INTRODUCTION

Animal ectoparasites are parasites that reside on the exterior of their animal hosts, presenting significant challenges to animal welfare across various hosts such as livestock, pets, and wildlife. Mites, eight-legged arthropods belonging to the class Arachnida, are among the notable ectoparasites affecting animals (Bowman, 2009). Mites of the genus Psoroptes are particularly notorious for causing severe dermatological issues. Psoroptes cuniculi, a prevalent ectoparasite found in rabbits, is responsible for causing ear mange (Ulutas et al., 2005) (Figure 1). This condition is characterized by intense itching, inflammation, and secondary bacterial infections. Infestation by P. cuniculi causes physical discomfort and poses health risks to affected rabbits. A study by Mohamad-Radzi et al. (2021) reported a prevalence of mite infestations exceeding 50% in rabbit farming in Selangor, Malaysia. Such infestations lead to economic losses in commercial rabbit farming by reducing production and increasing veterinary expenses.

Traditionally, chemical acaricides have been employed to control Psoroptes cuniculi infestations. However, excessive and improper use of synthetic acaricides has led to the development of resistance in mite populations, environmental pollution, and residues of chemicals in animal products (Seddiek et al., 2013). These challenges underscore the need to explore alternative control methods that are effective and environmentally sustainable. Plant-derived compounds have garnered attention as promising alternatives due to their bioactive properties and minimal environmental impact (Nwanade et al., 2022).

 

Senna alata, also known as Cassia alata, is a flowering shrub belonging to the Fabaceae family. Native to South America, it is commonly found in tropical regions, including Malaysia (Gritsanapan and Mangmeesri, 2009). Senna alata is known by various local names such as candle bush, ringworm bush, ‘ketepeng cina’, and in Malaysia, it is referred to as ‘gelenggang’. This shrub typically grows to a height of 1-2 meters, occasionally reaching up to 5 meters. Gelenggang is distinguished by its vibrant yellow inflorescence resembling candlesticks, which gives rise to its common name (Figure 2).

 

Senna alata, known for its medicinal properties, has been traditionally used to treat various ailments including typhoid, diabetes, malaria, asthma, ringworms, tinea infections, scabies, blotch, herpes, and eczema (Fatmawati, 2020). This plant exhibits a wide array of biological activities attributed to its components such as flowers, roots, leaves, stems, seeds, and bark. These activities include antioxidant, anti-inflammatory, abortifacient, antimicrobial, antiallergic, antilipogenic, antibacterial, antidiabetic, antifungal, anticancer, and antiviral properties (Oladeji et al., 2020). Locally in Malaysia, boiled Gelenggang leaves have been traditionally used to treat rabbits with ear mite infections.

The bioactive properties of S. alata are attributed to phytochemical constituents extracted from the plant, including flavonoids, phenolics, alkaloids, anthraquinones, tannins, terpenoids, and steroids (Angelina et al., 2021). A previous study has assessed the acaricidal efficacy of S. alata leaf extract against Rhipicephalus annulatus, a common tick species in cattle (Ravindran et al., 2012), and it was suggested that anthraquinone may contribute to the acaricidal activity of S. alata extracts due to its anti-mite properties (Phongpaichit et al., 2004).

Despite its extensive traditional medicinal use, the potential acaricidal effects of S. alata against P. cuniculi, particularly in rabbits, have not been extensively studied. An in-silico study has been conducted using phytochemicals from S. alata against two targeted proteins of the cattle tick Rhipicephalus (Boophilus) microplus, suggesting potential herbal acaricidal properties (Priyadarshini et al., 2019). However, further in vitro and in vivo studies are needed to validate these findings. Therefore, this study aims to evaluate the acaricidal activity of Senna alata leave extract against rabbit ear mites, Psoroptes cuniculi, using both in vitro and in vivo approaches.

MATERIALS AND METHODS

Ethical Approval

This study adhered to ethical guidelines approved by the UniSZA Animal and Plant Research Ethics Committee (UAPREC). The research strictly followed UAPREC’s protocols (UAPREC/008/020) to ensure minimal distress to the animals involved in the study.

Study Area

This study was conducted at the Hatchery and Laboratory facilities at the Universiti Sultan Zainal Abidin (UniSZA), Besut Campus, Terengganu, Malaysia.

Preparation of Senna Alata Leaves Extract

Senna alata leaves were harvested in Besut Terengganu (5°45’20.7”N 102°35’30.9”E) from March 2024 to May 2024. Old leaves were collected for this study. Maturity of the leaves was classified following a method by Dian Nashiela et al. (2016). After harvesting, the leaves were washed and air-dried for a week at 40°C, following the method described by Gritsnapan and Mangmeesri (2009). Subsequently, the dried leaves were finely ground into a powder. About twenty dry leaves of S. alata (n=20) were used to produce 10 g of powder form.

