(Euphorbiaceae) Against the Cattle Tick

Veterinary Parasitology 192 (2013) 292–295

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Short communication

Chemical composition and efﬁcacy of dichloromethane extract of

Croton sphaerogynus Baill. (Euphorbiaceae) against the cattle tick

Rhipicephalus microplus (Acari: Ixodidae)

a,∗ a b c

Adne A. Righi , Lucimar B. Motta , Guilherme M. Klafke , Paula C. Pohl ,

a a a d

Cláudia M. Furlan , Deborah Y.A.C. Santos , Maria L.F. Salatino , Giuseppina Negri ,

e a

Marcelo B. Labruna , Antonio Salatino

Botany Department, Institute of Biosciences, University of São Paulo, Brazil

Parasitology Department, Institute of Biomedical Sciences, University of São Paulo, Brazil

Biotechnology Center, Federal University of Rio Grande do Sul, Brazil

Psycobiology Department, Federal University of the State of São Paulo, Brazil

Preventive Veterinary Medicine and Animal Health Department, Faculty of Veterinary Medicine, University of São Paulo, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history:

The cattle tick Rhipicephalus microplus is widely distributed in tropical and subtropical

Received 12 April 2012

regions, causing high economic impact on cattle production. The control of tick infestations

Received in revised form 24 October 2012

is regarded worldwide as critical and has been based on the use of organophosphates, syn-

Accepted 6 November 2012

thetic pyretroids, amitraz and recently ivermectin and ﬁpronil. The present study reports

the analysis by gas chromatography/mass spectrometry of the constituents of leaf extracts

Keywords:

of Croton sphaerogynus and results of acaricidal activity against the cattle tick R. microplus.

Medicinal plants

Abietane The larval package test using the serial dilutions 0.625%, 1.25%, 2.5%, 5.0%, 10.0% and 20.0%

(v/v) gave mortality rates 2.25%, 8.26%, 8.81%, 24.80%, 83.66% and 99.32%, respectively.

Acaricidal activity

Euphorbiaceae Relevant constituents identiﬁed were abietanes, podocarpenes and clerodane type furano

Rhipicephalus microplus diterpenes. The present work may represent a possibility of attainment of natural sub-

stances useful for the control of R. microplus.

1. Introduction in Latin-America, considering direct production losses and

the cost of chemical control (FAO, 2004).

The cattle tick Rhipicephalus microplus is one of the most Chemical control has been the most used method for

important ectoparasites of cattle and is widely distributed controlling ticks. However, genetic resistance to acari-

in tropical and subtropical regions (Pirali-Kheirabadi and cides has been a serious problem in Brazil and other

Teixeira da Silva, 2010). It causes high economic impact on countries. Resistance has been conﬁrmed toward chlo-

cattle production by reducing weight gain, milk produc- rines, organophosphates, synthetic pyretroids, amitraz,

tion and by transmitting pathogens that cause babesiosis and recently ivermectin and ﬁpronil (Klafke et al., 2010).

and anaplasmosis (Castro-Janer et al., 2009). Parasitism by In the last few years, FAO has classiﬁed Brazil as the third

cattle tick is responsible for US$ 1 billion losses per year major country in the usage of pesticides. Coincidentally,

Brazil is the third country with the highest human mor-

tality rate due to cancer (Monteiro et al., 2009). Therefore,

the search for new strategies for tick control can be seen as

∗ a matter of public health. Plant compounds active against

Corresponding author. Tel.: +55 11 3091 7532; fax: +55 11 3091 7416.

E-mail address: [email protected] (A.A. Righi). ticks have been repeatedly reported (see e.g. Olivier et al.,

A.A. Righi et al. / Veterinary Parasitology 192 (2013) 292–295 293

2010), and are considered as an alternative to synthetic 10.0% and 20.0%, v/v) were prepared in a 2:1 (v/v) mixture

pesticides (Cetin et al., 2010). of trichloroethylene (TCE) and olive oil (OO). Each concen-

Croton is a genus of Euphorbiaceae, comprising over tration was considered a treatment. Each treatment and

1200 species, widespread in tropical regions of the new and control was performed with 3 replicates. A 75 mm × 85 mm

old worlds. Several species are widely used in traditional ﬁlter paper (Whatman No. 1, Whatman Inc., Maldstone,

medicine in Africa, Asia and South America. Compounds England) was impregnated with 0.67 mL of each solution

obtained from Croton aromaticus, Croton cajucara, Cro- using a micropipette. The material was left drying for 24 h

◦

ton californicus and Croton linearis have been pointed out at 25 C to allow TCE evaporation. After drying, the ﬁlter

as efﬁcient insecticides or growth inhibitors of Helio- papers were folded in the middle and sealed on the sides

this virescens (Lepidoptera) (Salatino et al., 2007). Croton with metal clips to form the packets. Approximately 100

sphaerogynus Baill. is a tree from the Southeast Brazilian larvae were transferred to each packet using a paintbrush.

