Journal of Parasitology Research

Journal of Parasitology Research / 2020 / Article

Research Article | Open Access

Volume 2020 |Article ID 7834026 | 10 pages | https://doi.org/10.1155/2020/7834026

In Vitro Acaricidal Activity of Selected Medicinal Plants Traditionally Used against Ticks in Eastern Ethiopia

Academic Editor: D. S. Lindsay
Received03 Sep 2019
Revised10 Dec 2019
Accepted28 Jan 2020
Published18 Feb 2020

Abstract

A study was carried out to evaluate the acaricidal activities of crude methanolic extract of leaves of six medicinal plants, namely, Vernonia amygdalina, Calpurnia aurea, Schinus molle, Ricinus communis, Croton macrostachyus, and Nicotiana tabacum, against Rhipicephalus (Boophilus) decoloratus and Rhipicephalus pulchellus using an in vitro adult immersion test. Five graded concentrations of the crude extracts, 6.25, 12.5, 25, 50, and 100 mg/ml, were tested at different time intervals, and temporal changes in tick viability were recorded for 24 hours. Diazinon (0.1%) and distilled water were used as positive and negative controls, respectively. Standard procedures were applied to screen the phytochemical constituents of the tested plant parts. Phytochemical screening showed the presence of a condensed amount of tannins in all extracts. Starting from 30 min post exposure, the 100 mg/ml concentration of C. aurea and R. communis extracts has caused significantly higher mortality () compared to the diazinon. A significant increase in tick mortality started 2 hr post exposure with diazinon and 50 and 100 mg/ml concentrations of S. molle. Vernonia amygdalina extract and diazinon showed a significant increase in tick mortality 3 hr post exposure with 100 mg/ml concentration. Similarly, a significant increase in tick mortality started 2 hr post exposure with diazinon and 100 mg/ml concentrations of C. macrostachyus and N. tabacum. At 24 hr post exposure, diazinon and 50 and 100 mg/ml concentrations of all the extracts have caused significantly higher tick mortality than the rest of the concentrations (). Higher concentrations of all the extracts had showed a comparable and strong acaricidal effect on Rhipicephalus (Boophilus) decoloratus and Rhipicephalus pulchellus having no significant difference with that of the positive control () at 24 hr post exposure period. Tick killing activity of all evaluated plant extracts increases with increasing exposure time and concentration as well. Thus, all the tested plants could be used against Rhipicephalus (Boophilus) decoloratus and Rhipicephalus pulchellus as a potential alternative to substitute commercially available drugs. We recommend further study on fractionating each component separately and validating the materials.

1. Introduction

Ticks are destructive blood-sucking ectoparasites of livestock and wild animals causing huge economic losses, thus creating food insecurity [1], with an estimated global cost of control and productivity losses of 7 billion USD per year [2]. Their effects are diverse, including reduced growth and milk production, paralysis/toxicosis, and transmission of tick-borne diseases (TBDs) that reduce production or cause mortality [3].

Worldwide tick control is based mainly on the repeated use of acaricides, which have resulted in problems related to environmental pollution, milk and meat contamination, and the development of resistance leading to increased cost of control [4]. In Ethiopia, over the past decades, ticks are mainly controlled by using a variety of synthetic acaricides [5]. However, ticks have developed resistance against commercial acaricides in Ethiopia with the widespread, under- or overconcentrated, and frequent use of these compounds [6, 7]. Thus, there is an urgent need for new tick control strategies to overcome the drawback associated with the use of synthetic drugs. One alternative control strategy could be phytotherapy, an important component of ethnoveterinary medicine [8].

The use of natural products, mainly acaricides from the botanical source used for the control of ticks, has been the focus of research in many countries, principally to withstand the noticeable increasing frequency of acaricide-resistant tick strains. The use of botanicals for the control of ticks is compatible with traditional practices in Africa, including Ethiopia where most resource-poor farmers use plant materials to treat ectoparasites and endoparasites of livestock [2]. Acaricidal activity of crude extracts from different plants against ticks has been reported [913]. The phytoextracts produce acaricidal properties through diverse mechanisms: killing adult ticks, reducing tick feeding, and inhibition of egg hatching, molting, fecundity, and viability of eggs [8, 14, 15].

