In vitro Evaluation of Nerium oleander Leaf Extract against Sarcoptes scabiei var. cuniculi Mite Isolated from Naturally Infested Rabbits

Mites of Sarcoptes scabiei (Family: Sarcoptidae) are usually regarded to be a single species. However, frequently are identified by a variety of names corresponding to the host species. The mite that causes scabies in rabbits is S. scabiei var. cuniculi and is an important species because of the possibility of zoonotic infection (Harrenstien et al., 1995). Diseases are characterized by intense pruritus, alopecia, erythema, scales and crusts around the head, neck, trunk, feet, ears, nose and genitals, resulting in hypertensive reactions, considerable losses in body weight, productivity, wool and death (Saha and Mukherjee, 1998) and is difficult to control in rabbits compared to other domestic animals (Aiello et al., 1998). All stages of the S. scabiei life cycle are found within the host and the entire life cycle lasts approximately 2 months (Suckow et al., 2002). Pregnant females of S. scabiei were approximately twice the size of males (Jofre et al., 2009) and lay eggs in tunnels in the stratum corneum of the skin, causing severe itchy rashes, hypersensitivity and inflammation. The six-legged larvae hatch within 3 to 10 days, move around in search of hair follicles on the skin and molt to the larval stage, where they mature into adult mites (Soulsby 1982). Adult mites live for 3-4 weeks within the skin of their host. Mites feed on lymphocytes and sloughed epithelial cells (Hofing and Kraus 1994). Demonstrating the presence of host IgG antibodies in the esophagus and midgut of fresh scabies mites removed from the host is evidence that these mites ingest host serum (Rapp et al., 2006). In general, acaricides such as pyrethroids and ivermectine are used for the control of infection (however, their efficacy is compromised. because of resistance, toxicity (O’Brien, 1999), environmental contamination and persistence (Halley et al., 1993) emphasizing the need for alternatives (Khater, 2011), medicinal plants. A study revealed the presence of mange mites through a microscopic examination of deep skin scrapings from infected sheep in the city of Riyadh (Murshed et al., 2022).
       
Nerium oleander, or oleander (locally called Défla), belongs to the family Apocynaceae (Beasley, 1999). It is mainly found on alluvium and rocky terrain, along wadis in the northern Sahara and the Tassili and Hoggar mountains (Chopra et al., 1960) and is frequently cultivated as an ornamental plant (Delille, 2007) to form hedges in parks and gardens. All parts of the oleander plant contain highly toxic and lethal glycosides and alkaloids (Barbosa et al., 2008) with antibacterial (Derwich et al., 2010), antimicrobials (Hussain and Gorsi, 2004), insecticides (Dinan, 2001), inflammatory (Aggarwal and Shishodia, 2006) and cytotoxic (Mijatovic et al., 2007) properties. The toxicity is selectively exerted on the nervous system, the reproductive system, or the digestive system of pests (Kumar et al., 2013). Humans, animals and some insects have been the subject of several studies relating to the toxic effects of N. oleander (Adom et al., 2003). The object of this study was to study the acaricidal activity of Nerium oleander extract against S. scabiei var. cuniculi mite in vitro.

Venue of the experiment
 
The study was carried out in July 2023 in Riyadh, Saudi Arabia the parasitological laboratory at the College of Science, King Saud University.
 
Preparation of extracts
 
The Nerium oleander extract is prepared from leaves obtained from one of the gardens in the city of Al-Kharj, Saudi Arabia. Leaves (100 g) were air-dried at 40°C, ground into powder and then extracted with 70% methanol for 24 h at 4°C. Then, the obtained extract was concentrated and dried using a vacuum rotary evaporator (Yamato RE300, Japan) at 40°C and reduced pressure. Once crude extracts were made, they were stored at -20°C until used in an experiment (Yang et al., 2014).
 
Phytochemical analysis of leaf extracts
 
GC-MS (gas chromatography-mass spectrometry) analysis of the extract was performed using the equipment 7000D Triple Quad GC-MS (Agilent Technologies, Thermo Scientific, Austin, Texas, United States). In Miami, California, in the United States, a Thermo Scientific Trace GC Ultra and an ISQ single quadruple MS were utilized. Both the Agilent 7890A and the Agilent 5975C inert XL EI/CI MSDs come equipped with a solitary quadrupole mass analyzer. The capillary employed was an HP-5MS UI (Ultra Inert) model with the following specifications: a length of 25 meters, an inner diameter of 0.25 millimeters, a film thickness of 0.25 millimeters, a stationary phase containing 5% phenyl and low-polar methyl polysiloxane. The split ratio was 50:50 and operated at 250°C using helium as the carrier gas at 1.0 mL/min. After running the GC-MS at 30°C for five minutes at a rate of five counts per minute (Co/min), the temperature was gradually raised to 250°C for 40 min. In the MS conditions, the ionization energy was measured to be 70 eV. We utilized the full scan detection mode and the mass range we looked at was from 50 to 500 Da. One minute passed during the solvent delay. Methanol was the liquid used to dissolve the sample. In order to identify the compounds, the Wiley 9 database, the answers and the libraries were utilized. Name, molecular weight, molecular formula and peak area were the metrics that were utilized in the analysis of the test substance’s constituent parts.
 
