Veterinary Parasitology 168 (2010) 299–303

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Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

Acaricidal activity of extracts from Petiveria alliacea (Phytolaccaceae)

against the cattle tick, Rhipicephalus (Boophilus) microplus
(Acari: ixodidae)
J.A. Rosado-Aguilar a,*, A. Aguilar-Caballero a, R.I. Rodriguez-Vivas a, R. Borges-Argaez b,
Z. Garcia-Vazquez c, M. Mendez-Gonzalez b
a
Departamento de Parasitologı´a, Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Yucatán, Km. 15.5 carretera Mérida-Xmatkuil,
CP 97100, Mérida, Yucatán, Mexico
b
Centro de Investigación Cientı´fica de Yucatán, Calle 43 No. 130 Colonia Chuburná de Hidalgo, CP 97200, Mérida, Yucatán, Mexico
c
Centro Nacional de Investigaciones en Parasitologı´a Veterinaria, Carretera Federal Cuernavaca Cuautla, 8534 Colonia Progreso, CP 62550, Jiutepec,
Morelos, Mexico

A R T I C L E I N F O A B S T R A C T

Article history: The acaricidal activity of crude extracts and fractions from stems and leaves of Petiveria
Received 28 July 2009 alliacea (Phytolaccaceae) was carried out on larvae and adults of the cattle tick
Received in revised form 25 November 2009 Rhipicephalus (Boophilus) microplus using the larval immersion test (LIT) and adult
Accepted 25 November 2009
immersion test (AIT), respectively. Methanolic extracts of stems and leaves of P. alliacea
showed 100% mortality on the LIT bioassay. On the other hand, methanolic extracts of
Keywords:
leaves and stem on the AIT test showed 26% and 86% of mortality, respectively, egg laying
Methanolic extracts
inhibition of 40% and 91%, respectively and hatchability inhibition of 26% and 17%,
Petiveria alliacea
Acaricidal activity respectively. Purification of the active stem methanolic extract showed that the activity
Mortality was present in the n-hexane non-polar fraction. Bioassay-guided purification of the n-
Benzyltrisulfide hexane fraction produced 10 semi-purified fractions; fraction B had the highest activity
Benzyldisulfide against tick larvae (100% mortality). Gas chromatography–mass spectrometry demon-
Rhipicephalus (Boophilus) microplus strated that the chemical composition of the active fraction B samples were mainly
composed of benzyltrisulfide (BTS) and benzyldisulfide (BDS). These metabolites might be
responsible for the acaricidal activity of stem extract of P. alliacea. However, further
experiments to evaluate the acaricidal activity of BTS and BDS on larvae and adults of R. (B.)
microplus are needed. Our results showed that P. alliacea is a promising biocontrol
candidate as acaricide against R. (B.) microplus resistant strains.
ß 2009 Elsevier B.V. All rights reserved.

1. Introduction pathogens to the host (Babesia bovis, Babesia bigemina and

Rhipicephalus (Boophilus) microplus (Acari: ixodidae) is
an endemic external parasite of cattle in tropical and
subtropical regions of the world, causing major economic
losses to cattle producers directly through feeding on cattle
and indirectly by transmitting several disease-causing
* Corresponding author. Tel.: +52 999 9423200; fax: +52 999 9423205.
E-mail address: alberto.rosadoaguilar@gmail.com
(J.A. Rosado-Aguilar).
Anaplasma marginale) (Rodrı́guez-Vivas et al., 2004;
Barros-Battesti et al., 2006).
Chemical acaricides such as synthetic pyrethroids (SP),
organophosphates (OP) and amitraz (Am) have played a
pivotal role in the control of R. (B.) microplus. However, as a
consequence of extensive use of chemicals this tick specie
has developed resistance to the major classes of acaricides
in different countries. In Mexico, tick resistance to
acaricide is recognized in several states mainly in the
Mexican tropics (Rodriguez-Vivas et al., 2007). Commer-
cial acaricides are usually toxic to humans and leave

0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2009.11.022
300 J.A. Rosado-Aguilar et al. / Veterinary Parasitology 168 (2010) 299–303
residues in the environment. However, acaricide from
plants are usually of low toxicity to mammals, water
soluble, producing non-residual effects and have a low
incidence of developing resistant strains (Chungsamar-
nyart et al., 1991; Sardá et al., 2007). Development of
resistance to commercial acaricides by R. (B.) microplus has
stimulated the search for new control strategies.
