Essential oil of parsley and fractions to in vitro control of cattle ticks and dengue mosquitoes

1 Programa de Pós-Graduação em Biotecnologia Aplicada à Agricultura. Universidade Paranaense, Umuarama, Praça Mascarenhas de Moraes, 4282, 87502-210 Umuarama, Paraná, Brasil. 2 Programa de Pós-Graduação em Promoção da Saúde e em Tecnologias Limpas. Centro Universitário de Maringá, UniCESUMAR, Av. Guerdner no 1610, Jd. Aclimação, Maringá, PR, Brasil. 3 Programa de Pós-Graduação em Ciência Animal. Universidade Paranaense, Umuarama, Praça Mascarenhas de Moraes, 4282, 87502-210 Umuarama, Paraná, Brasil.


INTRODUCTION
The veterinary industry is continuously challenged to develop new chemical solutions because of pests' resistance to anti-parasitic products.Acaricide resistance is a big issue to the Brazilian cattle livestock since ticks are responsible for significant economic losses and transmission of hemoparasites (Olivo et al., 2008).The continuous use of chemical acaricide, such as pyrethroid, organophosphate, amitraz, ivermectin and fipronil (Kafle et al., 2012), has caused tick resistance.The resistance to chemical larvicides is also a serious problem in the combat of Aedes aegypti L. (Diptera, Culicidae).For the culicidae larva control, chemical insecticides such as temephos ® , malathion ® and fenitrothion ® are the main products applied currently.These chemicals cause mosquito resistance repeatedly described in Brazil and worldwide (Magalhães et al., 2010).Furthermore, environmental impacts caused by these substances have demanded urgent alternatives for manufacturing commercial acaricides and larvicides with environmental appeal (Chagas et al., 2003).
The Petroselinum crispum (Mill.)Nym.ex A. W. Hill belongs to the Apiaceae family.It is a well-known spice plant widely cultivated in Brazil.The parsley is used in both the cosmetic industry and alternative medicine.The parsley's essential oil (EO) obtained from seeds and leaves has medicinal properties as anti-hepatotoxic, antihypertensive, anti-coagulant (Petropoulos et al., 2009), anti-microbial (Petropoulos et al., 2010) and anti-oxidant (Zhang et al., 2006).In the parsley's EO there are two main phenylpropanoids: apiole and myristicin (Petropoulos et al., 2009).Song et al. (2011) showed that apiole obtained from Petroselinum sativum seeds has high acaricidal activity against the house-dust and storedfood mites (Dermatophagoides farinae, Dermatophagoides pteronyssinus and Tyrophagus putrescentiae).A number of phenylpropanoid derivatives (isoeugenol, eugenol and safrole) also had acaricidal activity against D. farinae and D. pteronyssinus (Chu et al., 2011). Ferraz et al. (2010) suggested that phenylpropanoids of the EO obtained from Piper amalago, Piper mikanianum and Piper xylosteoides were the main reason for acaricidal activity against R. (B.) microplus.However, there was no conclusive study on the acaricidal and larvicidal activities of apiole and phenylpropanoids against cattle ticks (R. (B.) microplus) and dengue mosquitos (A. aegypti).
Therefore, the aim of this study was to isolate the main chemical compounds of the EO of P. crispum, which is a source of phenylpropanoides and apiole, and evaluate its acaricidal and larvicidal activities against R. (B.) microplus and A. aegypti.Alternative solutions like the insect and tick control from the parsley's EO reported in this study are highly demanded for the production of pro-environmental products to control pests as well as to avoid pest resistance.