To prepare the extract, 10 g of powdered S. alata leaves were macerated in 100 ml of distilled water. The maceration process was conducted at 55°C for 120 minutes, as detailed by Karthika et al. (2016). The extraction procedure was repeated until complete exhaustion of the plant material. The combined maceration extracts were then filtered and dried to completeness using a rotary evaporator.

Mites Collection

New Zealand white rabbits weighing approximately 3-4 kg, afflicted with Psoroptes cuniculi infections, were sourced from Expert Rabbitry Global Empire in Kampung Tempinis, Besut, Terengganu. These rabbits were subsequently housed in designated hatchery facilities at Besut Campus, UniSZA.

Mites were collected using a skin scraping method adapted from Abdisa (2018) with slight modifications. The affected skin areas were vigorously scraped six to seven times to remove the top layer of the papules. The collected scabs containing mites were transferred into petri dishes and examined under a stereo microscope to isolate the mites.

In vitro Acaricidal Activity

Mite bioassays were conducted in triplicate following the method described by Fichi et al. (2007). The assay included seven treatments:

• Positive control (Doramectin + distilled water).
• T0 (distilled water only as negative control).
• T1 (40 mg/ml of Senna alata extract in distilled water).
• T2 (80 mg/ml of S. alata extract in distilled water).
• T3 (120 mg/ml of S. alata extract in distilled water).
• T4 (160 mg/ml of S. alata extract in distilled water).
• T5 (200 mg/ml of S. alata extract in distilled water).

Each solution was evenly spread over the surface of a 10 cm diameter petri dish using a sterile swab. Twenty adults of Psoroptes cuniculi mites (n=20) were then placed in each petri dish. The plates were kept at room temperature, and mite mortality was recorded every two hours for 24 hours. Mites were considered dead if they showed no reaction when stimulated with a needle.

The efficacy of S. alata extract against Psoroptes cuniculi was evaluated by calculating the percentage of mite mortality following the methodology outlined by Ravindran et al. (2012).

In vivo Acaricidal Activity

For in vivo study, only two concentrations of Senna alata extract were prepared based on the in vitro study results. In vivo test were conducted in four treatments: 160 mg/ml (T1) and 200 mg/ml (T2) of S. alata extract. For comparison, a negative control group that received only distilled water (NC) and a positive control group that was treated with 10 mg/ml doramectin injections (PC) were also included (Seddiek et al., 2013).

To evaluate the acaricidal activity through in vivo testing, 20 domestic rabbits (n=20) that were naturally infected with Psoroptes cuniculi were selected. These rabbits were then randomly divided into four groups of five, ensuring a balanced representation. Each group received 2 ml of the treatment solution, which was applied topically to the both left and right of the infected ears of the rabbits (Yipel et al., 2016).

The degree of infestation was evaluated for seven days on the basis of the following scoring system by Guillot and Wright (1981) as shown in Table 1 and Figure 3.

 

Table 1: Degree of infestation scoring system.

Score	Infestation Stage
0	Absence of scabs and/or mites
0.5	Irritation in the ear canal but no mites observed
1	Small number of scabs in the ear canal, mites present
2	External ear canals filled with scabs, mites present
3	Scabs in the ear canal and proximal 1/4of the pinna, mites present
4	1/2pinna filled with scabs, mites present
5	3/4of the pinna filled with scabs, mites present
6	All internal surfaces of the pinna are full of scabs, and mites present

 

Table presents the list of degree of infestation for the scoring system of mite’s infestation on rabbit’s ears.

 

 

Statistical Analysis

Regression analysis was conducted in Microsoft Excel. Probit analysis and one-way analysis of variance (ANOVA) were conducted using Minitab software. The results are expressed as means ± standard error (SE). Group comparisons among the extract were performed using the Tukey test, with significances at P < 0.05. Graphs were created using Origin software.

RESULTS AND DISCUSSION

Acaricidal Activity of Senna Alata Leaves Extract In Vitro

Figure 4 illustrates the outcomes of the in vitro tests using Senna alata leaf extract. The effectiveness of the extract against Psoroptes cuniculi was evaluated by determining the percentage of mite mortality. The extract’s acaricidal activity was tested across various concentrations ranging from 40 mg/ml to 200 mg/ml.