Compounds from plants extracts provide a potential
alternative to existing acaricides, based on promising
results to control ticks susceptible and resistant to
acaricides obtained by researchers with other plant species
(Chagas et al., 2002; Fernandes et al., 2007). Plant products,
such as crude extracts from Petiveria alliacea (‘‘anamu’’,
‘‘zorrillo’’, ‘‘payche’’, etc.) (Alonzo, 1998), are widely used
as phytotherapeutic remedies with antimicrobial, antiin-
flammatory, antispasmodic and diuretic properties (Lor-
enzi and Matos, 2002; Carvalho, 2004). Pure metabolites
from roots extracts of P. alliacea have showed insecticidal
and acaricidal properties (Lyndon et al., 1997). However,
there is no information about the acaricidal activity of stem
and leaf extracts from P. alliacea. Therefore the aim of this
study was to evaluate the acaricidal activity of the crude
extracts of stem and leaf from P. alliacea against R. (B.)
microplus larvae and adults resistant to OP, SP and Am.
Additionally, the chemical composition of the active
fraction by gas chromatography–mass spectrometry was
also investigated.
2. Materials and methods
2.1. Plant material and extraction
Plant material (leaves and stems) of P. alliacea was
collected in Yaxcaba, Yucatan, Mexico in October 2007. The
Voucher specimen were authenticated and deposited at
the herbarium of the Centro de Investigación Cientı́fica de
Yucatan (CICY) under the following code numbers: P.
alliacea L. (Phytolaccaceae, MMendez 1417). Leaves and
stems were dried at 40 8C for 72 h and ground in a grinder
with a 5 mm diameter mesh. The ground material was
immersed in 100% methanol for 72 h (proportion of 1.0 ml
of methanol and 0.83 g of ground material). The extracted
methanol was filtered and evaporated at 45 8C by vacuum
rotary evaporator. The plant crude extracts were trans-
ferred to glass vials and kept at 4 8C until use (Borges-
Argáez et al., 2007).
2.2. Rhipicephalus (Boophilus) microplus ticks for bioassays
Engorged female R. (B.) microplus ticks were collected
from naturally infested cattle pastured on a ranch in the
municipality of Xmatkuil, in the Mexican state of Yucatan.
Cattle were free of acaricide treatments 45-day prior tick
collection. The ticks were previously diagnosed resistant to
three acaricide families (SP, OP and Am) (Rodriguez-Vivas
et al., 2006).
The engorged female ticks were washed with water and
dried in paper towelling. The average weight of ticks was
0.20 g. A group of females were used in the adult
immersion test (AIT) and another group was incubated
at 27  1.5 8C and 70–80% relative humidity (Cen et al., 1998)
for two weeks until the eggs were laid. These eggs provided
the larvae used for the larval immersion test (LIT).
2.3. Adult immersion test
The AIT (Drummond et al., 1976) was used to test the
acaricidal activity of crude extracts (leaf and stem) from P.
alliacea against R. (B.) microplus adult engorged females.
Ten engorged females of R. (B.) microplus weighting
approximately 2 g were deposited in 9 cm Petri dishes.