Atlantic Forest. The species is closely related to C. cajucara, The packets were sealed with a third clip and incubated at

◦

a species with bioactive substances and popularly known 27–28 C and 80–90% relative humidity. The control group

as “sacaca or sacaquinha” in the Amazon region (Caruzo was exposed to the ﬁlter paper impregnated with extract-

et al., 2011). free TCE-OO. After 24 h, larval mortality was determined by

The purpose of this study was to evaluate the acarici- counting the number of dead and alive individuals within

dal activity of dichloromethane extracts of C. sphaerogynus each packet. Larvae that were paralyzed or moving only

against R. microplus and to identify their main constituents. their appendages, with no walking capability, were con-

sidered dead. The mortality rate was obtained according to

2. Materials and methods the following formula:

dead larvae

2.1. Plant material

Mortality rate (%) = × 100

total larvae

Leaves of C. sphaerogynus were collected in April 2009, in

the Atlantic rainforest area in the municipality of Itanhaém,

2.5. Statistical analysis

state of São Paulo (southeast Brazil). A voucher specimen

(Cordeiro 3087) was deposited in the Herbarium “Maria

All data were subjected to statistical analysis including

Eneyda P.K. Fidalgo” of the Institute of Botany (SEMA, São

the calculation of the mean and standard error. Differences

Paulo, Brazil).

between control and treated groups were tested for signiﬁ-

cance using probit analysis with Polo-Plus (LeOra Software,

2.2. Preparation of plant extracts

2004). For this test the lethal concentration for 50% (LC50)

and for 95% (LC95) mortality was calculated.

Dried powdered leaves (30 g) were sequentially

extracted in Soxhlet with hexane, dichloromethane and

methanol for 6 h with each solvent. The extracts were 3. Results

concentrated under reduced pressure and evaporated to

dryness under nitrogen ﬂow. The dichloromethane exhibited a high diversity of

constituents. Major compounds detected were abieta-

2.3. Gas chromatography/mass spectrometry analyses 8,11-diene-3-one (20%), abieta-8,11,13-trien-12-ol

(11%) and podocarp-7-ene,13-methyl-13-vinyl-3-

Identiﬁcation of the extract constituents was one (12%) (Table 1). Most identiﬁed constituents

␮

performed with 1 L of 2 mg/mL solutions by gas are abietane derivatives, such as abieta-8,11,13-

chromatography/mass spectrometry, using a system triene, abieta-8,11-diene, abieta-8,11-diene-3-one,

Agilent 6890N GC/5975B MSD equipped with a 7683B abieta-8,11,13-triene-3-ol, abieta-8,11-diene-3-ol, abieta-

autosampler, DB-5HT fused silica capillary column 8,11,13-trien-12-ol, 13-hydroxy-abieta-8,11-dien-7-one,

× × ␮

(30 m 0.32 mm 0.10 m) and helium as carrier gas at 11-hydroxy-abieta-8,11,13-trien-7-one, abieta-6,8,11,13-

1 mL/min. The temperature program of the column started tetraene-3,12-diol and abieta-8,11,13-trien-6,14-diol.

◦ ◦ ◦

at 150 C (1 min), rising 6 C/min to 320 C; injector and Podocarpenes are also important constituents of the

◦

detector temperatures were 250 C. Mass spectra were extract, such as podocarp-7-ene,13-methyl-13-vinyl,

obtained at 70 eV and scans ranged from 40 to 450 Da at 2 podocarp-7-ene,13-methyl-13-vinyl-3-one and podocarp-

scans/s (modiﬁed from Tansakul and De-Eknamkul, 1998). 7-ene-3-ol,13-methyl-13-vinyl. Some clerodanes were

Identiﬁcation of the compounds followed comparisons of detected as minor constituents, such as a crotonin and a

mass spectra with NIST library and literature data. dehydrocrotonin derivatives (Table 1).