 

 

At the lowest concentration tested, 40 mg/ml, the mortality rate was 23%, which notably increased to 38% at 80 mg/ml. Further increments in concentration to 120 mg/ml and 160 mg/ml results mortality rates of 42% and 43%, respectively, suggesting a potential plateau effect. The highest concentration tested, 200 mg/ml, achieved a maximum mortality rate of 100%, confirming that higher concentrations are more effective in increasing mite mortality.

Results from the regression analysis demonstrated a significant positive correlation between the concentration of treatment and the mortality rate of Psoroptes cuniculi with the R2 value of 0.9492 as shown in Figure 5. This means that 94.92% of the variation in mite mortality is due to the treatment concentration. The findings show that as the dosage of the extract increases, so does the mortality rate of the mites, demonstrating the treatment’s efficacy in a dose dependent manner.

Figure 6 illustrates the relationship between the dosage of S. alata extract and mite mortality. The graph demonstrates that as the dosage of the extract increases, so does the mortality rate of the mites. The probit regression curve exhibits a steep slope significantly increasing in mite mortality at higher concentrations. The botanical extract from S. alata displays substantial acaricidal activity, with specific lethal concentrations for 50% and 90% mortality (LC50 and LC90) calculated at 282.45 mg/ml and 478.71 mg/ml, respectively.

 

Acaricidal Activity of Senna Alata Leaves Extract In Vivo

Figure 7 illustrates the degree of infestation observed across all four treatments over a seven-day period. In Figure 7a, the positive control group treated with doramectin, displaying a substantial decrease in mite infestation throughout the treatment period. The infestation level decreased progressively, indicating the efficacy of the standard treatment. By the end of the seven days, the infestation was nearly eradicated, validating doramectin as an effective benchmark for assessing the efficacy of Senna alata extracts.

The in vitro testing of Senna alata leaf extract in this study demonstrates its acaricidal properties against Psoroptes cuniculi. These findings are consistent with previous research, such as Ravindran et al. (2012), which showed the acaricidal activity of S. alata leaf extract against cattle ticks. The study reveals a dose-dependent acaricidal effect of S. alata extract, where higher concentrations lead to quicker mite mortality. Similar results were observed in a study by Magano et al. (2008) using Senna italica root extract, where increased extract concentrations correlated with higher mortality rates of the Mediterranean Hyalomma tick, Hyalomma marginatum rufipes.

 

Table 2: Degree of infestations score of Psoroptes cuniculi for different concentrations of Senna alata leaves extract

Concentration (mg/ml)	Mean Degree of Infestation ± SE
	D0	D1	D2	D3	D4	D5	D6	D7
Negative Control (Distilled water)	2.6± 0.31a	2.6± 0.31a	2.6± 0.31a	2.5 ± 0.25a	2.4 ± 0.23a	2.4 ± 0.25a	2.3 ± 0.22a	2.3 ± 0.22a
160	2.5± 0.34a	2.5± 0.34a	2.5± 0.34a	2.2 ± 0.25a	1.9 ± 0.23ab	1.8 ± 0.25ab	1.4 ± 0.22ab	1.1 ± 0.22b
200	2. ± 0.4a	2.3± 0.33a	2.2 ± 0.36a	1.8 ± 0.39a	1.7 ± 0.4ab	1.6 ± 0.4ab	1.35± 0.32ab	1.05± 0.23bc
Positive Control (Doramectin)	2.1 ± 0.18a	1.9 ± 0.18a	1.9 ± 0.18a	1.5 ± 0.17a	0.9 ± 0.07b	0.9 ± 0.07b	0.5 ± 0.17b	0.2 ± 0.13c

 

1/ Values are Mean ± SE, n = 10 in each group values in a column with different superscripts differ, (P>0.05). Table presents the results of degree of infestation of the ear mites at different concentration of Senna alata extract.

 

The probit regression analysis provides critical insight into the efficacy of S. alata as botanical pesticides, with the lethal concentration for 50% value (LC50) calculated at 282.45 mg/ml. However, the LC50 value of S. alata leave extracted in distilled water in this study was three times higher than the previous study by Sunil et al. (2013). In the study, the LC50 value calculated for Cassia fistula leaves extracted in ethanol to kill a tick species, Rhipicephlaus (Boophilus) annulatus was only 97.1 mg/ml. Halim-Lim et al. (2020) illustrate that the total phenolic content in S. alata leaves was greater in ethanolic extraction than in water extraction. This is because the solvents and extraction techniques utilized in plant extraction are critical factors that substantially affect the yield of bioactive compounds (Abubakar and Haque, 2020). For this reason, further research on various solvents for extraction is necessary to enhance the efficacy of S. alata as botanical insecticides, thus optimize its toxic effects on pests at lower lethal concentrations.