Five groups were made, four treated (leave and stem, all at
10% and 20%) and one control (tween 2%) with three
repetitions each. The treated group was immersed during
one minute in the diluted crude extract (tween 2%) and the
control group was immersed in tween 2% (with distilled
water) as recommended in a preliminary work (Rosado-
Aguilar et al., 2007). The ticks were placed individually
into a plate with 24 holes and incubated for a period of 15
days at the condition of temperature and humidity
described above. Ticks were observed with the aid of a
stereo-microscope and the mortality rate and weight of
produced eggs in each group was recorded. The mortality
rate was recorded daily by counting dead ticks. The dead
ticks were then transferred to Petri dishes and the extracts
effects on them were observed. Dead ticks were identified
by the presence of cuticular darkness, lack of malphigian
tube, movement and haemorrhagic skin lesions. After 15
days, the number of females laying eggs was recorded and
the eggs of each group were weighted by using an
analytical scale. After that 50 eggs were placed in
25 mm  95 mm glass vials at the same conditions. During
21 days the vials were observed and the hatching rates of
the different treatments were estimated and compared to
the controls. The egg laying inhibition (Drummond et al.,
1976) and the larval hatching inhibition percentage
(Rodrı́guez-Vivas and Cob, 2005) were determined for all
groups.
2.4. Larval immersion test
The modified LIT was used to test the acaricidal activity
of crude extracts (leaf and stem) from P. alliacea against R.
(B.) microplus larvae (Soberanes et al., 2002). Tween-20
was diluted in distilled water at 2% concentration and it
was used to dilute plant crude extracts (2.5%, 5.0%, 10% and
20%) and for the control group.
Tick larvae 7–14-day-old were used in this study
(Soberanes et al., 2002). Hatching vials with the highest
larval eclosion rate (90–100%) were selected and placed in
the centre of a Petri dish that was subsequently filled with
water and soap, which prevented their escape. The diluted
plant crude extract (3 ml) was transferred to Petri dishes
(60 mm  15 mm in diameter), and 300–500 larvae were
placed between two Whatman No. 1 papers and immersed
for 10 min. Approximately 100 larvae were picked with a
no. 4 paintbrush, and gently transferred to clean filter
paper packets.
The opening of the envelopes (treated and control with
larval ticks) was folded with metallic clip, with its
identification mark (tested solution and concentration)
on the outside. The packets were placed in an incubator at
 J.A. Rosado-Aguilar et al. / Veterinary Parasitology 168 (2010) 299–303
temperature of 27  1.5 8C and 70–80% relative humidity for
48 h. The envelopes were opened 48 h post-treatment (PT)
and observed using a stereoscopy. The number of live larvae,
mortality and any toxicological effects observed were
recorded. Larvae that were unable to walk forward were
considered dead. We breathed on the packet whilst counting
to stimulate live but inactive larvae. Larval mortality was
determinated by Abott’s formula (Abott, 1925) recom-
mended by FAO (2004):
Corrected mortality
% treated mortality  % controls mortality
¼  100
100  % controls mortality
None of the bioassays in control groups showed
mortality >5%.
2.5. Isolation of active fractions from the extracts
The active stem crude extract (g of crude extract) was
partitioned with n-hexane, ethyl acetate and methanol and
each fraction obtained was evaluated in the LIT using a 10%
concentration. The resulting acaricidal hexane extract
(1.88 g of hexane extract) was fractionated using a glass
column (4 cm  5 cm) packed with 140 g of sodic bentonite
and eluted with 200 ml each of n-hexane, and then, n-
hexane and acetone volumes of increasing polarity
(relation 100:0 up to 0:100). Based on similar Rf values
on TLC (silica gel 60F254 aluminium plates; Merck)
developed with the eluent n-hexane:acetone (8:2) and
sprayed with a solution of phosphomolybdic acid, ten
fractions (A–J) were grouped. Each fraction was evaluated
(10% concentration) against R. (B.) microplus larvae using
the LIT described above.
Fraction B had the highest acaricidal activity and it
was analysed, together with the hexane active extract,
on a Agilent Technologies 6890N Gas Chromatography
apparatus coupled with a mass selective detector 5975B,
using the following chromatographic conditions: split
injection of 1 ml of a 2% concentration sample; Ultra 1
column (100% dimethylpolisiloxane, 25 m  0.2 mm i.d.),
flow rate 1.0 ml/min (helium as carrier gas); samples
were analysed with the column held initially at 100 8C
for 4 min after injection, then increased to 200 8C with
a gradient of 10 8C/min heating programme. The injec-
tion was performed in split mode (split radio 50:1).
