.16% C. zeylanicum EO 0.31% C. zeylanicum EO 0.62% C. zeylanicum EO 1.25% C. zeylanicum EO 2.5% C. zeylanicum EO 5.0% C. zeylanicum EO 10.0% AcaCerulen Rsa Untreated control
5.88 16.67 69.66 96.77 100.00 100.00 100.00 100.00 100.00 100.00 100.00 5.16
Essential oil Table 1 shows the chemical composition of the essential oil of C. zeylanicum leaves. The composition of the essential oil of C. zeylanicum is quite variable, depending on the locality of growth and the organ used. However, many paper report eugenol as the main component of the oil of the leaves and cinnamaldehyde for the oil from the bark (Samarasekera et al., 2005; Variyar and Bandyopadhyay, 1989; Senanayake et al., 1978). This is in agreement with the sample used in this study (eugenol ¼ 76.1%). Other important constituents present in interesting concentration were linalool (3.7%), b-caryophyllene (6.7%), eugenyl acetate (2.8%), safrole (2.0%), benzyl benzoate (1,9%) and a-copaene (1.2%).
Acaricidal activity in vitro After 24 h of contact all concentrations of essential oil between 0.10 and 10% showed a good acaricidal efficacy respect to the untreated controls (po0:01) with a mortality of mites over 50%, but only the concentrations between 0.16 and 10% resulted so active as the reference drug (AcaCerulen Rs) with a mortality over 95% (Table 2). The activity of essential oil at 0.03% and 0.06% did not show significant differences respect to the untreated control group. The concentration of 0.0872% resulted the ED50 of used C. zeylanicum essential oil.
Composition
%
a-Pinene p-Cymene Limonene b-Phellandrene Linalool a-Terpineol Safrole Eugenol a-Copaene b-Caryophyllene (E)-Cinnamyl acetate a-Humulene Eugenyl acetate Caryophyllene oxide Benzyl benzoate
0.6 0.8 0.4 0.3 3.7 0.3 2.0 76.1 1.2 6.7 0.7 1.1 2.8 0.3 1.9
Table 2. Percentage of mortality of Psoroptes cuniculi treated with scalar concentrations (from 10% to 0.03%) of the essential oil of Cinnamomum zeylanicum, with Acacerulen Rs and untreated
a
Drug containing 25% of pyretrhins as active components.
Acaricidal activity in vivo The dose of C. zeylanicum (2 ml) at the concentration of 2.5% was selected in order to have a quantity of eugenol (40.5 mg) less than 100 mg/24 h, the toxic dose by topic use in the rabbits (http://ntp.niehs.nih.gov). Table 3 shows the main results of the in vivo trial. At the day 0 no difference appeared between the three groups, both for the parasitological (presence of mites) and clinical score (presence of scabs/crusts). The treatment with the essential oil gave a clinical cure in all rabbits in both ears. From day 7 after the beginning of the treatment, C. zeylanicum treated group and treated control group resulted negative for the presence of P. cuniculi mites and/or eggs and no difference resulted between their clinical scores. A reddening of the
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Table 3. Lesion scores found at 0, 7, 14 and 30 days after the beginning of the treatment, in Psoroptes cuniculi infected rabbits and treated with 2.5% of Cinnamomum zeylanicum essential oil or with Acacerulen Rs or left untreated Day
Ear
C. zeylanicum
AcaCerulen Rsc
Untreated control
0 7 14 30
L L L L
2.67 0.50a 0.25a 0.17a
2.83 0.17a 0.00a 0.00a
2.60 2.83b 2.83b 2.83b
0 7 14 30
R R R R
3.33 0.50a 0.17a 0.17a
3.33 0.17a 0.00a 0.00a
3.00 2.67b 2.00b 2.00b
R: right ear. L: left ear (after collection of all the scabs). a,b po0:05. c Drug containing 25% of pyretrhins as active components.