Collection of mites
 
S. scabiei var. cuniculi mites were collected from the toe of naturally infested rabbits using a scalpel blade. Scabs collected were examined under a stereomicroscope for the presence of mites and were incubated at 35°C for 30 min in Petri dishes. Live adult mites (male and female) released from scabs were picked up with a needle and put in Petri dishes for further use. It’s used in all experiments.
 
In vitro assay
 
The acaricidal activity of Nerium oleander leaf extract against adult mites was evaluated using a contact assay (immersion test). Mites (n=14) were immersed in different concentrations (100, 200 and 400 μg/ml), of extract at room temperature and examined under a stereomicroscope at 0.5, 1, 4, 6, 8 and 24 h post immersion. Mites immersedin PBS and ivermectin served as negative and positive controls, respectively.  Immobility of adult mites and a lack of reactions or persistent immobility within 1 minute following stimulation with a needle were considered indications of death. The test was repeated twice independently and three replicates were run concurrently for all tested treatments.

The GC-MS investigation uncovered a large number of bioactive compounds in the extract i.e., plant secondary metabolites with their retention time (RT) and peak area percentage. The components most available and potentially effective are as follows: 3-O-Methyl-d-glucose (69.8%), 5-Hydroxymethylfurfural (15.65%), Oleic Acid (5.85%), Linoleic acid (2.97%) and n-Hexadecanoic acid (1.14%) (Table 1 and Fig 1).
 

 

       
Scabiesarea very common dermatological condition and representa major public health burden globally (Hay et al., 2014). Due to acaricidal resistance, significant effort is devoted to developing new, safe and effective drugs for scabies.  After being submerged in concentrations, the mites, in general, exhibited excited behavior and moved in circles before slowing down and eventually passing away. In contact assay, Nerium oleander extract at different concentrations, exhibited potent acaricidal action against adult Sarcoptes scabiei var cuniculi (Fig 2). In fact, when the concentration of these extracts was increased to 400 mg/mL), the number of mortalities among adult mites was increased to (16.6±2) at 8 hrs (Table 2). It was very comparable to the standard medication, which included 1% ivermectin dosages, 200 and 100 mg/mL demonstrated a time-dependent acaricidal effect (Fig 3). At lower concentrations (100 mg/mL) the mortality of mites was 43% even after 8 h post-immersion. A low mortality rate was observed when adult mites were brought into contact at 43% for 8 h with a dose of 100 mg/mL. While the extract dose of 200 mg/ml showed a death rate of mites at a rate of 78% after 8 h. At all of the time intervals studied, the control group did not exhibit any acaricidal activity; however, the tick moved more activity than usual for more than a day. The obtained results showed that there were significant differences between the mortality mean of 400 mg/ml concentration after 1hr and at 200 mg/ml concentration after 8hrs and the remaining mortality means (Table 2). Also, there were significant differences between control and at 400% concentration after 1 hr (Fig 4). The highest mortality was after 8 hours at 100 and 200% concentrations. The lowest mortality was observed after 0.5 hr at all concentrations (Table 2). Significant differences (P<0.05) were obtained in S. scabiei var. cuniculi adults mortality percentage among plant extract concentrations (100, 200 and 400 mg/mL) and control (Fig 3). While there are no significant differences between the 400% concentration and the reference treatment (1% ivermectin). These results indicated that N. oleander leaf extract possesses in-vitro mange activity against mites. A similar finding has been reported by several authors who estimated the in-vitro N. oleander activity against adult S. mansoni (Morsi et al., 2022) and developmental stages of insects (Rathi and Al-Zubaidi, 2011; Kumar et al., 2012). The observed acaricidal effect could be due to the presence of active metabolites; terpenoids, alkaloids, flavonoids, saponins, tannin and carbohydrates which were known to be present in N. oleander through phytochemical screening, (Barbosa et al., 2008; Bhuvaneshwari et al., 2007; Chaudhary et al., 2015).
 

 

The current study concluded that the Nerium oleander showed a promising lethal effect against Sarcoptes scabiei var cuniculi mite. Therefore, this extract could be used in pest management applications against ticks. More in vivo studies are needed to better evaluate the possibility of this extract, or some of their pure components, as useful alternatives for the treatment of external parasites.

This work was supported by the Researchers Supporting Project (RSPD2023R1084), at King Saud University (Riyadh, Saudi Arabia).

The authors declare that there are no conflicts of interest.