Interfase and injector temperature was 250 8C. Identifi-
cation of components in the extract was performed by
computer searches in commercial reference libraries.
The fragmentation patterns of the mass spectra were
compared with those from the NIST05 Libraries. Com-
ponents identified from the active fraction B are listed in
Table 3.
2.6. Statistic analyses
Results of adults ticks (mortality, egg laying inhibition
and larval hatching inhibition) were analysed using
Kruskal Wallis test (p > 0.05). Lethal concentrations (LC)
to kill 50% and 99% of larvae and their respective 95%
confidence intervals (CI) were calculated by probit analysis
(Polo Plus software) (LeOra Software, 2004).
301
3. Results and discussion
Biological control is defined as the reduction of pest
populations by natural means including predators, para-
sitoids and pathogens. Application of plants or their
extracts to control microorganisms has been used for
hundreds of years by practitioners of traditional medicine.
For the past decades the acaricidal and insecticidal
properties of the plant extracts have been widely used
against phytophagous pests and mosquitoes (Calmasur et
al., 2006), ticks (Lori et al., 2005) and mites (Kim et al.,
2004). With the successful mass production of these plants
extracts the repellent properties of some have been
commercially exploited against a variety of insects
(Jaenson et al., 2006). Also the repellent properties of
these plant materials on ticks have been reported.
Different plant preparations including oils and extracts
have been used for a long time in traditional medicine in
Mexico on account of their pharmacological activity and
low toxicity.
The plant genera Petiveria have been considered in
regard to their biological control effects on living organ-
isms. The root extracts of P. alliacea have showed several
properties such as insectcidal, acaricidal and anti-fungal
(Lyndon et al., 1997; Coelho et al., 2001); however, the
acaricidal activity of stem and leaf of the P. alliacea extracts
against R. (B.) microplus has not been previously reported.
In this study we evaluated the acaricidal activity of crude
extracts (leaf and stem) from P. alliacea for the control of
adults and larvae of R. (B.) microplus ticks. The efficacy of
the extracts of P. alliacea against engorged females of R. (B.)
microplus was assessed by measuring mortality, egg laying
inhibition and larval hatching inhibition. The crude
extracts of leaf at 10% (100 mg/ml) and 20% (200 mg/ml)
concentration evaluated against R. (B.) microplus engorged
females showed accumulative percentage of mortality (15
days PT) of 23.3% and 26.6%, respectively, egg laying
inhibition (15 days PT) of 28.2% and 40.1%, respectively and
larval hatching inhibition (21 days after laying) of 26.0%
and 21.3%, respectively. On the other hand, the crude
extracts of steam at 10% (100 mg/ml) and 20% (200 mg/ml)
concentration evaluated against R. (B.) microplus engorged
females showed accumulative percentage of mortality of
40.0% and 86.6%, respectively; egg laying inhibition of
56.8% and 91.0%, respectively and larval hatching inhibi-
tion of 17.0% and 16.0%, respectively (Table 1). The
percentage of tick mortality of stem from P. alliacea was
eleven times greater (73.3%) than that leaf (6.6%) at 2 days
PT. It was also observed that both extracts did not affect the
eggs hatching. The hatching rates of the eggs in all
treatments were not significant difference from the
controls (p > 0.05). No previous studies have been reported
on the acaricidal properties of leaf and stem of P. alliacea
against resistant ticks. There is only one study on the
acaricidal activity of P. alliacea (Lyndon et al., 1997).
However these authors just evaluated a root pure
compound (dibenzyltrisulfide) of P. alliacea against R.
(B.) microplus adults reported efficacy of 50% in mortality
(0.001 mg/ml–4 days PT), egg laying inhibition
(0.0002 mg/ml–14 days PT) and larval hatching inhibition
(0.0002 mg/ml–56 days PT). The high concentration of
302 J.A. Rosado-Aguilar et al. / Veterinary Parasitology 168 (2010) 299–303
Table 1
Percent of mortality, inhibition of egg laying and inhibition hatchability of Rhipicephalus (Boophilus) microplus adults exposed to Petiveria alliacea extracts at
10% and 20% concentration.