pinnae, observed in two of six rabbits treated with C. zeylanicum essential oil up to 30 days after the treatment, represented the only side effect of the oil. However, no significant differences regard to the degree of infestation and lesion scores resulted respect to the treated control group. The untreated control group remained infected until the end of the trial and the conditions of the left ears of three rabbits get worse. From day 7, in this latter group the degree of infestation was significantly higher respect to C. zeylanicum treated group and treated control group in both ears.
cuniculi showed both in vitro and in vivo by the essential oil of C. zeylanicum investigated in this study. The in vitro efficacy of this oil resulted so good as AcaCerulen Rs, the reference drug used in the present study, at concentrations (from 10% to 0.16%) much lower in respect to that of pyretrhins (25%) contained in AcaCerulen Rs. The in vivo trial confirmed the good acaricidial activity of this essential oil and showed the same efficacy both in presence and in absence of scabs in the ears of infected rabbits. The method of application for topical treatments of psoroptic mange, as in the case of Acacerulen Rs, often suggests to collect the scabs prior the treatment; however, in this study both essential oil and Acacerulen Rs showed that the presence of scabs do not reduce their therapeutic efficacy. These results indicate that C. zeylanicum oil could represent a possible alternative for the topical treatment of psoroptic mange in rabbits and encourage further studies to evaluate its efficacy 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 more persistent brushing of the pinnae observed in two of six rabbits treated with C. zeylanicum essential oil, could be explained with the high topical toxicity of eugenol in this animal species (http://ntp.niehs.nih.gov). However, concentrations much lower (from 1.25 to 0.31%) than 2.5% of this essential oil resulted equally efficacious in vitro in the present study; these findings suggest to test in vivo also these concentrations of C. zeylanicum oil because they could be equally efficacious but safer for treated animals.
Discussion The chemical composition of the essential oil of C. zeylanicum leaves investigated in this trial shows that this essential oil consists of several compounds with known acaricidal or insecticidal activity. 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 linalool and eugenol on this mite species both by direct contact and by contact only with their vapour phase. The acaricidal activity against P. cuniculi of linalool was demonstrated also in vivo on rabbits and goats (Perrucci et al., 1997). In a recent study (Yang et al., 2005), linalool and cinnamyl acetate showed insecticidal activity against Pediculus humanus capitis, thus confirming previous reports on the insecticidal properties of cinnamyl acetate (Cheng et al., 2004). The presence of these active principles could be the reason of the good acaricidal activity against P.
Reference Adams, R.P., 1995. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Carol Stream, Allured. Alawa, C.B.I., Adamu, A.M., Gefu, J.O., Ajanusi, O.J., Abdu, P.A., Chiezey, N.P., Alawa, J.N., Bowman, D.D., 2003. In vitro screening of two Nigerian medicinal plants (Vernonia amygdalina and Annona senegalensis) for anthelmintic activity. Vet. Parasitol. 113, 73–81. Arlian, L.G., 1996. Immunology of scabies. In: Wikel, S.K. (Ed.), The Immunology of HostEctoparasitic Arthropod Relationship, vol. 10. CAB International, Wallinford, UK, pp. 232–259 (Chapter 10). Baltz, R., 2001. Il potere curativo degli oli essenziali. 223 Ediz. Il punto di incontro, first ed. Vicenza, Italy. Bates, P.G., 1999. Inter- and intra-specific variation within the genus Psoroptes (Acari: Psoroptidae). Vet. Parasitol. 83, 201–217. Beesley, W.N., 1973. Control of arthropods of medical and veterinary importance. Adv. Parasitol. 7, 115–192.