MATERIALS AND METHODS
Plant material and essential oil P. crispum was identified and the exsiccate (numbered 192) was maintained at the Educational Herbarium of the Paranaense University (Herbário Educacional da Universidade Paranaense -HEUP).The crop was cultivated in the Medicinal Garden of the Paranaense University (Horto Medicinal da Universidade Paranaense), Umuarama city, Paraná, Brazil, at coordinates S 23° 46, 225' and WO 53° 16 730', at 391 m high.The planting occurred in January, 2013.Non-tillage in lines was the crop system used, which is recommended for short-cycle and medium-to-small species (up to 1 m height) like P. crispum.Three planting lines were used and the spacing between them was 0.40 m.Spacing between plants was 0.30 m.Three seeds were planted per hill at a depth of 0.02 m (Kurowska and Galaska, 2006).Plants were harvested at 30 cm height and at the peak of flowering, when the inflorescence was visible (Kurowska and Galaska, 2006).Aerial parts were cut at 2 to 3 cm above soil, yellow and crushed leaves were removed.Plant material was harvested in the morning, the dew and rainy days were avoided.Plant material was subjected to hydrodistillation in Clevenger apparatus by 2 h (Stankovic et al., 2004) and then the EO was removed from equipment by a Pasteur pipette, filtered with anhydrous sodium sulfate (NA2SO4) and stored in amber vials at -20°C.

Chemical composition of essential oil
Chemical composition of EO and their FRs was obtained by GC-MS with 5973 N Mass Selective Detector and Agilent 6890N GC System equipped with a 5% phenylmethylsiloxane capillary column (DB-5) (30 m × 0.25 mm × 0.25 µm film thickness), using helium carrier gas at 4.8 ml/min and 2.1:1 split mode.The temperatures of transference line, ion source and quadrupole were maintained at 320, 230 and 150°C, respectively.The column was programmed to heat at 6°C/min, initially from 40°C to reach 300°C (maintained for 1 min).The total time of analysis was of 44.33 min.The mass spectra *Corresponding author.E-mail: cristianigazim@unipar.br.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License were obtained in a range of 50 to 550 (m/z) provided through scanmode with solvent delay time of 3.5 min.In addition to GC-MS results, eo chemical compounds were also identified by comparing retention indexes (RI) using a homologous series of nalkanes (C7-C26).Mass spectra were then compared with the Wiley 275 L mass spectral library and literature (Adams, 2007).

Acaricidal effect on the R. (B.) microplus
The acaricidal effect of EO and FRs (FR1, FR2, FR3 and FR4) of P. crispum on R. (B.) microplus was determined by the adult immersion test (AIT) originally developed by Drummond et al. (1973); and also by the larval pocket test (LPT) described by Stone and Haydock (1962).The EO of P. crispum and its FRs were diluted in water solution added with 2% of polysorbate 80 (v/v).The 2% polysorbate 80 (v/v) solution was used for negative control.The positive control was made with a commercial acaricide containing 150,000 µg/ml of cypermethrin, 250,000 µg/ml of chlorpyrifos and 100,000 µg/ml of citronellal.The commercial acaricide was prepared in 2% polysorbate 80 at 1,250 µg/ml.In the AIT, approximately 100 fully engorged females of R. (B.) microplus were collected from infested cattle in a farm at Umuarama, Parana, Brazil.Then, ticks were cleaned with water purified and selected according to their aspects of normal appearance, regular motility, integrity of body, and full engorgement.Groups of 30 females each were immersed in different concentrations of EO for 5 min.Each female was individually weighed and maintained in petri dishes at 28°C and 80% relative humidity (RH) for 14 days to monitor oviposition.Egg masses were obtained from each female, placed in sample tubes, and kept at 28°C and 80% (RH) for 21 days to hatching.Larvae were killed with sulfuric ether and counted with magnifying glass in order to obtain the rate of hatching.From the tick mass, egg mass and hatching rate data, the reproductive efficiency was computed as RE = [(egg mass (g) × % hatching × 20,000)/(tick mass)(g)]; and product efficiency as PE = [(RE (negative control) × RE (treatment group) × 100)/RE(negative control)] (Drumond et al., 1973).For the larval packet test (LPT), 100 larvae were placed between filter papers (2 × 2 cm) freshly impregnated with EO dilutions to form envelopes, which were sealed and kept in petri dishes.The envelopes were maintained at 28°C and 80% (RH).After 24 h, living larvae were isolated from dead with the help of a magnifying glass.All tests were performed in triplicate and larvae mortality was computed as the following: LM = [(dead larvae × 100)/(total larvae)]; and mean mortality as MM = (LM 1st repetition + LM 2nd repetition + LM 3rd repetition)/3.