P. alliacea extract % mortality
c c
Leaf 10% 23.3  15.2
Leaf 20% 26.6  5.7c
Stem 10% 40.0  20.0c
Stem 20% 86.6  15.2b
Control tween (2%) 0.0  0a
Means within a column followed by the same letter are not significantly different (p  0.05).
non-active metabolites in the crude extract of P. alliacea of
the present study might explain the general low efficacy
found. There are studies on the acaricidal activity of
extracts of other plants against R. (B.) microplus adults
measuring mortality, egg production and hatchability.
Srivastava et al. (2008) evaluated the ethanolic crude
extract (80 mg/ml–1 day PT) of Azadirachta indica, Prunus
persica, Mangifera indica, Psidium guajava and Curcuma
longa. A. indica seed extract was more effective (80%) than
P. persica seed (70%) and A. indica leaf (30%). The extracts
prepared from A. indica bark, P. persica leaf, and M. indica
bark had no effect on the adults of R. (B.) microplus, while
only 10% tested adults died when treated with the extract
of C. longa. In other study, the ethanolic crude extracts
(100 mg/ml–2 days PT) of Acanthus ebracteatus (leaf)
Acorus calamus (rhizome), Annona squamosa (seed and
leaf), Luffa acutángula (seed) and Stemona collinsae (root)
against engorged females of R. (B.) microplus showed
efficacies of 3%, 41%, 100%, 7%, 52% and 56%, respectively
(Chungsamarnyart et al., 1990). Crude methanolic extracts
(aerial parts) of Calea serrata and Hypericum polyanthemum
evaluated (50 mg/ml) against R. (B.) microplus females not
showed significant effect in egg laying inhibition (14 days
PT) and hatching inhibition (14 days after laying) in
relation to the controls (Sardá et al., 2007, 2008). In our
study we found high efficacy (91%–15 days PT) using stem
(200 mg/ml) of P. alliacea in egg laying inhibition. The eggs
laid by the treated females were incubated and no
significant effect in the hatching rates was observed.
On the other hand, In the LIT the leaf extracts of P.
alliacea showed acaricidal activity against larvae of R. (B.)
microplus with LC50 (CI) and LC99 (CI) values of 43 mg/ml
(39.0–47.1) and 122 mg/ml (103.8–155.6), respectively.
The activity for stem was 38 mg/ml (32.3–45.5) and
165 mg/ml (115.8–322.2), respectively (Table 2). No
mortality was observed in the control group. These results
showed that leaf extracts had higher efficacy to control R.
(B.) microplus larvae than stem extracts; however, for adult
the stem extracts showed the highest efficacy. There are
not studies on the acaricidal activity of extracts of P.
alliacea on R. (B.) microplus larvae; however, activities of
other plants against tick larvae have been reported (Prates
Table 2
Percent mortality, LC50, LC99 and CI of Rhipicephalus (Boophilus) microplus larvae exposed to different concentrations of Petiveria alliacea extracts.
P. alliacea 20.0% 10.0%
Leaf 100.0  0.0 95.5  4.1
Stem 100.0  0.0 94.7  1.3
LC50: lethal concentration 50%; LC99: lethal concentration 99%; CI: confidence intervals.
% inhibition of egg laying % inhibition hatchability
28.2  17.6 26.0  5.2a
40.1  10.2c 21.3  2.3a
56.8  9.5c 17.0  4.7a
91.0  9.1b 16.0  5.4a
0.0  0a 10.0  1.6a
et al., 1993). Chagas et al. (2002) obtained promising
results using commercial formulations of essential oils
from three Eucalyptus spp. as emulsified concentrates
against R. (B.) microplus larvae. The authors reported 100%
larval mortality when exposed to 10% (100 mg/ml)
concentration of the oils from E. staigeriana and E.
citriodora and 20% (200 mg/ml) from E. globules. Sardá
et al. (2007) evaluated the crude methanolic extract of H.
polyanthemum against larvae of R. (B.) microplus killing
100%, 96.7%, 84.7% and 52.7% at the concentrations of 50,
25, 12.5, and 6.25 mg/ml, respectively. The crude ethanol
extract of soapberry, Sapindus saponaria also had larvicidal
activity against R. (B.) microplus with LC50 and LC99 values
of 1.25 and 6.36 mg/ml, respectively (Fernandes et al.,
2005).
