In vitro, Evaluate Effectiveness of Allium cepa Ethanol Extract in Inhibiting the Growth of Toxocara canis Eggs

Dogs play a vital role in people life, their versatility makes it capable of performing a wide range of security scenarios such as detection dogs specialize in sniffing drugs, explosives and as guards for house and even personal protection. Besides, looking after dogs can contribute to children’s development, making them more confident and energetic, while also providing senior citizens with meaningful companionship (Waggoner et al., 1997; Jezierski et al., 2013). Dogs are susceptible to a number of zoonotic diseases, the two most common parasite illnesses being toxocariasis and echinococcosis. In various parts of the world, these disease are pose serious threats to public health and economic problems (El Maghrbi et al., 2019; Al Malki, 2021).
    
Toxocariasis is one of the most significant infectious disease, that is caused by T.canis  which primarily infect dogs with high zoonotic potential and these parasite belonging to the family Toxocaridae and the genus Toxocara (Rashid et al., 2022). The mature parasites inhabit within the small intestine of their definitive host (dogs), where females release eggs with feces into the external environment. These eggs can resist harsh conditions. Dogs become infected by consuming contaminated food that contains either eggs (which contain L2) or larvae (L2) encysted in the paratenic hosts tissues such as mice, birds and earthworms (Maqbool et al., 1998; Despommier, 2003; Hosin, 2008). Furthermore, puppies get up Toxocara larvae either through vertical lactation via infected milk or through transmission across the placenta from an infected mother. (Salazar et al., 2020; Jesudoss et al., 2021). In human, infection occurs following ingestion of eggs, the larvae released and travel to the major organs, such as liver, lungs, or brain causing damage while inducing inflammatory responses, this form of infection is known as visceral larva migrans. Otherwise, the larvae may migrate to the eye, where they can occasionally cause unilateral blindness; this type of infection is called ocular larva migrans (Magnaval et al., 2001; Öge et al.,2014; Kong et al.,2016). Flotation technique is the most dependable laboratory method for confirming T. canis eggs in clinically suspected cases because it gets more eggs than other techniques (Magnaval et al., 2001; Dryden et al., 2005).                                                                     
       
On the other hand, the benzimidazole carbamates group of anthelmintic medications is the most widely utilized for treating toxocariasis (Pawlowski, 2001). However, because to their exceedingly poor solubility, their low tissue bioavailability, these medications thus require relatively high doses to be administered over prolonged periods (Hrckova and Velebny, 2001). Furthermore, drug resistance could result by using these medications to treat different helminth infections (Geerts and Gryseels, 2000). Therefore, there is an urgent need for novel medications to treat helminthic diseases like the application of crude medicinal herbs has a beneficial influence on people as well as animals due to their anthelmintic efficacy without causing any negative effects. Recently, there has been an explosion of interest about discovering medicinal herbal that could potentially employed as novel anti-parasitic as a result of the increasing contraindications to the use of synthetic medications (Muhammed, 2015; Jasim et al., 2023). In veterinary medicine, natural treatments derived from plant extracts are anticipated to take the place of traditional parasite control techniques. Many plants can be employed as anti-parasitic agents like onions (Allium cepa) have been demonstrated in numerous investigations that they have nematocidal and trematocidal properties (Orengo et al., 2016). The objective of this research was to evalute the influence of A. cepa ethanol extract against T. canis eggs in vitro, in compare to negative control groups (Water) and positive control group (Drontal® Plus).

Study area
 
The study was conducted in the Parasitology Laboratory at the College of Veterinary Medicine, Al-Muthanna University, during the period from January, 2023 to the end of July, 2023.
 
