In vitro Study: Efficacy of Artemisia monosperma Leaves Extract on Eimeria papillata

Infection with apicomplexan parasites of the genus Eimera causes coccidiosis in chickens (Chapman et al., 2013; Witcombe and Smith, 2014) and ruminants (Khodakaram and Hashemnia, 2017). In ruminants, Eimeria sp. leads to poor weight, reduced production and increased mortality in younger stock. Infectious and parasitic illnesses like pneumonia and helminthosis can also made worse (Kanyari et al., 1993; Kusiluka et al., 1998; Etsay et al., 2020).
       
Globally, this disease has a significant impact on poultry production, resulting in annual economic losses of about US$ 3 billion (Noack et al., 2019; Blake et al., 2020). It affects the digestive system of the host, which develops in the small and large intestines (Dakpogan et al., 2019). It is characterized by diarrhea, dehydration, fluid loss, malabsorption of nutrients, inflammation, oxidative stress and heightened susceptibility to bacterial pathogens such as necrotic enteritis in chickens (Alnassan et al., 2014). Due to their negative effects on broilers’ final body weight, intestinal health and meat quality, microbial infections significantly affect chicken productivity (Mohamed et al., 2021; Swelum et al., 2021; Yaqoob et al., 2021). Therefore, antibiotics repressed and inhibited microorganisms until the emergence of antibiotic-resistant bacteria (Alagawany et al., 2021; Reda et al., 2021). This issue is typically resolved by the use of phytogenic substances, such as phenolic compounds in plant extracts (Abou-Kassem et al., 2021; El-Saadony et al., 2021a; Saad et al., 2021).
       
Many factors aid in the development of coccidiosis, including fecal-oral transmission, high oocyst reproductive potential, the presence of resistant oocysts, the direct life cycle and favorable environmental circumstances for infectivity (sporulation). Since 1939, many ionophore anticoccidial and chemical feed additives have been utilized to combat these potentially harmful parasites in chickens (Nogueira et al., 2009). However, drug resistance has emerged (Abbas et al., 2011; Hema et al., 2015). As a result of this, plant extracts are currently being assessed as viable, sustainable alternatives to new medications.
       
Recently, there has been a global interest in adopting herbal remedies as secure substitutes to treat different ailments with a reduced danger of the emergence of resistance (Abd El-Hack et al., 2020; Abdelnour et al., 2020; Ashour et al., 2020). Antiprotozoal activity in more than 1200 plants has been reported (Willcox and Bodeker, 2004; Muthamilselvan et al., 2016).
       
The Arabian Peninsula boasts a rich variety of plants and traditional medical practices centered on foods, spices and plants have been widely used in this region (Maideen, 2020). The Kingdom of Saudi Arabia represents one of the most biodiverse regions on the Arabian Peninsula due to its wide territory and diverse climatic and geographical circumstances (Almehdar et al., 2012). There are numerous significant crops and medicinal plants that are traditionally used for medical purposes among Saudi Arabia’s flora, such as A. monosperma, Acacia arabica, Artemisia judaica, Calligonum comosum, Lantana camara, Pulicaria glutinosa, etc. (Ullah et al., 2020).
       
Artemisia is a genus of shrubs and small herbs that includes more than 500 species, which are found in North America, Europe and Asia (Bora and Sharma, 2011). A. monosperma is a common desert plant that is used extensively in Saudi Arabian folk medicine to treat muscle spasms, hypertension and parasitic worms (Amin et al., 2019). This plant’s extracts and essential oils have reportedly demonstrated a range of biological properties, including antibacterial, antimalarial, antioxidant and insecticidal effects (Stavri et al., 2004; Stavri et al., 2005 and Guetat et al., 2017). Also, the medicinal importance of A. monosperma is attributed to its anticancer, antispasmodic and antihypertensive properties (Abu-Niaaj et al., 1996; Hijazi and Salhab, 2010 and Al-Soqeer, 2011).
       
Our study’s main objective is to assess the anticoccidial activity of Artemisia monosperma leaf extract (AMLE) against oocyst sporulation (Eimeria papillata) in vitro.

Between January 2023 and August 2023, this experiment was finished in the zoology department of King Saud University’s College of Sciences. The leaves of the wild plant A. monosperma were obtained from Tabuk (Saudi Arabia) and a taxonomist from the Botany and Microbiology Department at the College of Science, at King Saud University (Saudi Arabia) confirmed the plant’s botanical identity. Plant specimens were gathered and they were air-dried at room temperature. It was then sieved, ground in an electric mill and stored for extraction.
       
The method Dkhil (2013) suggested was used to prepare the crude extract. Samples (100 mg) were drenched in 1000 L of 70% methanol for 24 hours at 4°C and kept in a laminated bottle with a 1500 L capacity offered with secure stoppers and overnight shaking. The resulting extract was dried and concentrated in a Japanese Yamato RE300 rotary vacuum evaporator. The resulting crude extract was kept in the deep freezer until to perform various experiments.
       