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Chang, S.T., Cheng, S.S., 2002. Antitermitic activity of leaf essential oil and components from Cinnamomum osmophleum. J. Agric. Food Chem. 50 (6), 1389–1392. Cheng, S.S., Liu, J.T., Tsai, K.H., Chen, W.J., Chang, S.T., 2004. Chemical composition and mosquito larvicidal activity of essential oils from leaves of different Cinnamomum osmophloeum provenances. J. Agric. Food Chem. 52 (14), 4395–4400. Clark, A.M., Stephen, F.B., Cawley, G.D., Bellworthy, S.J., Groves, B.A., 1996. Resistance of the sheep scab mite Psoroptes ovis to propetamphos. Vet. Rec. 139, 451. Davies, N.W., 1990. Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicon and Carbowax 20M phases. J Chromatogr. 503, 1–24. Enan, E., 2001. Insecticidal activity of essential oils: octopaminergic sites of action. Comput. Biochem. Physiol. C Toxicol. Pharmacol. 130 (3), 325–337. Glantz, S.A., 2003a. Statistica Per Discipline Biomediche, fifth ed rev. Mc Graw-Hill, Milan, Italy. Glantz, S.A., 2003b. Statistica Per Discipline Biomediche. Mc Graw-Hill Companies s.r.l., Milan, Italy. Guillot, F.S., Wright, F.C., 1981. Evaluation of possible factors affecting degree of ear canker and number of Psoroptic mites in rabbits. Southwest. Entomol. 6 (3), 245–252. Halley, B.A., Vandenheuvel, W.J.A., Wislock, P.G., Herd, R., Strong, L., Wardhaugh, K., 1993. Environmental effects of the usage of avermectines in livestock. Vet. Parasitol. 48, 109–125 http://ntp.niehs.nih.gov/index.cfm?objectid ¼ 03610FA5-C828-304B-FE31F1182E8F764C10/7/2003. Jennings, W., Shibamoto, T., 1980. Qualitative Analysis of Flavor and Fragrance Volatiles by Glass Capillary Chromatography. Academic Press, New York. Lima, E.O., Gompertz, O.F., Giesbrech, A.M., Paulo, M.Q., 1993. In vitro antifungal activity of essential oils obtained from official plants against dermatophytes. Mycoses 36 (9–10), 333–336. Massada, Y., 1976. Analysis of Essential Oils by Gas Chromatography and Mass Spectrometry. Wiley, New York. O’Brien, D.J., 1999. Treatment of psoroptic mange with reference to epidemiology and history. Vet. Parasitol. 83, 177–185. Perrucci, S., Flamini, G., Cioni, P.L., Morelli, I., Macchioni, F., Macchioni, G., 2001. In vitro and in vivo efficacy of extracts of Artemisia verlotorum against Psoroptes cuniculi. Vet. Rec. 148, 814–815.
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Perrucci, S., Macchioni, G., Cioni, P.L., Flamini, G., Morelli, I., 1995. Structure/activity relationship of some natural monoterpenes as acaricides against Psoroptes cuniculi. J. Nat. Prod. 8 (58), 1261–1264. Perrucci, S., Cioni, P.L., Cascella, A., Macchioni, F., 1997. Therapeutic efficacy of linalool for the topic treatment of parasitic otitis caused by Psoroptes cuniculi in the rabbit and in the goat. Med. Vet. Entomol. 11, 300–302. Previtera, A., 1976. Elementi di Biostatistica Casa editrice Santini, vol. 2. Sarzana, La Spezia, Italy, p. 677. Raj, R.K., 1975. Screening of indigeneus plants for anthelmintic action against human Ascaris lumbricoides: part II. Indian J. Physiol. Pharmacol. 19 (1). Samarasekera, R., Kalhari, K.S., Weerasinghe, I., 2005. Mosquitocidal activity of leaf and bark essential oils of Ceylon Cinnamomum zeylanicum. S. J. Essent. Oil Res. 17, 301–303. Senanayake, U.M., Lee, T.H., Wills, R.B.H., 1978. Volatile constituents of cinnamon (Cinnamomum zeylanicum) oils. J. Agric. Food Chem. 26, 822–824. Singh, H.B., Srivastava, M., Singh, A.B., Srivastava, A.K., 1995. Cinnamomum bark oil fungitoxican against fungi causing respiratory tract mycoses. Allergy 50 (12), 995–999. Stenhagen, E., Abrahamsson, S., McLafferty, F.W., 1974. Registry of Mass Spectral Data. Wiley, New York. Swigar, A.A., Silverstein, R.M., 1981. Monoterpenes. Aldrich Chem. Comp., Milwaukee. Synge, B.A., Bates, P.G., Clark, A.M., Stephen, F.B., 1995. Apparent resistance of Psoroptes ovis to flumethrin. Vet. Rec. 137, 51. Tabak, M., Armon, R., Neem, I., 1999. Cinnamomum extracts inhibitory effect on Helicobacter pylori. J. Ethnopharmacol. 