effect of ethanolic extract of capsicum frutescens l. on adult female of rhipicephalus microplus...

ORIGINAL PAPER

Effect of ethanolic extract of Capsicum frutescens L. on adultfemale of Rhipicephalus microplus (Ixodidae)

Viviane O. Vasconcelos & Maria Alice D. Martins &

Neide J. F. de Oliveira & Eduardo R. Duarte

Received: 23 November 2013 /Accepted: 10 January 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract This study evaluated the effects of ethanol extractof Capsicum frutescens L. (Solanaceae), colloquially knownas malagueta pepper, on egg production and hatching rate oflarvae of Rhipicephalus microplus. Plant samples were col-lected in Montes Claros, Minas Gerais, Brazil. Selected ma-ture fruits were washed, dehydrated in a forced air oven at 40±5 °C to constant weight. The material was incubated in abso-lute ethanol during 10 days, and the extract was filtered, dried,and stored in amber vials under refrigeration at 4 °C.Engorged adult female ticks were immersed in 10ml solutionsof ethanol extracts at 25, 50, 75, 100, or 150 mg ml−1 drymatter, solubilized in dimethyl sulfoxide (DMSO) at 1 % v/v.These concentrations were compared to distilled water or 1 %v/v DMSO in distilled water as negative controls and a com-mercial product as positive control. The extract resulted insignificantly lower oviposition at all tested concentrationswhen compared to the negative controls. On days 2 and 3posttreatment, mortality rates of female ticks ≥55 % wereobserved for concentrations ≥75 mg ml−1. These concentra-tions resulted in a significantly lower hatchability mean, andthe LC90 on hatching inhibition of R. microplus, estimated byprobit analysis, was 91.8 mg ml−1. High acaricidal in vitroeffect was verified, and toxicological tests and analysesin vivo are important to determine appropriate dosages andfrequency of the application necessary to promote this extractas safe and effective alternative for control of R. microplus.

Introduction

Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) canreduce bovine productivity through anemia associated withblood loss, release of toxins at the bite location, and transmis-sion of possibly lethal parasitic diseases such as anaplasmosisand babesiosis (Grisi et al. 2002; Campos Júnior and Oliveira2005). The climate in many tropical regions is suitable forcompletion of two or three life cycles per year (Magalhães andLima 1992; Grisi et al. 2002).

The tick is frequently controlled with chemical acaricides.However, continual use, and misuse, of these products canresult in resistant populations and toxicity to animals and canlead to accumulation of toxic residue in animal products andthe environment (Furlong et al. 2004).

Acaricide resistance of R. microplus is widespread, andalternatives to conventional control should be evaluated(Clemente et al. 2008; Fernandez-Sala et al. 2012).Developing new drugs is expensive, and it is not alwayspossible to keep pace with the evolution of resistance(Santos et al. 2009). Plant extracts promoted wide beneficialuses in pharmaceutical applications as insecticides or acari-cides (Amer and Mehlhorn 2006). Phytochemicals derivedfrom plant sources can act as larvicides, growth regula-tors, repellents, and oviposition attractive and can playan important role in interrupting the transmission ofdiseases to their hosts (Bagavan et al. 2008; Mathewet al. 2009). Therefore, research into plants with acari-cide potential is scientifically, practically, and commer-cially relevant (Chagas 2004; Fernandes et al. 2005).Effective plant extracts could contribute to a reductionin resistant tick populations, preserving the efficacy ofconventional acaricides (Chagas 2004).

Capsicum frutescensL., the malagueta pepper (Fig. 1), is asmall shrub of the family Solanaceae, native to tropicalAmerica, and widely grown in Brazil, Portugal, Africa, and

V. O. Vasconcelos :M. A. D. Martins :N. J. F. de Oliveira :E. R. Duarte (*)Instituto de Ciências Agrárias, Universidade Federal de MinasGerais, Av Universitária 1000, Bairro Universitário, Montes Claros,MG 39400-006, Brazile-mail: duartevet@hotmail.com

Parasitol ResDOI 10.1007/s00436-014-3779-y

South Asia (Oliveira 2000). Solanaceae are important in thefood industry and medicine (Reifscheneider 2000).