KBr pellet method, the plant leaf extract was analyzed using a NICOLET 6700 (Thermo Scientific, Waltham, USA) spectrometer FT-IR with a 400-4000 cm^-1 range (Abu Hawsah et al., 2023).
       
The phenolic content overall of AMLE was decided by the method according to Singleton et al., (1999), with a few modifications. To create a standard curve (25-150 µg/mL), gallic acid solutions were used. Briefly, Folin-Ciocalteu reagent (0.1 mL), ultrapure water (Milli-Q) (1.5 mL) and gallic acid or 0.1 mL of the plant extract (1 mg/mL) were mixed and left for 8 min., then a 20% sodium carbonate (0.3 mL) solution was blended and mixed by a vortex in darkness for 2 h and the mixture was incubated. The absorbance of the resulting blue color was measured with a UV-visible spectrophotometer at 765 nm. Utilizing the equation based on the calibration of the curve, the extracts’ overall content of phenolics was calculated as gallic acid equivalent (mg/g DW):
   
Where,
(y) = Absorbance.
(x) = Gallic acid equivalent concentration (mg/g).

The total flavonoids in AMLE were determined using a method reported by Ordonez et al., (2006). Briefly, a 2% AlCl3 (1.0 mL) water solution was mixed with 1.0 mL of plant extract (1 mg/mL). At 420 nm, absorbance was measured following an hour of incubation at room temperature. 50-800 g/mL of quercetin solution was used to prepare the standard solution and create a standard curve (R2 = 0.9996). Using the equation for the calibration curve:
   
Where,
y = Absorbance.
x = Quercetin equivalent concentration (mg/g).
       
The flavonoids in the extracts were expressed as quercetin (mg/g DW).
       
From the feces of mice that had caught the infection on their own, E. papillata oocysts were isolated.
       
The non-sporulated oocysts (1×10⁵) were incubated in each group, which contained the following: a positive control group (5 ml of 2.5% potassium dichromate), distilled water (5 ml) as the negative control group, a treatment group (5 ml of potassium dichromate 2.5%) with one of the following: AMLE (50, 100, 200 and 300 mg/mL), amprolium (8.3 mg) (Veterinary Agriculture Products Company [VAPCO], Jordan), phenol (25 µL), Dettol TM (109 µL) and formalin (5%). A compound Olympus microscope (Olympus Co., Tokyo, Japan) was used to examine, photograph and track oocyte sporulation. For each treatment, three replicates were carried out and all Petri dishes were incubated for 72 and 96 h at 25 to 29°C (Gadelhaq et al., 2018). The sporulation % and sporulation inhibition percentage were computed following Daiba et al., (2022) and Cedric et al., (2018), respectively. One-way analysis of variance (ANOVA) was used to examine the data in Sigma Plot ® version 11.0 (Systat Software, Inc., Chicago, IL, USA). Differences between groups were considered significant at p-value <0.01.

Infrared spectroscopy
 
Data in Table 1 show that the major bands of FT-IR were used to analysis of AMLE. at 3383.5 7cm^-1, 2930.29 cm^-1, 2231.66 cm^-1,1653.48 cm^-1,1598.80 cm^-1, 1513.75cm^-1, 1495.72 cm^-1, 1435.09 cm^-1, 1361.44 cm^-1, 1282.76 cm^-1, 1160.39 cm^-1, 1123.21 cm^-1, 1074.98 cm^-1, 820.68 cm^-1 and 613.27 (Fig 1 and Table 1). N-H stretching was indicated by the band at 3383.5 7 cm^-1 confirming the presence of aliphatic primary amine. The band at 2930.29 cm^-1 implied C-H stretching in the presence of alkane. C=N stretching at 2231.66 cm^-1 confirms the presence of nitrile. The band at 1653.48 cm^-1 corresponds to C=N stretching in the presence of conjugated imine/oxime. N-H bending at the band 1598.80 cm^-1 confirmed the presence of amine. The band at 1513.75 cm^-1 implied (N-O stretching) for the presence of nitro compound and the band at 1495.72 cm^-1 corresponds to C-H bending for the presence of alkane. 1435.09 cm^-1 (O-H bending), 1361.44 cm^-1 (S=O stretching), 1282.76 cm^-1 (C-O stretching), 1160.39 cm^-1 (C-O stretching), 1123.21 cm^-1 (C-O stretching), 1074.98 cm^-1 (C-O stretching), 820.68 cm^-1 (C=C bending) and 613.27 cm^-1 (C-I stretching) assigned to carboxylic acid, sulfonate, aromatic ester, ester, tertiary alcohol, primary alcohol, alkene and halo compound respectively (Table 1).
 