67 (3), 269–277. Variyar, P.S., Bandyopadhyay, C., 1989. On some chemical aspects of Cinnamomum zeylanicum. PAFAI J. 11, 35–38. Wrenn, W.J., 1996. Immune response to mange mites and chiggers. In: Wikel, S.K. (Ed.), The Immunology of Host–Ectoparasitic Arthropod Relationship. CAB INTERNATIONAL, Wallingford, UK., pp. 290–315 (Charter 11). Yang, Y.C., Lee, H.S., Lee, S.H., Clark, J.M., Ahn, Y.J., 2005. Ovicidal and adulticidal activities of Cinnamomum zeylanicum bark essential oil compounds and related compounds against Pediculus humanus capitis (Anoplura: Pediculidae). Int. J. Parasitol. Sept., 23. Yust, J., Fung, D.Y., 2002. Inactivation of Listeria monocitogenes Scott A 49594 in apple juice supplemented with cinnamom. J. Food Prot. 65 (10), 1663–1666.
Phytomedicine 14 (2007) 227–231 www.elsevier.de/phymed
SHORT COMMUNICATION
Efficacy of an essentifal oil of Cinnamomum zeylanicum against Psoroptes cuniculi G. Fichia, G. Flaminib, L.J. Zarallia, S. Perruccia, a
Dipartimento di Patologia Animale, Profilassi ed Igiene degli Alimenti–Faculty of Veterinary Medicine of Pisa University, Italy Dipartimento di Chimica Bioorganica e Biofarmacia–Faculty of Pharmacy of Pisa University, Italy
b
Abstract The aim of the present study was to investigate the in vitro and in vivo acaricidal effects of an essential oil of Cinnamomum zeylanicun leaves on Psoroptes cuniculi, a mange mite. In vitro, 2.5 ml of the essential oil diluted at different concentrations, from 10% to 0.03%, in paraffin oil were added to Petri dishes containing all motile stages of P. cuniculi. Mites mortality observed in these dishes was compared with that observed in untreated and treated (AcaCerulen Rs) control plates. In vivo, one group of six P. cuniculi infected rabbits was topically treated two times at seven days interval with two ml of the essential oil at the concentration of 2.5% in paraffin oil and compared with untreated and treated (AcaCerulen Rs) control groups of six rabbits each. After 24 h of contact, all concentrations of essential oil between 0.10 and 10% showed a good in vitro acaricidal efficacy if compared with the untreated controls (po0:01), but only the concentrations between 0.16 and 10% turned out as active as the drug. In vivo, the treatment with the essential oil cured all infested rabbits and no statistical differences were observed with the treated control group. r 2006 Elsevier GmbH. All rights reserved. Keywords: Psoroptes cuniculi; Cinnamomum zeylanicum; Essential oil; In vitro and in vivo acaricidal activity
Introduction Psoroptes cuniculi is a worldwide obligate ectoparasite mainly of rabbits, goats, horses, and sheep. It 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 crusts and scabs, that in the untreated animals can completely fill the external ear canal and the internal surface of the pinna (Bates, 1999). Therapy and control of both human scabies and animal mange are based mainly on the use of effective drugs and chemicals. At present, however, the use of drugs to Corresponding author. Tel.: +39 50 22 16949; fax: +39 50 22 16949. E-mail address: [email protected] (S. Perrucci).
