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Laboratory assessment of acaricidal activity of Cymbopogon winterianus, Vitex negundo and Withania somnifera extracts against deltamethrin resistant Hyalomma anatolicum Nirbhay Kumar Singh Jyoti Bhaskar Vemu Abhijit Nandi Harkirat Singh Rajender Kumar V. K. Dumka Received: 26 September 2013 / Accepted: 5 March 2014 / Published online: 20 March 2014 Ó Springer International Publishing Switzerland 2014 Abstract Larval packet test was used for detection of resistance levels against cyper- methrin and deltamethrin, the most commonly used synthetic pyrethroids, in the multi-host tick Hyalomma anatolicum collected from district Moga, Punjab (India). Results indicated the presence of level I resistance against deltamethrin (RF = 2.81), whereas the tick isolate was susceptible to cypermethrin (RF = 0.2). The aqueous and ethanolic extracts of leaves of Cymbopogon winterianus, Vitex negundo and Withania somnifera along with roots of Vitex negundo were assessed for their acaricidal activity against the larvae of deltamethrin resistant H. anatolicum. The efficacy was assessed by measuring per cent larval mortality and determination of LC 50 values. The various ethanolic extracts produced a concentration dependent increase in larval tick mortality, whereas the aqueous extracts exhibited a much lower mortality. The highest mortality (93.7 ± 0.66 %) was observed at the 5.0 % con- centration of ethanolic extract of leaves of C. winterianus and the lowest LC 50 value (0.011 %) was recorded for ethanolic extracts of leaves of V. negundo. The results indi- cated that these plant extracts have potential to be developed as herbal acaricides. Keywords Acaricidal activity Á Cymbopogon winterianus Á Hyalomma anatolicum Á Vitex negundo Á Withania somnifera N. K. Singh (&) Á Jyoti Á A. Nandi Á H. Singh Department of Veterinary Parasitology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141004, India e-mail: [email protected] B. Vemu Á V. K. Dumka Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana 141004, India R. Kumar Department of Agronomy, Punjab Agricultural University, Ludhiana 141004, India 123 Exp Appl Acarol (2014) 63:423–430 DOI 10.1007/s10493-014-9791-1

Laboratory assessment of acaricidal activity of Cymbopogon winterianus, Vitex negundo and Withania somnifera extracts against deltamethrin resistant Hyalomma anatolicum

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Page 1: Laboratory assessment of acaricidal activity of Cymbopogon winterianus, Vitex negundo and Withania somnifera extracts against deltamethrin resistant Hyalomma anatolicum

Laboratory assessment of acaricidal activityof Cymbopogon winterianus, Vitex negundo and Withaniasomnifera extracts against deltamethrin resistantHyalomma anatolicum

Nirbhay Kumar Singh • Jyoti • Bhaskar Vemu • Abhijit Nandi •

Harkirat Singh • Rajender Kumar • V. K. Dumka

Received: 26 September 2013 / Accepted: 5 March 2014 / Published online: 20 March 2014� Springer International Publishing Switzerland 2014

Abstract Larval packet test was used for detection of resistance levels against cyper-

methrin and deltamethrin, the most commonly used synthetic pyrethroids, in the multi-host

tick Hyalomma anatolicum collected from district Moga, Punjab (India). Results indicated

the presence of level I resistance against deltamethrin (RF = 2.81), whereas the tick isolate

was susceptible to cypermethrin (RF = 0.2). The aqueous and ethanolic extracts of leaves

of Cymbopogon winterianus, Vitex negundo and Withania somnifera along with roots of

Vitex negundo were assessed for their acaricidal activity against the larvae of deltamethrin

resistant H. anatolicum. The efficacy was assessed by measuring per cent larval mortality

and determination of LC50 values. The various ethanolic extracts produced a concentration

dependent increase in larval tick mortality, whereas the aqueous extracts exhibited a much

lower mortality. The highest mortality (93.7 ± 0.66 %) was observed at the 5.0 % con-

centration of ethanolic extract of leaves of C. winterianus and the lowest LC50 value

(0.011 %) was recorded for ethanolic extracts of leaves of V. negundo. The results indi-

cated that these plant extracts have potential to be developed as herbal acaricides.