In far rural parts of the country including eastern Hararghe, modern veterinary drugs including synthetic acaricides are not affordable to the majority of poor livestock owners. Even when accessible, the owners tend to treat their livestock with synthetic products in a haphazard way, and misuse of chemicals illegally imported at a high cost from neighboring countries is increasing year after year. Furthermore, there is a continuous complain from the livestock keepers over the poor efficacy of most of the existing acaricidal drugs. As a result, livestock raisers continue to rely on ethnoveterinary knowledge and practices for socially acceptable, inexpensive, and locally available remedies for managing ticks and other ectoparasites affecting their livestock. Additional fact that desires due attention is that livestock owners claim a number of botanicals having acaricidal effect which need scientific evaluation by using standardized parasitological procedures.

Thus, it is quite rightly convincing that evaluating acaricidal efficacy of natural remedies can greatly contribute to control tick invasion in a cheaper and more sustainable way. Moreover, there is a great potential to develop sources of acaricides from the available medicinal plants which can be easily produced by livestock producers themselves or processed by cottage industries and used as cheap and efficient natural biocide for tick control [1]. Implementing an effective tick control strategy suitable to a specific livestock production system is a dual harvest, controlling ticks and also the TBDs. Therefore, it is necessary to undertake an acaricidal efficacy evaluation of botanicals traditionally used by the livestock owners as an alternative tick management strategy. The present work was aimed at evaluating the acaricidal activity of crude extracts of leaves of six medicinal plants traditionally used by livestock raisers as an alternative tick control strategy in eastern Hararghe, Ethiopia. The plants tested in the current biological activity assay include Vernonia amygdalina, Calpurnia aurea, Schinus molle, Ricinus communis, Croton macrostachyus, and Nicotiana tabacum leaf extracts against Rhipicephalus (Boophilus) decoloratus and Rhipicephalus pulchellus for antitick activity.

2. Materials and Methods

2.1. Study Design

This study employed an experimental study design: an in vitro immersion method as described by Vongkhamchanh et al. [15].

2.2. Collection and Identification of the Plant Materials

The plants were selected based on the scientific and ethnomedical information in the literature complemented with a preliminary ethnobotanical survey during collection from their natural habitats. The candidate plants that were included for the experiment were V. amygdalina (Girawa/Dhebichaa), S. molle (Mirmir/Muka libaanataa), C. aurea (Digita/Ceekaa), R. communis (Gulo/Qobboo), C. macrostachyus (Bisana/Makannisa), and N. tabacum (Tumbahoo/Tamboo). The selected plants were collected, identified, and verified with taxonomical studies as reported by [16]. To reduce possible contamination, especially by fungi, latex gloves were worn during leaf collection.

2.3. Crude Extract Preparation

Fresh leaves of the plants were separately cleaned with tap water to remove dirt and soil particles, shade dried at room temperature for two weeks, mechanically grinded, and coarsely powdered using an electric grinder. The powdered specimen was then subjected to extraction using 80% methanol by a cold maceration technique. A total of 500 g of the pounded materials was separately soaked in each extraction solvent (100 g of powder in 400 ml of solvent) followed by shaking periodically for three days and then filtered. This was repeated three times to allow the solvents extract substantial quantities of the chemical constituents from the pounded plant materials. The mixture was first filtered using gauze, and then, the filtrate was passed through sterile filter paper (Whatman No. 1, Whatman Ltd. England). Then, the filtered extract was kept overnight in a hot air oven at a temperature of 60°C to obtain the pure crude extracts. The extraction rate (%) was calculated as follows:

The resulting extracts were then transferred into well-labeled vials and kept in a refrigerator until required for use.

2.4. Phytochemical Screening

Phytochemical screening was carried out to assess the qualitative chemical composition of crude extracts using commonly employed precipitation and coloration reaction to identify the major natural chemical groups and secondary metabolites present in the plants. Combinations of several methods were used to identify the phytochemicals of the medicinal plants. Standard screening tests were conducted using a conventional protocol and reagents on the methanolic extracts of herbs to identify the constituents as described by Sofowora [17]. The screening was done to detect the presence of bioactive principle believed to have acaricidal activities: saponins, tannins, flavonoids, steroids, phenolic compounds, alkaloids, glycosides, and triterpenes.

2.5. Study Parasite Collection, Transportation, and Identification

Ticks were collected from naturally infested cattle in Haramaya (Finkille, Adelle) and Harar (Erer, Dire Teyara), Eastern Ethiopia. For collection of ticks, the entire body surface of the animals was examined thoroughly, and adult ticks were collected from the body of the animals where they were available. Collected ticks were put in vials and were wrapped in cotton net gauze for oxygen supply and transported and identified in Haramaya University College of Veterinary Medicine, Parasitology Laboratory. All collected ticks from naturally infested cattle were examined under a stereomicroscope and identified according to [18].