0944-7113/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2006.01.004
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). Concern over toxicity (O’Brien, 1999) of many acaricides limits their use and reduces the number of safe effective products available. These problems have led to research efforts to discover new effective compounds. The identification of novel active plant derived natural compounds could increase the number of available chemotherapeutic agents, thereby reducing the frequency of resistance phenomena of pathogen arthropods and providing alternative drugs with greater acceptance, especially in terms of environmental safety (Beesley, 1973; Alawa et al., 2003). The human folk medicine reports the activity of the essential oil of Cinnamomum zeylanicum against bacteria, viruses and parasites and suggested its use for the
ARTICLE IN PRESS 228
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treatment of intestinal worms and skin parasites (Baltz, 2001). Modern studies showed that C. zeylanicum and its extracts have really anti-microbial and antiparasitic activities. The anti-bacterial activity of this drug was demonstrated against Helicobacter pylori (Tabak et al., 1999) and Listeria monocytogenes (Yust and Fung, 2002), while some studies reported the activity of essential oil on dermatophytes (Lima et al., 1993) and fungi involved in respiratory tract mycoses (Singh et al., 1995). The anti-helmintic activity of C. zeylanicum essential oil was demonstrated in vitro on Ascaris lumbricoides (Raj, 1975). In a recent study (Yang et al., 2005), C. zeylanicum oil and its main constituents showed ovicidal and adulticidal activities against the human head louse, Pediculus humanus capitis. The essential oil of another plant of the same genus, C. osmoploeum Kaneh, also showed a good insecticidal activity on mosquito larvae and termites (Chang and Cheng, 2002; Cheng et al., 2004). Thus this study was performed in order to evaluate the acaricidal properties of an essential oil of C. zeylanicum leaves on Psoroptes cuniculi.
Materials and methods Essential oil The essential oil of Cinnamomum zeylanicum leaves used in this study was a commercial sample by FLORA srl (Lorenzana, Pisa, Italy). GC/EIMS analyses were performed with a Varian CP-3800 gas-chromatograph equipped with a DB-5 capillary column (30 m 0.25 mm; coating thickness 0.25 mm) interfaced with a Varian Saturn 2000 ion trap mass detector. Analytical conditions were: injector and transfer line temperatures 220 and 240 1C respectively; oven temperature programmed from 60 1C to 240 1C at 3 1C/min; carrier gas helium at 1 ml/min; injection volume, 0.2 ml of a10% hexane solution; split ratio 1:30. The identification 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; Massada, 1976; Stenhagen et al., 1974; Swigar and Silverstein, 1981). Moreover, the molecular weights of all the identified substances were confirmed by GC/CIMS, using MeOH as CI ionizing gas.
Acaricidal activity in vitro P. cuniculi mites were isolated from naturally infected rabbits. The scabs and the cerumen, collected from 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. The essential oil of leaves of C. zeylanicum was diluted from the concentration of 10% to 0.03% (10%, 5%, 2.5%, 1.25%, 0.62, 0.31%, 0.16%, 0.010% 0.06%, 0.03%) in paraffin oil and 2.5 ml of each solution were added to Petri dishes. Six replications were made for each concentration. As untreated control, six Petri dishes containing only 2.5 ml of paraffin oil were used, while six Petri dishes containig 2.5 ml of a pyrethrum extract containing 25% of pyretrhins (AcaCerulen Rs, Teknofarma s.p.a., Torino), a drug used for topically treatment of psoroptic mange of the rabbit, represented the treated control. Plates were placed in a humidity chamber in saturated humidity conditions at 22 1C (High Performance Incubator 2800, Galli, Milan, Italy). After 24 h, mites were placed in clean Petri dishes containing 2.5 ml of paraffin oil and after further 24 h all the motionless mites were stimulated with a needle; lack of reactions and persistent immobility indicated their death (Perrucci et al., 2001). Data were statistically elaborated with the exact test of Fisher between the three groups (Glantz, 2003a) using Mc Graw Hill software (Glantz, 2003b). In addition, the Efficacy Dose 50 (ED50) was calculated (Previtera, 1976).