The hexanic, methanolic and ethylene acetate parti-
tions of crude extract of P. alliacea at 10% against R. (B.)
microplus larvae showed mortalities of 93.6%, 6.4% and
5.2%, respectively. The hexanic partition showed the
highest efficacy against R. (B.) microplus larvae, showing
that the non-polar compounds are responsible for the
acaricidal activity of this plant extract. This partition was
purified in 10 fractions and the B fraction showed the
highest acaricidal activity (100%) against R. (B.) microplus
larvae. However, due to the small amount of the B
extracted fraction, it was not tested against adult ticks.
In future studies the evaluation of the B fraction (with
different concentrations) against adult ticks are recom-
mended. The CG/MS analysis of fraction B produced six
components (Table 3). Benzyltrisulfide (BTS) and benzyl-
disulfide (BDS) were found to be the highest abundant
constituents (63.1% and 32.5%, respectively) of P. alliacea
stem. This finding is in agreement with Lyndon et al. (1997)
who identified BTS in the root extract of P. alliacea as one of
their main constituents with a high efficacy against R. (B.)
microplus adults.
In conclusion, the crude extracts of P. alliacea produced
high acaricidal activity (larvae and adults) and reduced the
ability to lay eggs (engorged females) of R. (B.) microplus.
BTS and BDS were identified as their main constituents and
might be responsible for the acaricidal activity of this plant
extract. However, complementary experiments to evaluate
5.0% 2.5% LC50 (CI) LC99 (CI)
69.8  8.3 7.7  3.1 4.30 (3.90–4.71) 12.29 (10.38–15.56)
65.3  20.8 26.2  7.6 3.88 (3.23–4.55) 16.52 (11.58–32.22)
 J.A. Rosado-Aguilar et al. / Veterinary Parasitology 168 (2010) 299–303 303

Table 3 Chungsamarnyart, N., Jiwajinda, S., Jansawan, W., 1991. Larvicidal effect
Individual components, with retention times and percent yield, identified of plant crude-extracts on the tropical cattle tick (B. microplus).
in stem extract of Petiveria alliacea from GC:MS analyses used in bioassays Thailand. Kasetsart J. Nat. Sci. 25, 80–89.
against larvae. Coelho, B.P.J., Young, M.C., Giesbretch, A.M., Roque, N.F., Bolzani, V.S.,
2001. Antifungal polysulphides from Petiveria alliacea L. Phytochem-
Components Retention % istry 57, 743–747.
times (min) Drummond, R.O., Graham, O.H., Ernest, S.E., 1976. Evaluation of insecti-
cides for the control of B. Annulatus and B. microplus (Acarina:
Cis-stilbene 12.53 0.3 ixodidae) on cattle. Proc. Second Int. Cong. Acarol. 493–498.
Hexadecanoic acid, methyl ester 16.74 2.8 Fernandes, F.F., Leles, R.N., Silva, I.G., Freitas, E.P.S., 2007. Study of the
Dibenzyldisulfide 18.44 32.5 activity of Sapindus saponari (Sapindaceae) on larvae of the brown dog
Octadecadienoic acid, methyl ester 19.70 0.4 tick Rhipicephalus sanguineus (Latreille 1806) (Acari: Ixodidae). Arq.
Octadecenoic acid, methyl ester 19.93 0.7 Bras. Med. Vet. Zootec. 59, 145–149.
Dibenzyltrisulfide 25.38 63.1 Food Agriculture Organization of the United Nations (FAO), 2004. Resis-
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Jaenson, T.G.T., Garboui, S., Palsson, K., 2006. Repellency of oils of lemon,
the acaricidal activity of BTS and BDS are need. Our results eucalyptus, geranium, and lavender and the mosquito repellent
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