The mortality rates of the serial dilutions of the

2.4. Bioassays dichloromethane extract are shown in Fig. 1. Mortality

rates increased with the concentration of the extract: the

Unfed larvae (14–21 days old) of R. microplus of the mean lowest rate (2.25% mortality) corresponded to the

strain POA were used in the bioassays. The acaricidal activ- 0.625% concentration, while at the 20.0% concentration the

ity was evaluated with the larval package test according to mean rate observed was 99.32% (Fig. 1). Taking into consid-

Ducornez et al. (2005) with adaptations. Six serial dilutions eration the mean rates of mortality corresponding to 10%

of the dichloromethane extracts (0.625%, 1.25%, 2.5%, 5.0%, and 20% concentrations (82% and 99.32%, respectively), the

294 A.A. Righi et al. / Veterinary Parasitology 192 (2013) 292–295

Table 1

Main constituents of leaf extracts of Croton sphaerogynus and respective GC/EIMS data. RT, retention time (min); MM, molecular mass; MF, molecular

formula; N.C., non-characterized.

RT MM (MF) MS data: m/z (intensity, %) Characterization Relative amount (%)

6.48 272 (C20H32) 272 (70), 257 (90), 243 (40), 229 (920), 187 Podocarp-7-ene, 0.5

(50), 133 (60), 119 (70), 109 (100) 13-methyl-13-vinyl

7.23 270 (C20H30) 270 (40), 255 (100), 185 (60), 173 (80), 159 Abieta-8,11,13-triene 0.5

(80), 143 (40), 129 (50)

7.56 272 (C20H32) 272 (100), 257 (50), 229 (80), 187 (40), 148 Abieta-8, 11-diene 2.2

(60), 136 (80), 105 (90)

8.71 286 (C20OH30) 286 (60), 271 (100), 135 (90), 119 (70), 107 Abieta-8,11-diene-3- 20.2

(70), 91 (100), 79 (70) one

8.91 286 (C20OH30) 286 (60), 271 (100), 135 (80), 119 (70), 107 Abieta-8,11,13-triene- 6.2

(70), 105 (60), 93(80), 91 (90), 79 (70) 3-ol

9.04 288 (C20OH32) 288 (20), 270 (30), 255 (30), 135 (100), 119 Abieta-8,11-diene -3-ol 3.8

(30), 107 (40), 91 (40)

9.32 286 (C20OH30) 286 (100), 271 (50), 257 (55), 243 (40), 229 Podocarp-7-ene,13- 12.9

(50), 215 (40), 201 (60), 187 (50), 133(60), methyl-13-vinyl-3-one

119 (80), 105 (100), 91 (100), 79 (80)

10.4 286 (C20OH30) 286 (100), 271 (30), 253 (30), 243 (50), 136 Abieta-8,11,13-trien- 11.9

(50), 105 (70), 91 (60) 12-ol

10.79 288 (C20OH32) 288 (100), 273 (20), 255 (30), 245 (30), 227 Podocarp-7-ene-3-ol, 4.5

(60), 136 (60), 105 (70), 91 (60) 13-methyl-13-vinyl

11.7 302 (C20O2H30) 302 (40), 287 (50), 246 (50), 231 (40), 175 13-Hydroxy-abieta- 4.8

(70), 147 (40), 135 (60), 119 (80), 105 (70), 8,11-dien-7-one

91 (100)

11.91 300 (C20O2H28) 300 (60), 282 (20), 257 (30), 225 (80), 197 11-Hydroxy-abieta- 3.4

(100), 183 (60), 162 (50), 141 (50), 105 8,11,13-trien-7-one

(40), 91 (50)

12.23 300 (C20O2H28) 300 (20), 285 (100), 267 (30), 243 (20), 225 Abieta-6,8,11,13- 0.7

(40), 183 (20), 143 (20), 105 (20), 91 (30) tetraene-3,12-diol

12.72 302 (C20O2H30) 302 (50), 287 (100), 284 (90), 269 (40), 227 Abieta-8,11,13-trien- 0.8

(30), 213 (50), 185 (50), 121 (60), 105 (70), 6,14-diol

91 (80)