Evaluation of acaricidal activity through in vivo studies demonstrated significant reductions in the degree of infestation for both concentrations of S. alata leaf extract. The progression of mite infestation over seven days highlighted the temporal dynamics of the extract’s effectiveness. The slight superiority of the 200 mg/ml concentration over the 160 mg/ml concentration is statistically significant, highlighting its potential as the optimal concentration for practical use as presented in Table 2. These findings align with studies involving Senna italica root extract, emphasizing that higher concentrations of botanical extracts enhance their efficacy against mite infestations due to increased potency of phytochemicals (Ravindran et al., 2012). The infestation level in the 200 mg/ml group decreased from 2.5 ± 0.4 on Day 0 to 1.05 ± 0.23 by Day 7, indicating prolonged efficacy of S. alata extract over time.

The notable decrease in mite infestation during the treatment period indicates that S. alata extract may function as a sustainable and efficacious acaricidal agent. The maintained efficacy over seven days suggests the potential for extended protection, decreasing the necessity for frequent reapplications and alleviating stress on treated animals. The temporal efficacy patterns indicate that strategically scheduling applications may enhance outcomes, especially when synchronized with the critical stages of the mite life cycle. These findings emphasize the necessity of evaluating both the immediate impacts and the long-term impact of treatments to comprehensively understand its potential.

Both in vitro and in vivo results indicate that crude extracts of Senna alata leaves possess potent acaricidal properties, attributed to their diverse phytochemical constituents. Previous studies have identified various bioactive components in S. alata, including flavonoids, phenolics, alkaloids, anthraquinones, tannins, terpenoids, and steroids, contributing to a range of pharmacological activities (Angelina et al., 2021). Characterization of these constituents is crucial for understanding the biological activities of S. alata extracts and exploring their potential as sources for new acaricidal agents.

Anthraquinones, such as rhein identified in Senna alata, are noted for their anti-mite properties (Phongpaichit et al., 2004). Rhein has been documented for its activities against lepidopteran pests (Duraipandiyan et al., 2011) and human cancer cells through mechanisms like apoptosis (Chang et al., 2012). Based on these previous studies reporting the effect of anthraquinone against several arthropods, the acaricidal efficacy observed in this study may be attributed to anthraquinones present in S. alata leaf extract. However, this can be concluded with further in vitro and in vivo studies using only anthraquinone compounds against mites.

In conclusion, this study underscores the potential of S. alata leaf extract as a natural acaricide. It promotes sustainable alternatives to synthetic acaricides, supporting safer pest management practices in rabbit farming and aligning with animal welfare concerns. However, further research is required to investigate the long-term effects of frequent applications, the possible emergence of mite resistance, and the environmental impacts of these treatments. Furthermore, refining extraction techniques and solvents may improve the stability and efficacy of bioactive chemicals such as anthraquinones, which is thought to contribute to the reported acaricidal effects.

CONCLUSIONS AND RECOMMENDATIONS

This research underscores the efficacy of Senna alata leaf extract as a natural acaricide, presenting a sustainable alternative to synthetic solutions. Its application promotes safer pest management in rabbit farming while resolving animal welfare issues. Additional research is necessary to identify further bioactive chemicals, examine long-term benefits, and assess the danger of resistance development in mite populations.

ACKNOWLEDGEMENTS

The authors would like to offer particular thanks to the Universiti Sultan Zainal Abidin (UniSZA) for the facilities provided for this study. This project is supported by Universiti Sultan Zainal Abidin (UniSZA) under Dana Penyelidikan Universiti 1.0 (UniSZA/2022/DPU1.0/05).

NOVELTY STATEMENTS

This research is the first to comprehensively evaluate the in vitro and in vivo acaricidal activity of Senna alata leaf extract against Psoroptes cuniculi in rabbits. Our data indicate that S. alata may serve as a viable natural alternative to synthetic acaricides, evidenced by substantial reductions in mite mortality and infestation. This study enhances sustainable pest management approaches in rabbit farming, simultaneously tackling issues of chemical resistance and animal welfare. Moreover, our findings facilitate additional investigation into bioactive substances and their modes of action against parasitic mites.

AUTHOR’S CONTRIBUTIONS

Nur Athiqah designed the study and drafted the manuscript, Nur Elisa conducted the in vitro study, Lananan helped in data analysis for in vitro study, Muhammad Erfan Faiq conducted the in vivo study, Ghazali and Chai helped in data analysis for in vivo study.

Conflict of Interest

The authors declare that there is no conflict of interests regarding the publication of this article.

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