Capsaicinoids of this family are associated with caustic orspicy characteristics (Reifscheneider 2000). The speciesC. frutescens also contains diterpenoids, flavonoids, saponins,and phenolic compounds having lethal effects, antifeedanteffects, and parasite repellency (Iorizzi et al. 2000;Madhumathy et al. 2007). Little is known about acaricidalactivity of C. frutescens extract. There are informal reports byorganic dairy cattle breeders in Brazil on the effectiveness ofsolutions containing C. frutescens fruit for tick control. Theaim of this study was to investigate the effects of extracts ofC. frutescens fruits on R. microplus.

Material and methods

Mature C. frutescens fruits were obtained fromMontes Claroscity in northern Minas Gerais, Brazil. The plants were collect-ed in March 2012, identified, and deposited at the Herbariumin the Universidade Estadual de Montes Claros (HMC-3611).

Selected fruits were washed in running water, dried onabsorbent paper, dehydrated in a forced air oven at 40±5 °Cto constant weight, and stored at −4 °C. For preparation of theethanol extract, fruits were incubated in absolute ethanol PA99.5 °GL in 1:10 10 days at room temperature (22 to 33 °C).The extract was filtered through a gauze-lined glass funnel anddried in a forced air circulation oven at 40±5 °C to constantweight (Pires et al. 2001). The dried extract was removed andstored in amber vials under refrigeration at 4 °C until requiredfor analysis. Subsamples of the dried extract were taken for thedetermination of dry matter at 105 °C (AOAC 1990).

The extract was evaluated at 25, 50, 75, 100, or 150 mg drymatter per milliliter, solubilized in dimethyl sulfoxide(DMSO) at 1 % v/v. Solutions of the extract were preparedwith distilled water and with 1 % v/v of DMSO in distilled

water as negative controls. A commercial acaricide containing1.87 mg ml−1 cypermethrin, 3.12 mg ml−1 chlorpyrifos, and0.12 mg ml−1 citroneal, diluted as recommended by the man-ufacturer, was used as positive control. The commercial prod-uct was successfully used for tick control on the farm fromwhich studied ticks were obtained.

Engorged adult R. microplus females were collected fromnaturally infested Zebu x Holstein cows in Francisco Sá, MinasGerais, Brazil. Ticks were placed in aerated plastic containers.Ticks larger than 4 mm were selected, washed with distilledwater, placed on paper towels, and divided into 32 homoge-neous groups of ten based on the degree of engorgement andweight. The collection of engorged female ticks was conductedat least 60 days after the most recent acaricide application.

The acaricide effectiveness of the test solutions was eval-uated by an immersion test following Drummond et al.(1973). Ticks were immersed in 10 ml of test solution for5 min. Excess solution was removed with a paper towel, andticks were placed in a Petri dish and maintained at 28 °C and70 % relative humidity in an BOD incubator. All procedureswere performed with four repetitions.

Fifteen days after the onset of egg laying by surviving ticks,the mass of eggs for each group was determined on an ana-lytical scale and transferred to 3-ml plastic syringes. Thirtydays after the start of hatching, syringe contents were trans-ferred to Petri dishes, and the larvae and eggs were countedunder a stereoscopic microscope to determine the hatchingrate of each group.

A modified formula described by Bennett (1974) was usedfor the analysis of the oviposition capacity (CO) of femaleticks:

CO ¼ weightof eggmass=initialweightof femaleð Þ � 100

The efficacy of treatment (product efficacy) was estimatedusing the equation of Drummond et al. (1973):

Product efficacy was calculated for each replicateconsidering the ER of the negative controls. A randomizeddesign was used to compare the four extract concentrationswith two negative controls and the commercial acaricidepositive control. The tests were repeated four times, and thedata were transformed and subjected to analysis of variance.

ER (reproductive efficacy) egg weight x hatching x 20,0001

Initial weight of females

EP (product efficacy) (ER control group – ER treated group) x 100

ER control group

Fig. 1 Fruits of Capsicum frutescens L. (the malagueta pepper)

1 Represents the constant of 20,000 eggs/g of mass.

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The means were compared by the Scott–Knott test (P≤0.05).The concentration of the extract sufficient to inhibit 90 % ofthe hatching (LC90) was calculated by probit analysis usingthe statistical package Saeg 9.1 (SAEG 2007). Solutions witha minimum efficacy of 95 % were considered to be effective(Brasil 1990).