15.43 316 (C19O4H24) 316 (10), 222 (90), 177 (40), 121 (30), 107 Crotonin derivative 5.2

(40), 95 (100), 81 (70)

17.47 314 (C19O4H22) 314 (50), 299 (60), 220 (60), 134 (60), 105 Dehydrocrotonin 0.5

(40), 95 (100), 94 (70), 81 (70) derivative

20.58 400 (C28OH48) 400 (50), 382 (40), 367 (30), 315 (30), 289 Ergost-5-en-3-ol 0.5

(30), 255 (30), 105 (60), 95 (100), 81 (90)

21.48 414 (C29OH50) 414 (50), 396 (40), 329 (40), 273 (30), 213 Sitosterol 1.5

(60), 145 (70), 119 (60), 105 (100), 91 (90),

81 (90)

N.C. 19.9

120.0

calculated LC95 and LC50 concentrations are 17.6% and 6.7%, 100.0 respectively.

80.0

4. Discussion

60.0

40.0 The dichloromethane extract from C. sphaerogynus

Mortality rate (%) leaves was shown to have a potent acaricidal activity. This

20.0

species is rich in terpenoids, a class of metabolites known

0.0 to exert acaricidal properties, among which triterpenes

stand out (Ribeiro et al., 2010). The prevalence of diter-

control 0.625% 1.25% 2.5% 5.0% 10.0% 20.0%

penes in C. sphaerogynus extract (Table 1) is coherent with

this observation.

Fig. 1. Mortality rate (%) of Rhipicephalus microplus larvae exposed to

different concentrations of Croton sphaerogynus dichloromethane leaf The present results indicate that the dichloromethane

extracts. extract has acaricidal activity higher than the aqueous

A.A. Righi et al. / Veterinary Parasitology 192 (2013) 292–295 295

extracts from leaves of Lippia javanica. According to relationships of Croton section Cleodora (Euphorbiaceae). Mol. Phylo-

genet. Evol. 60, 193–206.

Madzimure et al. (2011), 10% leaf aqueous extract of L.

Castro-Janer, E., Rifran, L., Piaggio, J., Gil, A., Miller, R.J., Schumaker, T.T.S.,

javanica showed potent acaricidal effect on cattle ticks.

2009. In vitro tests to establish LC (50) and discriminating con-

Our results seem to be roughly comparable with acari- centrations for ﬁpronil against Rhipicephalus (Boophilus) microplus

(Acari: Ixodidae) and their standardization. Vet. Parasitol. 162 (1–2),

cidal activity obtained with extracts from other plants.

120–128.

Fernadez-Salas et al. (2011) reported the effect of extracts

Cetin, H., Cilek, J.E., Oz, E., Ayﬁn, L., Deveci, O., Yanikoglu, A., 2010.

prepared with leaves of Lysiloma latisiliquum and Piscidia Acaricidal activity of Satureja thymbra L. essential oil and its major

components, carvacrol and ␥-terpinene against adult Hyalomma

piscipula. The LC50 values of these plants extracts were

marginatum (Acari: Ixodidae). Vet. Parasitol. 170, 287–290.

nearly 6% and 2%, respectively. Silva et al. (2009) reported

Ducornez, S., Barré, N., Miller, R.J., de Garine-Wichatitsky, M., 2005. Diag-

70.42% mortality for cattle tick larvae exposed to 20% of nosis of amitraz resistance in Boophilus microplus in New Caledonia

hexane extract from Piper aduncum. Rosado-Aguilar et al. with the modiﬁed larval packet test. Vet. Parasitol. 130, 285–292.

FAO, 2004. Resistance Management and Integrated Parasite Control in

(2010) reported 99% larval mortality using methanolic

Ruminants – Guidelines, Module 1 – Ticks: Acaricide Resistance:

extracts of leaves and stems of Petiveria alliacea at 12.2%

Diagnosis, Management and Prevention. Food and Agriculture Orga-

and 16.5%, respectively. nization, Animal Production and Health Division, Rome, p. 53.