Toxocara canis eggs preparation
 
Shortly after the stray dogs’ deceases, 46 fully developed female T. canis were removed from the intestines .The identification of the adult female T. canis worms was based on their morphological characteristics as described by Soulsby (1982). In order to get rid of the tissue debris, the adult female worms had been washed with phosphate-buffered saline (PBS). Subsequently, the female worms’ uterus was dissected, eggs of T. canis had been eliminated from the adult worms, washed many times in PBS and then centrifuged at 3,000 rpm to 5 minutes. Then discarding the supernatant, the precipitate eggs were put into an opaque glass filled with normal saline to be kept at 40°C until needed again (El-Sayed, 2017).
 
McMaster’s technique for counting eggs
 
The number of eggs that had been collected from the uteri of adult worms was calculated employing the McMaster technique, as described by Taylor, (2016) and Jasim and Al-Amery, (2023).
 
Preparation of onions (Allium cepa) ethanol extract                                                                         
                                       
The fresh onions (Allium cepa) bulbs had been bought from the herbal market in Muthanna province/ Iraq. The onions were cut into slices, then left to dry in the shade for a week. After that, they were mechanically powdered using electric blender, then packed in in a glass container and maintained in a dry, cool environment. One hundred gram of dry onion powder and 400 ml of 99.5% ethanol were mixed together and gently shaken with a magnetic stirrer for an hour in order to obtain the ethanol extract. The product solution was mixed again and filtered through Whatman cellulose filter paper after being left at room temperature for 72 hours. Above a sand bath heated to 80°C, the solvent evaporated. In order to avoid the dry extract adhering to the container walls, the final step of the drying process was carried out in an oven heated to 40°C. Following, a maroon paste was obtained from the extraction process of A. cepa  was placed in a sterile container of glass and stored at 4°C for subsequent use (Abhijeet et al., 2012; Akintobi et al., 2013; Orengo et al., 2016).
 
Onions (Allium cepa) extract: An experimental study on eggs of T. canis in vitro
 
After dissolving A. cepa extracts in the water bath at 40°C with vigorous agitation for 15 minutes, various concentrations (5, 10, 20, 30, 40 and 50 mg/ml) prepared. Unembryonated T. canis eggs were exposed in vitro to A. cepa extracts at different concentrations and for different period of time (24 and 48 hrs.). A total of 50ìl of rich sediment (500) unembryonated eggs was added into an eppendroff tube containing 450 μl of A. cepa extract with different concentration. The eppendroff tubes were put in an incubator adjusted to 28°C for periods of 24 and 48 hours. After the incubation period, the extract solution’s supernatant was removed with a pipette and eggs are washed several times with PBS. After that, each tube’s eggs were transported into new eppendroff tubes containing 750 μl tap water (dechlorinate) and they have been incubating for two weeks at 28°C. At the same time, 50ìl of egg rich sediment was incubated at 28°C without exposure to the extract as a negative group (Orengo et al., 2016; El-Sayed, 2017). In order to make Drontal® Plus (5 mg/ml), 25 mg of the drug was dissolved in 5 ml of distilled water containing 5% DMSO as the positive control. The presence of larvae inside eggs, developing cells, or dead eggs was used to investigate the influence of A. cepa extracts on the T. canis egg embryogenesis. Ovicidal activity had been calculated as described by Jasim and Al-Amery, (2023). 

The experience was accomplished to evaluate the influence of A. cepa extract against T. canis eggs development through different concentrations (5, 10, 20, 30 and 50 mg/ml) for different periods (24 and 48 hours), in compare to negative control groups (Water) in vitro. According to the results of microscopic investigations of T. canis eggs subjected to different A. cepa extract doses and intervals in vitro, revealed that eggs are sensitive to A. cepa extract at different concentrations and their activities are concentration and time dependent, as shown in Fig 1. The highest efficacy, reaching 100% was recorded with concentrations of 30 and 50mg/ml after 24 and 48 hours, respectively. But, the eggs exposed to 5 and 10 mg/ml of A. cepa extract after 24 and 48 hours demonstrated ovicidal activity by 88.4, 93.9, 94.3 and 98.6%, respectively, in compared with Drontal® Plus (praziquantel/pyrantel pamoate/febantel;5 mg/ml) and negative control group were 100 and 11.8 % respectively, as showed in Table 1, 2.  Also, Table 1 and 2 show that the extract’s efficacy at concentrations of 20 mg/ml for 24 and 48 hours is 98.8 and 100%, respectively.    