Results and discussion

The ethanol extract of C. frutescens resulted in a significantlylower oviposition of R. microplus at all tested concentrationswhen compared to the negative controls (Table 1, P≤0.05).The probable mechanism was the interference with the con-version of blood ingested by ticks in eggs (Vasconcelos et al.2004).

On days 2 and 3 posttreatment, mortality rates of femaleticks ≥55 % were observed for concentrations of 75 mg ml−1

and higher (Table 1). In contrast, Lucini et al. (2010) reportedthat an aqueous extract of the dedo-de-moça chili pepper(Capsicum baccatum) was not associated with the mortality ofTetranychus ludeni (red mite), but oviposition was significantlyreduced for the highest concentrations (4.0 and 8.0 %) tested.

The single exposure to the extract at concentrations≥75 mg ml−1 resulted in a significantly lower mean hatchabil-ity than seen in negative controls, and the acaricide efficacy ofC. frutescens extract was greater than 96 %, similar to thecommercial acaricide, and higher than the 95 % (Table 1)minimum efficacy required for conventional acaricides(Brasil 1990). The LC90 of C. frutescens extract on the hatch-ing inhibition of R. microplus, estimated by probit analysis,was 91.8 mg ml−1 (Fig. 2).

Capsicum chili peppers are widely used as food ingredi-ents, and Capsicum oleoresins are constituents of pharmaceu-ticals and of self-defense repellent sprays (Schweiggert et al.

2006). Studies have demonstrated that the aqueous extract ofC. frutescens has potential for the control of parasites in fish(Lin et al. 2012). Results of in vitro trials have shown aqueousextract ofC. frutescens at dilution ratios of 1:32 and 1:64 to beassociated with more than 70 % mortality of the protozoaIchthyophthirius multifiliis with 4 h of exposure (Lin et al.2012).

Capsicum frutescens has also been investigated for antimi-crobial properties. Abdou et al. (1972) reported that its crudejuices were active on Escherichia coli, Salmonella typhi, andBacillus subtilis. Plain and heated aqueous extracts of freshC. frutescens showed varying degrees of inhibition of fivespecies of bacteria (Cichewicz and Thorpe 1996).

Water extracts of accessions of Capsicum annuum, ofC. baccatum, and of C. frutescens were highly toxic to thecabbage lopper larvae Trichopulsia ni (Hubner) and spidermite, Tetranychus urticae Koch (Antonius et al. 2007), themost difficult pest of crucifer crops to control (Hines andHutchison 2001). Mortality was greatest (94 %) when the fruitextract of C. annuum was sprayed on larvae of the cabbagelooper, while crude extracts of C. frutescens) and C. annuumwere repellent to the spider mite (74 % mortality) (Antoniuset al. 2007).

The volatile fractions of C. frutescens were characterizedusing headspace solid phase micro-extraction, gas chromatog-raphy, and mass spectrometry as reported by Bogusz-Junioret al. (2012). Eighty-three compounds, mostly esters andalcohols, have been identified in C. frutescens. The predom-inant and most potent chemical in Capsicum sp. was capsai-cin. It possesses anti-inflammatory and antioxidant propertiesand has been used in the treatment of arthritis and cystitis. Inaddition, capsaicin has a negative effect on predators of her-bivores (Newall et al. 2002; Lee et al. 2005). Capsaicin maybe the active component producing the acaricidal results ob-tained in the present research, a topic for further investigation.