Larvicidal effect on A. aegypti
The third stage larvae of A. aegypti were used for bio-tests.They were originated from the Control Center of Endemic Diseases Transmitted by Vectors -Umuarama City, Paraná.The EO and FRs from P. crispum were diluted on 2% polysorbate 80 (v/v) solutions.The negative control was a 2% polysorbate 80 solution and the positive control was made with an organophosphate-based temephos ® .Ten third stage larvae of A. aegypti were taken on Pasteur pipette and placed into 250 ml vials with 10 ml solution containing different concentrations of EO or the isolated.The number of dead larvae was counted after 24 h (Cavalcanti et al., 2004).

Anticholinesterase activity
The method described by Yang et al. (2009) was used to determine the anticholinesterase activity through an autobiographic assay.The EO and FRs obtained by CC were solubilized in methanol indifferent concentrations from 100,000 to 0.012 µg/ml.The samples were applied to aluminum chromatoplates (10 × 10 cm, silica Gel 60 F254 with 0.2 mm thickness) where a decreasing series of spots with different amounts of EO and FRs was obtained.For chromatographic development, a mixture of 9:1 dichloride methane/methanol was utilized.After sample migration and drying to eliminate solvents, an enzyme solution (Acetylcholinesterase in Tris buffer solution) was sprayed on the chromatoplate, followed by a solution of α-naphthyl acetate.Partial drying was done in the plate that was kept in water bath (37°C) for 20 min for enzyme incubation.Then, the chromatoplate was sprayed with colorimetric Fast Blue B salt reagent resulting in a purple color surface on the chromatoplate for 5 min.By analyzing the absence of purple color, the anticholinesterase activity was determined.

Statistical analysis
Tests were performed in triplicate.Acaricidal and larvicidal activities were analyzed by frequency distribution with Bioestat ® (version 5.3) (Kamirauá Institute for Sustainable Development, Manaus, Tefé).Values for LC50 and LC99.9 were obtained in Microsoft Excel ® (versionExcel ® 2010) (Microsoft, Redmond, Washington, United States) by linearization of the response curve.The confidence intervals were calculated using Assistat (version 7.7) INPI 0004051-2 developed by prof.Dr. Francisco de A. S. e Silva from Federal University of Campina Grande, Brazil distributed for free.

Acaricidal and larvicidal effect on R. (B.) microplus
Results to acaricidal activity (Table 2) indicate that the EO of P. crispum at 100,000 µg/ml caused 100% mortality in the tick females; similar results were obtained with the positive control that is a commercial mixture of 150,000 µg/ml of cypermethrin, 250,000 µg/ml of chlorpyrifos and 10,000 µg/ml of citronellal.The hatching rates were reduced by the increasingly oil concentrations: at 100,000 µg/ml of EO no hatching larvae was found (Table 2).The product efficiency ranged from 100 to 58.18% as the concentrations varied from 100,000 to 50,000 µg/ml.Within the concentration range from 40,000 to 12,500 µg/ml, product efficiency was lower than 50%.
Lethal concentrations (LC 50 and LC 99.9 ) of parsley's EO on engorged female were of 73,400 µg/ml (71,240 to 78,450) and of 109,760 µg/ml (106,540 to 117,320), respectively.The EO and FRs of P. crispum were active in the control of R. (B.) microplus larvae (Tables 3 and 5).The FR4, which presented apiole as the main compound (96.80%),showed the highest larvicidal activity with LC 50 of 0.82 µg/ml and LC 99.9 of 9.62 µg/ml.These results were similar to the positive control that presented LC 50 and LC 99.9 of 0.45 and 4.62 µg/ml, respectively.

Anticholinesterase activity
Preliminary lab tests for EO and FRs demonstrated that these substances have inhibited the acetyl cholinesterase activity (Table 6).The quantitative analysis of acetylcholinesterase (AChE) activity revealed that the highest enzymatic inhibition occurred for EO and FR4 at 0.012 µg/ml, which agrees with the highest activities against A. aegypti larvae.The FR3 showed low potential for enzyme inhibition, suggesting that the larvicide action against A. aegypti is related to another mechanism as perhaps the opening of the sodium channels (Soderlund et al., 2002).