Keywords Acaricidal activity � Cymbopogon winterianus � Hyalomma

anatolicum � Vitex negundo � Withania somnifera

N. K. Singh (&) � Jyoti � A. Nandi � H. SinghDepartment of Veterinary Parasitology, College of Veterinary Science, Guru Angad Dev Veterinaryand Animal Sciences University, Ludhiana 141004, Indiae-mail: [email protected]

B. Vemu � V. K. DumkaDepartment of Veterinary Pharmacology and Toxicology, College of Veterinary Science, Guru AngadDev Veterinary and Animal Sciences University, Ludhiana 141004, India

R. KumarDepartment of Agronomy, Punjab Agricultural University, Ludhiana 141004, India

123

Exp Appl Acarol (2014) 63:423–430DOI 10.1007/s10493-014-9791-1

Page 2: Laboratory assessment of acaricidal activity of Cymbopogon winterianus, Vitex negundo and Withania somnifera extracts against deltamethrin resistant Hyalomma anatolicum

Introduction

Hyalomma anatolicum, a three-host tick species, is widely distributed over most geo-

graphical regions in India infesting cattle, buffaloes, sheep and goat and transmitting

Theileria annulata, T. buffeli, T. lestocardi (T. hirci) (Ghosh et al. 2007). Along with

vectorial potential, the direct effect of this tick on livestock production leads to significant

economic losses. Tick control now is exclusively dependent on a large scale and repeated

use of synthetic acaricides, however, application of these chemical acaricides has had

limited efficacy in reducing tick infestations and is often accompanied by serious draw-

backs, including the development of acaricide resistant ticks, environmental contamina-

tion, and even contamination of milk and meat products with insecticide residues (Graf

et al. 2004). To reduce the utilization of acaricides other approaches including use of hosts

with natural resistance to ticks, use of biological control agents and tick vaccines has been

meagerly evaluated against multi host ticks (Ghosh et al. 2008). In order to offer an eco-

friendly acaricide, there is a need to assess botanical extracts, especially from plants which

are rustic, perennial, easily cultivable and have a better potential of extension, in addition

to their inherent acaricidal properties.

Cymbopogon winterianus (Citronella grass) a tall perennial grass, native to India and

tropical Asia is a medicinal plant from the Poacea family with insecticidal and repellent

activity (Shasany et al. 2000). Vitex negundo L. (Nirgundi) belongs to family Verbenaceae

is a hardy plant, flourishing mainly in the Indian subcontinent and is used as a bio-pesticide

and mosquito repellent (Singh et al. 2011). Withania somnifera (Ashwagandha) is a plant

of Solanaceae family cultivated in many of the drier regions of India with insecticidal

activity (Bansal et al. 2011). Although acaricidal activity against cattle tick Rhipicephalus

(Boophilus) microplus of C. winterianus (Martins 2006; Olivo et al. 2008) and V. negundo

(Kamaraj et al. 2010; Singh et al. 2014) has been reported, the acaricidal activity of these

plants has not been reported against H. anatolicum. Therefore, an attempt has been made in

this study to assess the acaricidal effect of V. negundo, W. somnifera and C. winterianus

against the larvae of H. anatolicum.

Materials and methods

Ticks

Fully engorged females ticks were collected from cattle sheds of Moga, Punjab (India).

After identification the ticks were kept individually in labelled plastic tubes covered with

muslin cloth and kept in desiccators placed in BOD incubator maintained at 28 ± 1 �C and

85 ± 5 % relative humidity for oviposition. The eggs laid were allowed to hatch to larvae

under similar conditions of incubation.

Acaricides

Technical grade (100 % pure) cypermethrin and deltamethrin (AccuStandard�, USA) were

used for conducting Larval Packet Test (LPT). The acaricides were dissolved in methanol

for preparation of stock solutions and different concentrations of cypermethrin and del-

tamethrin were prepared in distilled water from the stock solutions for testing against H.

anatolicum.

424 Exp Appl Acarol (2014) 63:423–430

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Larval packet test (LPT)

The LPT was conducted according to FAO (1971) guidelines with minor modifications.

Briefly, 0.5 ml of different concentrations of cypermethrin (50, 100, 200, 400 and

800 ppm) and deltamethrin (12.5, 25, 50, 100 and 200 ppm) in water were used to

impregnate 7 9 7 cm filter paper (541 Whatman). The aqueous solution of acaricide was

dried by keeping the filter paper for 30 min in incubator at 37 �C. The filter papers were

then folded in half diagonally and sealed on one side with adhesive tapes, forming an

open-ended triangular packet to place tick larvae. After insertion of approximately 100

larvae, the open end of each packet was sealed with adhesive tape and the packets were

placed in a desiccator placed in BOD incubator maintained at 28 ± 1 �C and 85 ± 5 %

RH. For each concentration of acaricide the test was conducted in triplicate and in

control group distilled water was used. The packets were removed after 24 h, and larval

mortality was calculated.