2.6. Acaricidal Activity Evaluation
2.6.1. Preparation of Concentrations of Crude Methanolic Extracts

The dried extracts were diluted in distilled water at the concentrations required for the bioassays (6.25 mg/ml, 12.5 mg/ml, 25 mg/ml, 50 mg/ml, and 100 mg/ml) for all tested plants. The concentrations were used for the acaricidal efficacy test. Distilled water was used as the negative control while 0.1% diazinon was used as the positive control. The positive control, 0.1% diazinon 60 EC (Adamitulu Pesticides Processing, Ethiopia), was diluted in water according to the manufacturer’s recommendation (1 : 1000) before being used for further experiment [19].

2.6.2. Adult Immersion Test

The in vitro tests were started within one hour after tick collection [19]. Ten active live adult ticks in three replications were put into the Petri dish, and 3 ml of each concentration was directly added to the three replicated Petri dishes for 2 min of exposure. After immersion, the ticks were filtered with filter paper and placed in separate Petri dishes [20]. Three millilitres of distilled water and 0.1% diazinon 60 EC were used as the negative and positive controls, respectively. The Petri dishes were incubated at 28°C with 80% relative humidity, and each tick in each Petri dish was closely observed for any death under a stereomicroscope at 30 min, 1 hr, 2 hr, 3 hr, 6 hr, 12 hr, and 24 hr time intervals [21]. The viability of ticks was checked regularly by stimulation with a needle, and ticks were recorded as dead if no reaction was shown. The percentage mortality was calculated by using a formula given by Krishnaveni and Venkatalakshmi [22] as follows:

2.7. Data Analysis

Collected raw data was stored in a Microsoft Excel database system used for data management. SPSS Windows version 20 was used for data analysis. Mean tick mortality and related results of the study were expressed using descriptive statistics (, percentage, and graph). One way analysis of variance (ANOVA) followed by Tukey’s HSD multiple comparisons was used to compare differences between different in vitro groups. All significant levels are set at .

3. Result

3.1. Percentage Extraction Yield and Phytochemical Constituents

Table 1 summarizes the percentage extraction yields of 500 g powder of leaves of each plant. Each extract had a dark brown color and sticky and semisolid consistence. The highest and lowest percentage yields obtained were 30.67% and 12% for R. communis and V. amygdalina, respectively. The phytochemical constituents detected in the resultant crude extracts are shown in Table 2. Phytochemical screening showed the presence of condensed tannins and alkaloids in all extracts.


Plant nameYield in gramYield in %

C. aurea6220.67
S. molle5819.33
V. amygdalina3612
R. communis7230.67
C. macrostachyus6829.3
N. tabacum4622


IngredientsC. aureaS. molleV. amygdalinaR. communisC. macrostachyusN. tabacum

Saponin++++
Tannin++++++
Phenolic compounds++++
Steroids+++
Flavonoids++++
Phlobatannin+++
Glycosides+++
Triterpenes
Alkaloids+++++

Note: +: present; −: negative.
3.2. In Vitro Acaricidal Activity of the Plant Extracts against Rh. decoloratus and pulchellus

A significant increase in tick mortality started 3 hr post exposure with 100 mg/ml concentration of C. aurea extract and diazinon and 12 hr post exposure with 50 mg/ml concentration of the C. aurea extract. Starting from 30 min post exposure, the 100 mg/ml concentration of C. aurea extract has caused significantly higher mortality compared to the diazinon (). At 24 hr post exposure period, diazinon and 50 and 100 mg/ml concentrations of the extract have caused significantly higher tick mortality compared with the rest of the concentrations below 25 mg/ml (). The least concentration (6.25 mg/ml) has caused significantly higher mortality when compared with the negative control (distilled water) at 24 hr exposure time.

A significant increase in tick mortality started 2 hr post exposure with diazinon and 50 and 100 mg/ml concentrations of S. molle and 12 hr post exposure with 6.25, 12.5, and 25 mg/ml concentrations of S. molle extract. At 24 hr post exposure period, 25, 50, and 100 mg/ml concentrations of the extract and diazinon had a comparable tick killing effect compared with the rest of the concentrations below 12.5 mg/ml. However, the three higher concentrations of the extracts and diazinon had no significant difference in their effect on the parasite ().