DISCUSSION
In this work, the main compound of parsley's EO was the apiole.Kurowska and Galazka (2006) found α-pineno (32.0%) as the major constituent of the EO of P. crispum cultivated in Poland, followed by β-pineno (19.0%), myristicin (18.3%) and apiole (10.1%).Stankovic et al.  (7.65) as the main compounds of parsley's EO from the local market in Beirut, Lebanon.This diverse chemical composition could be a result of genetic and environmental factors that can affect the secondary metabolism.Among these factors, it is worth mentioning the plant germplasm diversity, their ages and development stages, as well as their interactions with microorganisms and insects.In addition, abiotic factors like luminosity, temperature, rainfall, nutrition, time of the day for harvest and post-harvest techniques are also important to form the chemical composition of Eos (Morais and Castanha, 2012).Although many variations in the EO composition of P. crispum were already reported in literature, the apiole and myristicin have always appeared as the major constituents of parsley's EO, which agrees with the results of this work.
Literature has shown a series of advantages in the use of EO for the control of ticks.Among them, low toxicity to mammalian, rapid degradation and slow development of resistance are common benefits of acaricides extracted from plants.Such benefits make bio-acaricides commercially attractive to control R. (B.) microplus with reduced hazard to environment.Essential oils of Eucalyptus citriodora, Eucalyptus globules and Eucalyptus staigeriana (Myrtaceae) have showed in vitro 100% control over cattle ticks at concentrations of 175,000, 150,000 and 125,000 µg/ml, respectively (Chagas et al., 2002).Olivo et al. (2008) observed in vitro 89.5% control at 100,000 µg/ml of EO of Cymbopogon nardus (Poaceae) on the cattle tick larvae.In our work, the efficiency of P. crispum EO over females of R. (B.) microplus (100% with 100,000 µg/ml) was higher than those reported in literature.Thus, the EO of P. crispum presented considerable acaricide activity on the cattle tick observed in vitro and such activity should be deeply investigated for field applications to control R. (B.) microplus.
In our work, apiole had the highest larvicidal activity with LC 50 of 0.82 µg/ml and LC 99.9 of 9.62 µg/ml.The EO of Pipper mikanianum and Pipper xylosteoides (with 67.89 and 48.53% of phenylpropanoides, apiole and safrole, respectively) showed LC 50 of 2.33 µg/ml and 6.15 µg/ml against R. (B.) microplus larvae (Ferraz et al., 2010).For EO of Pipper aduncum (with 94.84% of dillapiole) just 0.1 µg/ml induced 100% mortality in larvae of R. (B.) microplus (Silva et al., 2009).Single chemical control is a concern, since populations of R. (B.) microplus are acquiring resistance to chemical acaricides.The chemical resistance is often found in regions favorable to tick development, as the major countries of Latin America, Central America, Australia and South Africa.In the Brazilian States of São Paulo, Mato Grosso do Sul, Rio Grande do Sul and Paraná, where the largest livestock producers are located, the tick populations are becoming increasingly resistant to different conventional acaricides (Madalena et al., 2012).Therefore, the EO of parsley and its main constituent apiole could be useful as part of an integrated pest management.Future studies are in progress by our group to search for synergistic effects of essential oils and chemical acaricides to increase the efficacy of these compounds in the control of R. (B.) microplus.
Research results on larvicidal activity of essential oils and isolated compounds to control A. aegypti are scarce.Rafael et al. (2008) isolated the dillapiole from leaves of Piper aduncum and the insecticidal activity against larvae and pupae of A. aegypti occurred at 200 and 400 µg/ml, respectively.In our work, the FR4 (with 96.80% of apiole) presented a small LC 99.9 value of 0.07 µg/ml, which was lower than that reported by Rafael et al. (2008).So apiole seems to have a real potential to help the A. aegypti control; note that Rafael et al. (2008) also found that apiole performed better than the positive control.For EO of Citrus limon (with high limonene content) was reported low insecticidal activity against A. aegypti larvae with LC 50 of 95.8 mg/ml and LC 90 of 102.7 mg/ml (Furtado et al., 2005).Also, for limonene obtained from EO of Clausena excavate, Cheng et al. (2009) reported LC 50 of19.4µg/ml.In our work, the FR1 (in which p-mentha represents 80.13%) showed LC 99.9 of 1.28 µg/ml, which Table 5. Means of lethal concentrations (LC50 and LC99.9 in µg/ml) of the essential oil of Petroselinum crispum and its fractions applied to larvae of R. (B.) microplus and Aedes aegypti followed by confidence interval by probit analysis.