Estimation of resistance status

Dose response data were analyzed by probit method (Finney 1962) using GraphPad Prism

4 software. The lethal concentration for 50 % (LC50) and 95 % (LC95) values of cyper-

methrin and deltamethrin against H. anatolicum were determined by applying regression

equation analysis to the probit transformed data of mortality. Resistance factors (RF) was

worked out by the quotient between LC50 of field ticks and LC50 of susceptible line of H.

anatolicum (Castro-Janer et al. 2009). The LC50 values of cypermethrin and deltamethrin

against acaricides susceptible reference IVRI-II line of H. anatolicum were used as per

Shyma et al. (2012) and were 245.91 and 11.7 ppm, respectively. On the basis of RF, the

resistance status was classified as susceptible (RF \1.4), level I (RF = 1.5–5.0), level II

(RF = 5.1–25.0), level III (RF = 25.1–40) and level IV (RF[40.1) (Sharma et al. 2012).

Plant material

The leaves of V. negundo, W. somnifera and C. winterianus along with roots of V. negundo

were collected from Herbal garden, Department of Agronomy, Punjab Agricultural Uni-

versity, Ludhiana, India.

Preparation of plant extract

The leaves of V. negundo, W. somnifera and C. winterianus (both tender and old) along

with the roots of V. negundo were cleaned, shade dried at room temperature and were

finely pulverized using a grinder. The plant extraction was carried out by maceration

wherein 100 g each of the powdered material was added with the solvent in the ratio of

1:20 (distilled water) and 1:10 (ethanol) separately. The flasks were agitated for frequent

mixing over a period of 24 h. The mixtures were filtered using muslin cloth followed by

filter paper (No. 5 B—Advantec�) and the filtrates were completely air dried at 40 �C and

yields were measured. The required quantity of aqueous and ethanolic extracts were

weighed and dissolved in distilled water and 10 % DMSO, respectively for preparing the

working dilutions of 0.1, 1 and 5 % to test their acaricidal potential against H. anatolicum.

Exp Appl Acarol (2014) 63:423–430 425

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Bioassay

For evaluation of anti-tick activity of these extracts LPT as described above was adopted.

Various concentrations (0.1, 1.0 and 5.0 %) of aqueous extracts of leaves of C. winterianus

(CLA), W. somnifera (WLA), V. negundo (VLA) and roots of V. negundo (VRA), as well

as ethanolic extracts of leaves of C. winterianus (CLE), W. somnifera (WLE), V. negundo

(VLE) and roots of V. negundo (VRE) were used and the larval mortality in H. anatolicum

was determined against each concentration after exposure of 24 h.

Analysis of data

Dose response data were analyzed by probit method using GraphPad Prism 4 software and

LC50 values of various plant extracts against H. anatolicum were determined.

Results

Resistance status

The ticks collected were identified as H. anatolicum. The slope, LC50, LC95, RF values and

the level of resistance to cypermethrin and deltamethrin are shown in Table 1. Results

indicate presence of level I resistance to deltamethrin (RF = 2.81), whereas the tick isolate

was susceptible to cypermethrin (RF = 0.2).

In vitro efficacy of aqueous extracts

Yield, mortality, slope and LC50 values of aqueous extracts of leaves of C. winterianus, W.

somnifera, V. negundo and roots of V. negundo against deltamethrin resistant H. anatol-

icum are shown in Table 2. The efficacy of various extracts against H. anatolicum larvae

were assessed by estimating mortality. Larval mortality caused by the aqueous extracts of

different plants varied from 0.0 to 28.97 %, when tested at concentrations ranging from 0.1

to 5.0 %. Further, because of the low mortality the LC50 values of aqueous extracts against

H. anatolicum were impracticable except WLA with LC50 value of 20.3 %.

In vitro efficacy of ethanolic extracts

Yield, mortality, slope and LC50 values of ethanolic extracts of leaves of C. winterianus,