                                  
       
Because there is currently no approved vaccination for any parasitic disease and the growing contraindications associated with synthetic medications stemming from their adverse effects, or the increase of resistance to parasites, it is crucial to require alternative anti-parasitic pharmaceuticals (Thakur et al., 2022; Barua et al., 2023). Consequently, there has been significant recent interest in identifying plants with biological properties that can utilized as new anti-parasitic drugs without causing harmful side effects (Wink, 2012). Moreover, successful natural product research requires careful selection of herbs based on diverse criteria, such as chemotaxonomic data, field observation and information from conventional medicine (Queiroz et al., 2009). This study was designed to evaluate the influence of A. cepa extract on the viability and embryogenesis of T. canis eggs due to their significant role in the transmission of human toxocariasis. The findings of ovicidal activity for A. cepa ethanol extract against T. canis eggs at different concentrations (5, 10, 20, 30 and 50 mg/ml) for varying periods (24 and 48 hrs.), were agreement with the obtained results by Orengo et al., (2016), which employed the A. cepa ethanol extract of  as an anti-parasite. It was demonstrated that 100% of T. canis eggs were suppressed by A. cepa ethanol extract at doses of 10,000-1,250 ug/ml. Furthermore, our investigation findings indicated that the efficiency of A. cepa ethanol extract increases with duration of exposure and concentration. Several studies have suggested that the biological and medical functions of A. cepa can be attributed to its phytochemical constituents. The anthelmintic properties of A. cepa extract are primarily due to sulfur compounds like Allyl propyl disulfide and Allicin, as well as flavonoids such as Quercetin (Githiori et al., 2006; Chakraborty et al., 2022). Allicin exhibits its antimicrobial activity primarily by rapidly and completely inhibiting the synthesis of DNA, RNA and proteins (Feldberg et al., 1988; Etewa and Abaza, 2011; Al-Zayyadi, 2020). Moreover, onion-derived sulfur compounds particularly diallyl trisulfide (DTS), inhibited the growth the parasites and damage to the cell membrane of parasites, resulting in reduced viability and destruction (Krstin et al., 2018). Besides, Gomes et al., (2016) Zingiber officinale contains phytochemical constituents (saponin) is act on destabilization of the cell membrane and lead to increased permeability, which causes embryonic lysis. Onion extract contains modest amounts of the same components (saponin) that may be responsible for T.canis egg destruction. On the other hand, the results of the current study regarding Drontal®Plus (praziquantel/pyrantel pamoate/febante) effectiveness against T. canis eggs in vitro were consistent with the findings of Orengo et al., (2016), who revealed that eggs of T. canis are sensitive to Vermic TotalTM (praziquantel/pyrantel pamoate/febante) in vitro, which has the same composition as Drontal® Plus but different concentrations. Also, the drug’s effectiveness is dependent on time and concentration. Drontal® Plus is a widely used veterinary dewormer designed to treat various intestinal parasites in dogs. While, it effectively targets adult parasites such as tapeworms, roundworms, hookworms and whipworms, but, its ability to kill parasite eggs is not clear (Yadav et al., 2007).

Based on the data, the results indicated that eggs of T. canis treated with A. cepa extract in vitro are sensitive to the extract at the different concentrations and times in vitro and that their activities are time- and concentration-dependent.

The facilities were provided by the College of Veterinary Medicine parasitology laboratory at Al-Muthanna University, for which the authors are grateful.
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All procedures for experiments were approved by the Committee of Experimental Animal care.

The researchers have not declared any conflicts of interest.