Starting from 3 hr post exposure with 100 mg/ml concentration of V. amygdalina leaf extract and diazinon, there showed a significant increase in tick mortality. Starting from 12 hr post exposure, the 6.25 mg/ml concentration of V. amygdalina extract has caused significantly higher mortality compared to the distilled water (negative control) (). At 24 hr post exposure period, diazinon and 25, 50, and 100 mg/ml concentrations of the extract have caused significantly higher tick mortality than the rest of the concentrations below 12.5 mg/ml () (Figure 1).

A significant increase in tick mortality started 2 hr post exposure with 100 mg/ml concentration of R. communis extract and diazinon and 3 hr post exposure with 100 mg/ml concentration of the extract. At 24 hr post exposure period, diazinon and 50 and 100 mg/ml concentrations of the extract have caused significantly higher tick mortality than the rest of the concentrations below 25 mg/ml (). The least concentration (6.25 mg/ml) has caused significantly higher mortality when compared with the negative control at 24 hr exposure time (Figure 1).

Tick mortality was significantly increased starting from 2 hr and in 3 hr post exposure of 100 mg/ml concentrations C. macrostachyus and diazinon, respectively. At the 24 hr post exposure period, 50 and 100 mg/ml concentrations of the C. macrostachyus extract and diazinon had a comparable tick killing effect than the rest of the concentrations below 25 mg/ml (Figure 1).

At the 24 hr post exposure period, diazinon and 50 and 100 mg/ml concentrations of the N. tabacum extract have caused significantly higher tick mortality than the rest of the concentrations below 25 mg/ml (). However, the three higher concentrations of the extract and diazinon had no significant difference in their effect on the parasite. Moreover, there is no significant difference between the three higher concentrations tested (≥50 mg/ml) (Figure 1).

The of Rh. (B). decoloratus and Rh. pulchellus at minute/hour post exposure with different concentrations of C. aurea, S. molle, V. amygdalina, R. communis, C. macrostachyus, and N. tabacum was showed in Tables 38. The findings indicated that both C. aurea and S. molle showed mortality whereas V. amygdalina, R. communis, C. macrostachyus, and N. tabacum showed , , , and , respectively, mortality effect after 24 hr of exposure to 100 mg/ml concentrations (Tables 38).


Extract concentration (mg/ml)Mean number of ticks dead () at minute/hour post exposure
30 min1 hr2 hr3 hr6 hr12 hr24 hr

100bbbbcdecde
50aabababcdeccde
25aaababbcdcbcd
12.5aaababccbc
6.25aaaaabbb
0.1% diazinonaabbcede
Distilled wateraaaaaaa

Means followed by the same letter on the same column are not significantly different by ANOVA ().

Extract concentration (mg/ml)Mean number of ticks dead () at minute/hour post exposure
30 min1 hr2 hr3 hr6 hr12 hr24 hr

100abbcdcdcd
50aabbcccbc
25aaabbccbc
12.5aaabbcbcb
6.25aaababbb
0.1% diazinonabcddd
Distilled wateraaaaaa

Means followed by the same letter on the same column are not significantly different by ANOVA ().

Extract concentration (mg/ml)Mean number of ticks dead () at minute/hour post exposure
30 min1 hr2 hr3 hr6 hr12 hr24 hr

100aaabcdcddebc
50aaababcbccdbc
25aaabcbbcb
12.5aaaababbcb
6.25aaaaabb
0.1% diazinonaabddec
Distilled wateraaaaaaa

Means followed by the same letter on the same column are not significantly different by ANOVA ().

Extract concentration (mg/ml)Mean number of ticks dead () at minute/hour post exposure
30 min1 hr2 hr3 hr6 hr12 hr24 hr

100abbabbcb
50ababcabcb
25aaabbcabcb
12.5aaabbab
6.25aaaaabcb
0.1% diazinonaaabcabcb
Distilled wateraaaaaa

Means followed by the same letter on the same column are not significantly different by ANOVA ().

Extract concentration (mg/ml)Mean number of ticks dead () at minute/hour post exposure
30 min1 hr2 hr3 hr6 hr12 hr24 hr

100aababababa
50aaaabbaba
25aaaabbaba
12.5aaaaabba
6.25aaaaaba
0.1% diazinonaaabababa
Distilled wateraaaaaaa

Means followed by the same letter on the same column are not significantly different by ANOVA ().

Extract concentration (mg/ml)Mean number of ticks dead () at minute/hour post exposure
30 min1 hr2 hr3 hr6 hr12 hr24 hr

100abbababab
50aababababab
25aaababbb
12.5aaabababab
6.25aaabababab
0.1% diazinonaababbabab
Distilled wateraaa