W. somnifera, V. negundo and roots of V. negundo against deltamethrin resistant H.

anatolicum are shown in Table 3. The larval mortality caused by the ethanolic extracts

showed a concentration dependent increase and for different extracts varied from 9.7 to

93.7 % at concentrations of 0.1–5.0 %. Among all ethanolic extracts the best acaricidal

Table 1 The results of larval packet test to various acaricides performed on Hyalomma anatolicum

Acaricide Slope (95 % CL) R2 LC50 (ppm)

(95 % CL)

LC95 (ppm)

(95 % CL)

RF RL

Cypermethrin 2.87 ± 0.39 (1.62–4.13) 0.946 49.26 (48.77–49.75) 183.21 (176.50–190.17) 0.2 S

Deltamethrin 3.56 ± 0.75 (1.17–5.95) 0.882 32.89 (32.40–33.38) 94.9 (92.13–97.74) 2.81 I

RF resistance factor, RL resistance level

426 Exp Appl Acarol (2014) 63:423–430

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property was exhibited by the VLE with the minimum LC50 values of 0.011 %, followed

by CLE as 0.14 %, VRE as 1.27 % and maximum for WLE as 10.12 % (Table 3).

Discussion

The problem of ticks and tick borne diseases is particularly relevant in India because of the

congenial environmental conditions for tick survival throughout the most parts of the year

and maintenance of susceptible cross bred animals to improve the production of milk and

other animal products. Among the various ixodid ticks H. anatolicum is one of the

important ticks infesting dairy animals of India (Ghosh et al. 2007) particularly Punjab

state (Haque et al. 2011; Singh and Rath 2013). The use of acaricides as the principal

means for tick control has resulted in the selection of chemical resistant ticks. Presence of

wide spread resistance to cypermethrin and deltamethrin in H. anatolicum by LPT has been

recently reported from different places of India (Shyma et al. 2012; Singh et al. 2013). In

bioassays (LPT), technical grade cypermethrin and deltamethrin were selected over

commercial formulation as commercial products are prepared with many proprietary

ingredients and it is difficult to assess the responses due to active ingredients (Shaw 1966).

Further, the stock solutions were prepared by dissolving in 100 % methanol and the

working concentrations were prepared with distilled water. Use of organic solvent facili-

tates adsorption of compound over the surface area of target biological materials and also

enhances penetration of active ingredients of the acaricide across the exoskeleton (Sharma

et al. 2012).

Acaricides of herbal origin stand out as an important possible alternative as future

acaricide (s) as they are environment friendly and the problem of resistance development is

minimal. During the last few years, many plant extracts have been tested for their acari-

cidal effects against R. (B.) microplus (Chungsamarnyart and Jiwajinda 1992; Martins

2006; Olivo et al. 2008; Kamaraj et al. 2010; Singh et al. 2014). However, studies on

Table 2 The effect of various aqueous plant extracts on larvae of Hyalomma anatolicum

Plant Yield(%)

Conc.(%)

Mortality (%)(mean ± SE)

Slope (95 %CL)

R2 LC50 (%)

Vitex negundo (root) 8.97 0.1 5.06 ± 0.81 0.103 ± 0.091(-1.05 to1.26)

0.562 7.61 9 1010

1.0 7.84 ± 1.96

5.0 7.11 ± 1.22

Vitex negundo (leaves) 18.17 0.1 6.26 ± 2.41 0.194 ± 0.054(-0.50 to0.89)

0.926 3.82 9 1018

1.0 9.69 ± 4.93

5.0 11.24 ± 2.55

Withania somnifera(leaves)

20.48 0.1 0 ± 0 1.035 ± 0.130(-0.61 to2.68)

0.984 20.25

1.0 6.85 ± 2.23

5.0 28.97 ± 1.7

Cymbopogonwinterianus (leaves)

16.32 0.1 0 ± 0 0.630 ± 0.455(-5.16 to6.42)

0.656 985.4

1.0 0 ± 0

5.0 12.28 ± 1.17

Control (DW) – – 0 ± 0 – – –

Exp Appl Acarol (2014) 63:423–430 427

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herbal acaricides against H. anatolicum tick are limited in number (Abdel-Shafy and Zayed

2002) and as per our knowledge no published reports are available on the effect of C.

winterianus, W. somnifera, V. negundo extracts on deltamethrin resistant H. anatolicum

ticks.