Acaricidal activity in vivo In order to assess the in vivo efficacy of C. zeylanicum essential oil, 18 New Zealand rabbits were experimentally infected with P. cuniculi with the method reported by Perrucci and collaborators (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 different efficacy 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 paraffin oil. Another group of six rabbits was treated with two ml of a mixture composed by 98% of saline solution and 2% of paraffin oil (untreated control group), and the last group of six
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rabbits was treated with two ml of Acacerulens (treated control group). All rabbits were treated two times, at the beginning of the treatment (0 day) and seven days later. 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 ¼ absence of scabs and/or mites; 0.5 ¼ irritation in ear canal but no mites observed; 1 ¼ small number of scabs in the ear canal, mites present; 2 ¼ external ear canal filled with scabs, mites present; 3 ¼ scabs in ear canal and proximal 14 of pinna, mites present; 4 ¼ 12 pinna filled with scabs, mites present; 5 ¼ 34 of the pinna filled with scabs, mites present; 6 ¼ all internal surface of the pinna full of scabs, mites present (Guillot and Wright, 1981). 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).
Table 1. Principal components of Cinnamomum zeylanicum essential oil (leaves)
Results
Treatment
Dead mites (%)
C. zeylanicum EO 0.03% C. zeylanicum EO 0.06% C. zeylanicum EO 0.10% C. zeylanicum EO 0.16% C. zeylanicum EO 0.31% C. zeylanicum EO 0.62% C. zeylanicum EO 1.25% C. zeylanicum EO 2.5% C. zeylanicum EO 5.0% C. zeylanicum EO 10.0% AcaCerulen Rsa Untreated control
5.88 16.67 69.66 96.77 100.00 100.00 100.00 100.00 100.00 100.00 100.00 5.16
Essential oil Table 1 shows the chemical composition of the essential oil of C. zeylanicum leaves. The composition of the essential oil of C. zeylanicum is quite variable, depending on the locality of growth and the organ used. However, many paper report eugenol as the main component of the oil of the leaves and cinnamaldehyde for the oil from the bark (Samarasekera et al., 2005; Variyar and Bandyopadhyay, 1989; Senanayake et al., 1978). This is in agreement with the sample used in this study (eugenol ¼ 76.1%). Other important constituents present in interesting concentration were linalool (3.7%), b-caryophyllene (6.7%), eugenyl acetate (2.8%), safrole (2.0%), benzyl benzoate (1,9%) and a-copaene (1.2%).
Acaricidal activity in vitro After 24 h of contact all concentrations of essential oil between 0.10 and 10% showed a good acaricidal efficacy respect to the untreated controls (po0:01) with a mortality of mites over 50%, but only the concentrations between 0.16 and 10% resulted so active as the reference drug (AcaCerulen Rs) with a mortality over 95% (Table 2). The activity of essential oil at 0.03% and 0.06% did not show significant differences respect to the untreated control group. The concentration of 0.0872% resulted the ED50 of used C. zeylanicum essential oil.
Composition
%
a-Pinene p-Cymene Limonene b-Phellandrene Linalool a-Terpineol Safrole Eugenol a-Copaene b-Caryophyllene (E)-Cinnamyl acetate a-Humulene Eugenyl acetate Caryophyllene oxide Benzyl benzoate
0.6 0.8 0.4 0.3 3.7 0.3 2.0 76.1 1.2 6.7 0.7 1.1 2.8 0.3 1.9
Table 2. Percentage of mortality of Psoroptes cuniculi treated with scalar concentrations (from 10% to 0.03%) of the essential oil of Cinnamomum zeylanicum, with Acacerulen Rs and untreated
a
Drug containing 25% of pyretrhins as active components.