 

       
Essential oils and plant extracts have gained attention as possible anticoccidial agents in the poultry sector, where coccidial prevention and control are top priorities (Remmal et al., 2011; Orengo et al., 2012) because they lead to production losses, treatment costs and disease prevention  (Quiroz-Castañeda and Dantán-González, 2015). Some factors contribute to the development of coccidiosis, including the presence of resistant oocysts, elevated oocyst reproductive potential and conducive environmental factors for infection (sporulation) (Remmal et al., 2011). To effectively manage this parasite, it is essential to block the sporulation process (Mai et al., 2009). In addition to increasing the risk of coccidiosis, it can also increase the risk of other contagious diseases and parasitic like pneumonia and helminthosis (Kusiluka et al., 1998; Etsay et al., 2020). This study’s goal was to assess the anticoccidial efficacy in vitro of various AMLE doses.
 
Total phenolics and flavonoid
 
The amounts of some secondary metabolites in the AMLE were measured, like phenolics and flavonoids. Fig 2 shows that the phenols concentration (39.7±0.3) was high compared to the flavonoids concentration (33.4±0.2). Flavonoids, alkaloids, tannins and phenolic compounds are the most important plant bioactive compounds (Mehmood et al., 2015). Several studies have shown that plant extracts consisting of phenolic compounds have inhibitory properties. Components of natural polyphenolic components derived from medicinal plants in vitro have been shown to inhibit E. tenella sporozoite cell invasion (Arlette et al., 2019). These researchers also noted that extracts with compounds of polyphenolic may have the power to inhibit the enzymes necessary for the coccidian oocysts’ sporulation process. Some flavonoids affect host-parasite interactions, whereas others disrupt protozoan parasite development or metabolism (Kerboeuf et al., 2008), including Leishmania sp. and Trypanosoma sp. (Fotie, 2008).
 

 
Oocyst sporulation
 
In vitro tests on AMLE and a few other materials revealed sporulation of the oocyst (%) and sporulation inhibition (%) at 72 and 96 h for E. papillata. Also, a few changes such as oocyst wall deformation and nuclear material distortion, were noted in oocysts treated with 300 mg/ml AMLE (Fig 3). A significant degree of oocyst sporulation (%) in distributed H₂O was found to be (66.6%) when compared to the AMLE, which had sporulation levels of 0%, 18.83%, 37.04% and 47.5% at 72 h (Fig 4), while at 96 h, were 0%, 67.1%, 78.57% and 81.2% at concentrations of 300, 200, 100 and 50 mg/mL, respectively (Fig 5). Also, the rate of sporulation (%) varied in each of the DettolTM, phenol and formalin 5% were 23.08%, 7.7% and 0%, respectively, at 72 h (Fig 5), while at 96 h, were 18.67%, 10.67% and 0%, respectively (Fig 6). On the other hand, the highest sporulation inhibition (100%) was obtained for AMLE at a concentration of 300 mg in 72 and 96 h (Fig 6 and 7). While the levels of sporulation inhibition for amprolium, Dettol^TM, phenol and formalin were 37. 33%, 81.33%, 89.33% and 100%, respectively, at 96 h (Fig 7) and at 72 h it was 34.61%, 76.92%, 92.30% and 100% (Fig 6). The findings of this experiment demonstrated that the AMLE ethanol leaf extract has an in vitro anticoccidial effect on unsporulated oocysts of E. papillata in a concentration-dependent manner, which is attributable to numerous bioactive phytochemical constituents studied (Abdulrahman et al., 2023). Likewise, the methanolic extract for AMLE significantly prevented the growth of Blastocystis in vitro and changes in Blastocystis shape under the influence of the extract were observed. This may be attributed to Artemisinin has also been shown to affect the operation of oocyst wall construction, resulting in a deficient oocyst wall, mortality of developing oocysts and a decrease in sporulation rate (Del et al., 2010). This alteration is produced by a decrease in the expression of SERCA (Sarco/endoplasmic reticulum calcium ATPase) in macrogametes, which plays a role in calcium homeostasis by influencing the production of wall-forming bodies, a calcium-mechanism dependent (Del et al., 2010). Furthermore, the disinfectant formalin (5%) prevented the sporulation of E. papillata, which coincided with Thagfan et al., (2020) and Abu Hawsah et al., (2023). This extremely reactive compound is said to interact with proteins in vitro and hinder sporulation. In addition, Dettol^TM and phenol have been reported to prevent sporulation at 96 h by 81.33% and 89.33%, respectively, which is consistent with Mai et al., (2009), Gadelhaq et al., (2018) and Abu Hawsah et al., (2023) who found that the oocyst wall is impermeable to water-soluble substances and resistant to proteolysis.
 

 

 

 

 

It could be concluded that AMLE has anticoccidial and anthelmintic efficacy in vitro. More research should be done to determine the in vivo effectiveness of AMLE. This will help guide ongoing studies into the development of AMLE as a novel medication that can be used to treat coccidian infections that affect animals.

This work was supported by the Researchers Supporting Project (RSPD2023R1079) at King Saud University (Riyadh, Saudi Arabia).

The authors declare that there are no conflicts of interest.