(10.49-11.55) 3,703.55 (3,595.00-3,958.80) 40,125.42 (38,949.35-42,890.87) 3,753.17 (3,643.16-4,011.84) 20,777.31 (20,168.33-22,209.29) 247.37 (240.12-264.42 was lower than the values reported in the literature.So the larvicidal activity of FR1 to A. aegypti seems to be related to the presence of pmentha.However, there is no report on the larvicidal activity of the p-mentha against A. aegypti.The EO of Pink Chablis bluebeard (Caryopteris × clandonensis 'Durio') compounded by 4.6% of trans-p-mentha-2,8-dien-1-ol, 4.5% of trans-p-mentha-1( 7),8-dien-2-ol and 4.0% of cis-pmentha-2,8-dien-1-ol exhibited weak activity against mosquito larvae (Blythe et al., 2015).Although the larvicidal effect of FR1 (80.13% of pmentha compounds) to A. aegypti was 18 times less effective than FR4 (96.80% of apiole).Simas et al. (2004) showed that phenylpropanoids and sesquiterpene alcohols have higher larvicidal effect against A. aegypti.Myristycin, the main constituent of FR3, is the phenylpropanoid responsible by the larvicidal activity of this fraction.The other phenylpropanoids showed larvicidal activity to A. aegypti with LC 50 values as the following: 28 µg/ml for anethole (Morais et al., 2006), 67 µg/ml for E-methyl-cinnamate and 60 µg/ml for eugenol.Knio et al. (2008) evaluated the Parsley's seed oil in the control of the seaside mosquito (Ochlerotatus caspius).The EO results showed LC 50 values of 34.3 and 23.4 mg/ml and LC 90 values of 62.2 and 42.2 mg/ml, for 24 and 48 h, respectively.
In this present work, an efficient in vitro control of larvae of A. aegypti was observed for the reduced concentrations of P. crispum EO and its FRs, mainly for FR4 (apiole).Thus, these substances are promising to substitute or complement synergistically chemical insecticides such as temephos ® , malathion ® , fenitrothion ® on the control of A. aegypti mosquitoes.Importantly, the use of these commercial insecticides is the main action adopted by the Public Health Programs for mosquito control.However, chemical products cause pollution in the environment and also insect resistance to conventional insecticides was already observed in different regions of the world, including Brazil (Magalhães et al., 2010).
In this study, the high acaricide and larvicide potential of the parsley's EO and its FRs to control R. (B.) microplus and A. aegypti may be related to the inhibition of AChE (Souza et al., 2012).This enzyme hydrolyzes the acetylcholine neurotransmitter, being the major action of commercial acaricides, such as organophosphates chlorpyrifos.
Differently, pyrethroids can prevent the closure of the voltagegated sodium channels in the axonal membranes so that the nerves cannot repolarize, leaving the axonal membrane permanently depolarized and thereby paralyzing the organism (Soderlund et al., 2002).Many essential oils and primarily monoterpenes (Picollo et al., 2008), sesquiterpenes (Fujiwara et al., 2010) and phenylpropanoids (Dohi et al., 2009) have AChE inhibitory activity.This could explain the high anticholinesterase activity for FR4, composed mostly for phenylpropanoid apiole.The phenylpropanoids were also the major compounds of EO (52.07%).

Conclusion
The EO and the FRs containing apiole and myristicin as the major compounds of P.crispum presented larvicidal and acaricidal activitiy on the R. (B.) microplus (cattle tick) and high larvicide potential to control the A. aegipty (dengue mosquito).The parsley's EO is promising to be used in the bio-control of R. (B.) microplus and A.aegipty.Further studies in living animals at field conditions are needed to confirm the acaricide and larvicide potential of the parsley's EO.In addition, proper concentrations and mode of application should be tested by exploring surfactant and synergistic effects from the mixture with other acaricides and larvicides.
da Universidade Federal do Rio de Janeiro UFRJ-IQ) for their support.

Table 1 .
Chemical composition of the essential oil obtained from leaves and stems of P. crispum.