Acaricidal activity in vivo The dose of C. zeylanicum (2 ml) at the concentration of 2.5% was selected in order to have a quantity of eugenol (40.5 mg) less than 100 mg/24 h, the toxic dose by topic use in the rabbits (http://ntp.niehs.nih.gov). Table 3 shows the main results of the in vivo trial. At the day 0 no difference appeared between the three groups, both for the parasitological (presence of mites) and clinical score (presence of scabs/crusts). The treatment with the essential oil gave a clinical cure in all rabbits in both ears. From day 7 after the beginning of the treatment, C. zeylanicum treated group and treated control group resulted negative for the presence of P. cuniculi mites and/or eggs and no difference resulted between their clinical scores. A reddening of the
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Table 3. Lesion scores found at 0, 7, 14 and 30 days after the beginning of the treatment, in Psoroptes cuniculi infected rabbits and treated with 2.5% of Cinnamomum zeylanicum essential oil or with Acacerulen Rs or left untreated Day
Ear
C. zeylanicum
AcaCerulen Rsc
Untreated control
0 7 14 30
L L L L
2.67 0.50a 0.25a 0.17a
2.83 0.17a 0.00a 0.00a
2.60 2.83b 2.83b 2.83b
0 7 14 30
R R R R
3.33 0.50a 0.17a 0.17a
3.33 0.17a 0.00a 0.00a
3.00 2.67b 2.00b 2.00b
R: right ear. L: left ear (after collection of all the scabs). a,b po0:05. c Drug containing 25% of pyretrhins as active components.
pinnae, observed in two of six rabbits treated with C. zeylanicum essential oil up to 30 days after the treatment, represented the only side effect of the oil. However, no significant differences regard to the degree of infestation and lesion scores resulted respect to the treated control group. The untreated control group remained infected until the end of the trial and the conditions of the left ears of three rabbits get worse. From day 7, in this latter group the degree of infestation was significantly higher respect to C. zeylanicum treated group and treated control group in both ears.
cuniculi showed both in vitro and in vivo by the essential oil of C. zeylanicum investigated in this study. The in vitro efficacy of this oil resulted so good as AcaCerulen Rs, the reference drug used in the present study, at concentrations (from 10% to 0.16%) much lower in respect to that of pyretrhins (25%) contained in AcaCerulen Rs. The in vivo trial confirmed the good acaricidial activity of this essential oil and showed the same efficacy both in presence and in absence of scabs in the ears of infected rabbits. The method of application for topical treatments of psoroptic mange, as in the case of Acacerulen Rs, often suggests to collect the scabs prior the treatment; however, in this study both essential oil and Acacerulen Rs showed that the presence of scabs do not reduce their therapeutic efficacy. These results indicate that C. zeylanicum oil could represent a possible alternative for the topical treatment of psoroptic mange in rabbits and encourage further studies to evaluate its efficacy 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 more persistent brushing of the pinnae observed in two of six rabbits treated with C. zeylanicum essential oil, could be explained with the high topical toxicity of eugenol in this animal species (http://ntp.niehs.nih.gov). However, concentrations much lower (from 1.25 to 0.31%) than 2.5% of this essential oil resulted equally efficacious in vitro in the present study; these findings suggest to test in vivo also these concentrations of C. zeylanicum oil because they could be equally efficacious but safer for treated animals.
Discussion The chemical composition of the essential oil of C. zeylanicum leaves investigated in this trial shows that this essential oil consists of several compounds with known acaricidal or insecticidal activity. 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 linalool and eugenol on this mite species both by direct contact and by contact only with their vapour phase. The acaricidal activity against P. cuniculi of linalool was demonstrated also in vivo on rabbits and goats (Perrucci et al., 1997). In a recent study (Yang et al., 2005), linalool and cinnamyl acetate showed insecticidal activity against Pediculus humanus capitis, thus confirming previous reports on the insecticidal properties of cinnamyl acetate (Cheng et al., 2004). The presence of these active principles could be the reason of the good acaricidal activity against P.
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