Table 1 Effect of Capsicum frutescens fruits ethanol extract on oviposition, hatchability, and mortality of Rhipicephalus microplus

Treatment (mg ml−1) Mortality (%) Oviposition capacity Hatchability (%) Effectiveness (%)c

150 55.0a 3.1a 34.1b 94.4a

100 72.5a 1.0a 2.1a 99.9a

75 85.0a 3.1a 41.8b 91.8a

50 10.0b 25.3b 84.6c 31.4b

25 10.0b 27.5b 86.8c 23.4b

Distilled water 0.0b 39.7c 98.1c 0c

Negative controla 0.0b 38.7c 85.8c 0c

Positive controlb 95.0a 1.0a 5.2a 99.7a

Variation coefficient (%) 41.5 31.2 42.8 27.8

Means followed by same lowercase letter are similar statistically by Scott–Knott’s test at 5 % probability (P≤0.05)a Negative Control with 1 % dimethyl sulfoxideb Commercial acaricide containing 1.87 mg ml−1 cypermethrin, 3.12 mg ml−1 clorpyrifos, and 0.12 mg ml−1 citronealcMeans obtained by the equation from Drummond et al. (1973)

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However, gas chromatography–mass spectrometry spec-trometric analysis of fruits from different Capsicum speciesrevealed that capsaicin and dihydrocapsaicin, the pungentcomponents of pepper fruit, were not correlated with toxicityor repellency, indicating that the two capsaicinoids are notlikely related to the efficacy of pepper fruit extracts (Antoniuset al. 2007). Major compounds in hot pepper fruit extractswere detected and identified as pentadecanoic acid methylester, hexadecanoic acid methyl ester, and octadecanoic acidmethyl ester. It was also indicated that capsaicin anddihydrocapsaicin, the major capsaicinoids in hot pepper, werenot toxic to Trichopulsia ni (Antonius et al. 2007).

Plants produce a vast array of volatiles and tannins that playan important role in plant defense (Aharoni et al. 2003). Hotpepper also contains significant amount of tannins(Malgorzata and Perucka 2005; Antonious et al. 2006) thatbreak down and behave as toxins (Antonious et al. 1999).

Due to its low toxicity to humans, fruit extracts of chilipepper can be used as an agricultural alternative (Pires et al.2004). The ethanolic extract of C. frutescens can be easilyused by farmers since the plant is widely cultivated. The use ofthis extract could reduce synthetic acaricide contamination ofthe environment and of foods of animal origin, and eliminatethe need for discarding milk of treated bovines as is necessarywith synthetic acaricides.

Peppers belonging to other families have also been used inthe control of R. microplus. The extracts of Pipper aduncumleaves are effective for the control of R. microplus, yieldingsimilar results to those reported in the literature for a variety ofsynthetic and other natural agents. The LC50 of a hexaneextract of P. aduncum was 9.30 mg ml−1 for larvae andreduction in reproduction ranged from 12.48 to 54.22 %,while 0.1 mg/ml of the essential oil induced 100 % mortalityin larvae (Silva et al. 2009). The leaves and stems ofP. aduncum contain an essential oil composed mainly ofdillapiole (Pino et al. 2004; Walia et al. 2004) which has been

demonstrated to have synergistic effects with several naturalinsecticides (Maia et al. 1998).

Ethanolic extracts of other plants have also showed efficacyagainst R. microplus, but at higher concentrations. The extractof Ricinus communis leaf at 95 % concentration was shown tosignificantly increase mortality rate in a dose-dependent man-ner, ranging from 35.0±5.0 to 95.0±5.0 %. This extract alsoinhibited 36.4–63.1 % of oviposition of R. microplus (Ghoshaet al. 2013). The crude ethanolic extract of Leucas asperatested against Rhipicephalus (Boophilus) annulatus obtainedsignificant adult tick mortality at 100 mg ml−1. Inhibition offecundity was concentration dependent and significantly dif-fered from the control (Ravindran et al. 2011).

The vegetal extracts can potentially be used in tick popula-tions resistant to synthetic products, contributing to an effectivealternative control. In this study of the ethanolic extract ofC. frutescens, reduced oviposition was verified at 25 mg ml−1

and high acaricidal efficacywas observed at 75mgml−1, throughtick mortality and egg-hatching inhibition. Toxicological testsand analyses in vivo are important to determine appropriatedosages and frequency of application necessary to promote thisextract as an alternative acaricide for control of R. microplus.

Acknowledgments The authors thank the Coordination of Improve-ment of Higher Education Personnel (CAPES), the National Council forScientific and Technological Development (CNPq), Foundation for Re-search Support of Minas Gerais (FAPEMIG).

Conflict of interest The authors of this manuscript have no financial orpersonal relationship with individuals or organizations that could influ-ence or bias the content of the paper.

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