Fernadez-Salas, A., Alonso-Diaz, M.A., Acosta-Rodriguez, R., Torres-Acosta,

It is worth mentioning that podocarpane-type diter-

J.F.J., Sandoval-Castro, C.A., Rodriguez-Vivas, R.I., 2011. In vitro

penes do not occur extensively in nature and are known to

acaricidal effect of tannin-rich plants against the cattle tick Rhipi-

occur only in a few genera. An example is Azadirachta (Meli- cephalus (Boophilus) microplus (Acari: Ixodidae). Vet. Parasitol. 175,

113–118.

aceae) (Ara et al., 1990). The high diversity of abietane-type

Giglioti, R., Forim, M.R., Oliveira, H.N., Chagas, A.C.S., Ferrezini, J., Brito,

diterpenes in C. sphaerogynus might be related to the

L.G., Falcoski, T.O.R.S., Albuquerque, L.G., Oliveira, M.C.S., 2011. In vitro

high activity observed against R. microplus. According to acaricidal activity of Neem (Azadirachta indica) seed extracts with

known azadirachtin concentrations against Rhipicephalus microplus.

Biswas et al. (2002), neem (Azadirachta indica) extract con-

Vet. Parasitol. 181 (2–4), 309–315.

tains tricyclic diterpenoids, such as abietane derivatives.

Klafke, G.M., Albuquerque, T.A., Miller, R.J., Schumaker, T.T.S., 2010. Selec-

Interestingly, it has been reported that neem extracts are tion of an ivermectin-resistant strain of Rhipicephalus microplus (Acari:

Ixodidae) in Brazil. Vet. Parasitol. 168, 97–104.

effective in controlling ectoparasites (Giglioti et al., 2011).

LeOra Software, 2004. Polo-Plus Probit and Logit Analysis: User’s Guide,

Berkeley, p. 36.

5. Conclusions Madzimure, J., Nyahangare, E.T., Hamudikuwanda, H., Hove, T., Stevenson,

P.C., Belmain, S.R., Mvumi, B.M., 2011. Acaricidal efﬁcacy against cattle

ticks and acute oral toxicity of Lippia javanica (Burm F.) Spreng. Trop.

To our knowledge, this is the ﬁrst report of abietanes,

Anim. Health Prod. 43, 481–489.

podocarpenes and clerodane furano-type diterpenes in a

Monteiro, C.M., Daemon, E., Clemente, M.A., Rosa, L.S., Maturano, R., 2009.

plant extract with acaricidal effect. C. sphaerogynus leaves Acaricidal efﬁcacy of thymol on engorged nymphs and females of

Rhipicephalus sanguineus (Latreille, 1808) (Acari: Ixodidae). Parasitol.

contain podocarpane-type diterpenes, a relatively rare

Res. 105, 1093–1097.

class of terpenoids. The results of the present study are

Olivier, D.K., Shikanga, E.A., Cambrinck, S., Krause, R.W.M., Regmier, T.,

consistent with previous evidences indicating that Croton Dlamini, T.P., 2010. Phenylethanoid glycosides from Lippia javanica. S.

Afr. J. Bot. 76 (1), 58–63.

species are likely sources of substances useful for the devel-

Pirali-Kheirabadi, K., Teixeira da Silva, J.A., 2010. Lavandula angustifolia

opment of new drugs (Salatino et al., 2007). Further studies

essential oil as a novel and promising natural candidate for tick (Rhipi-

are needed to isolate active compounds from plants, hope- cephalus (Boophilus) annulatus) control. Exp. Parasitol. 126, 184–186.

Ribeiro, V.L.S., dos Santos, J.C., Bordignon, S.A.L., Apel, M.A., Henriques,

fully contributing to the development of new molecules for

A.T., von Poser, G.L., 2010. Acaricidal properties of the essential oil

tick control, with low or no environmental impact.

from Hesperozygis ringens (Lamiaceae) on the cattle tick Rhipicephalus

(Boophilus) microplus. Bioresour. Technol. 101, 2506–2509.

Acknowledgments Rosado-Aguilar, J.A., Aguillar-Caballero, A.J., Rodriguez-Vivas, R.I., Borges-

Argaez, R., Garcia-Vazques, Z., Mendez-Gonzalez, M., 2010. Acaricidal

activity of extracts from Petiveria alliaceae (phytolaccaceae) against

The authors are grateful to FAPESP, CAPES and CNPq for the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

ﬁnancial support. Vet. Parasitol. 168, 299–303.

Salatino, A., Salatino, M.L.F., Negri, G., 2007. Traditional uses, chemistry

and pharmacology of Croton species (Euphorbiaceae). J. Braz. Chem.

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