Various species of the genus Cymbopogon (Poaceae) showed high acaricidal activity

against the larvae and adult females of R. (B.) microplus (Chungsamarnyart and Jiwajinda

1992; Olivo et al. 2008), Amblyomma cajennense (Soares et al. 2010), R. sanguineus

(Heimerdinger et al. 2006). Further, C. winterianus has also been investigated against R.

(B.) microplus (Martins 2006; Singh et al. 2014). These properties are attributed to the

presence of volatile substances in its leaves such as citronellal, eugenol, geraniol and

limonene, among others, denominated in general as monoterpenes (Shasany et al. 2000).

Results of the current study indicate that the ethanolic extracts of leaves of C. winterianus

(LC50 = 0.14 %) can prove to be a promising herbal acaricide against deltamethrin

resistant H. anatolicum.

The leaves, flowers, fruit, roots and bark of V. negundo have great medicinal value and

has proven insecticidal property against various agricultural pests. The acaricidal property

of leaf extracts of V. negundo against cattle tick R. (B.) microplus has been studied

(Kamaraj et al. 2010; Singh et al. 2014). The current study report a LC50 value of 0.011 %

against deltamethrin resistant H. anatolicum. The acaricidal property may be due to the

number of constituents that are present in the plant as terpenoids, irridoids, steroids,

phenolic compounds, lignane derivatives, amino acids, fatty acids and aliphatic alcohol

(Singh et al. 2011). Similarly, in recent past W. somnifera has also been evaluated for its

larvicidal property against larvae of Anopheles stephensi, Aedes aegypti and Culex quin-

quefasciatus (Bansal et al. 2011). The acaricidal property against R. (B.) microplus has also

been reported (Singh et al. 2014) but effects against H. anatolicum has not been reported

and the current study reports LC50 values of 20.25 and 10.12 % of the aqueous and

ethanolic extracts of leaves of W. somnifera against deltamethrin resistant H. anatolicum.

The acaricidal activity may be because of the presence of biologically active chemical

constituents as alkaloids (isopelletierine, anaferine), steroidal lactones (withanolides,

Table 3 The effect of various ethanolic plant extracts on larvae of Hyalomma anatolicum

Plant Yield(%)

Conc.(%)

Mortality (%)(mean ± SE)

Slope (95 %CL)

R2 LC50 (%) (95 %CL)

Vitex negundo (root) 1.9 0.1 9.74 ± 2.76 1.045 ± 0.314(-2.94 to5.03)

0.917 1.27 (1.21–1.33)

1.0 58.48 ± 14.48

5.0 66.66 ± 12.27

Vitex negundo(leaves)

9.34 0.1 66.01 ± 1.60 0.381 ± 0.131(-1.28 to2.04)

0.894 0.011 (0.010–0.013)

1.0 73.05 ± 22.11

5.0 85.66 ± 4.81

Withania somnifera(leaves)

10.46 0.1 11.23 ± 1.81 0.566 ± 0.241(-2.49–3.62)

0.846 10.12 (9.21–11.11)

1.0 36.81 ± 7.08

5.0 37.78 ± 2.22

Cymbopogonwinterianus(leaves)

6.35 0.1 40.57 ± 9.42 1.064 ± 0.149(-0.83 to2.95)

0.980 0.14 (0.13–0.15)

1.0 84.82 ± 4.05

5.0 93.68 ± 0.66

Control (10 %DMSO)

– – 4.05 ± 0.48 – – –

428 Exp Appl Acarol (2014) 63:423–430

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withaferins), saponins containing an additional acyl group (sitoindoside VII and VIII), and

withanolides with a glucose at carbon 27 (sitoindoside IX and X) (Mishra et al. 2000).

The beneficial medicinal effects of plant materials typically result from active com-

pounds present in the plant and can be due to effects of a single compound or combination

of one or more. The chemical constituents of the plants may exert acaricidal effects in

different ways; therefore, the development of resistance seems difficult against botanical

acaricides in combination. Further, studies are needed to identify the active ingredients

present in these plant that caused the mortality of tick larvae. Analyzing the data obtained

in the present study, it can be concluded that the ethanolic extract of leaves of V. negundo

and C. winterianus possesses acaricidal properties which may provide an effective eco-

friendly herbal formulation for the control of deltamethrin resistant tick infestation on

animals.

Acknowledgments Authors are thankful to the Director of Research, GADVASU, Ludhiana for providingfacilities to carry out the research work.

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