Efficacy of Teucrium polium leaves extract as anticoccidial and anthelmintic: In vitro study

ABSTRACT Natural sources are microbiological species and medicinal plants, which could be potential new sources for development of drugs against different diseases. Coccidiosis affects many animals and leads to great economic losses. Drug-resistant strains of Eimeria species have emerged because of overuse and misuse of drugs. In vitro, using Eimeria papillata oocyst and earthworm (Eisenia fetida), we evaluated the anticoccidial and anthelmintic effect of Teucrium polium leaves extract (TPLE). Using infrared spectroscopy showed the presence of thirteen compounds for TPLE. Mebendazole (10 mg/mL) caused paralysis and earthworm death by 13.91±0.373 and 18.2±0.980 min, respectively, while, for TPLE (100 mg/ml) were 4.23±0.077 and 4.817±0.386 min. Also, the histological study revealed obvious surface architecture abnormality for treated worms. Moreover, TPLE (300 mg/mL) and formalin (5%) at 72, 96, and 120 hrs led to inhibition of sporulation by approximately 100% with marked deformities, while TPLE (200, 100, 50, and 25 mg/mL), amprolium, DettolTM, and phenol at 120 hr approximately 65.9%, 23.6%, 4.8%, 3.2%, 12.6%, 68.4%, and 46.6%, respectively. This pilot investigation revealed that TPLE possesses anticoccidial and anthelmintic activity, encouraging additional testing in vivo to create a new medication for the treatment of coccidiosis and helminthiasis.


INTRODUCTION
Intestinal coccidiosis is one of the most important parasitic diseases affecting many animals (Mehlhorn, 2014), which is caused by a protozoan parasite belonging to the genera Eimeria species that develops in the small and large intestines and has devastating effects on the younger animals (Kanyari et al., 1993;Bakunzi et al., 2010;Dakpogan et al., 2019).ubclinical manifestations are often associated with poor weight gain, reduced production, and increased mortality in younger animals (Khodakaram-Tafti and Hashemnia, 2017;Macedo et al., 2019).Coccidiosis could facilitate the occurrence of other parasitic diseases, such as pneumonia and helminthiasis (Kanyari et al., 1993;Kusiluka et al., 1998;Etsay et al., 2020).
The direct life cycle, fecal-oral transmission, presence of resistant oocysts, lack of crossprotection between Eimeria species, high oocyst reproductive potential, high stocking density, and conductive environmental factord for infectivity (sporulation) are effective factors that facilitate the development of coccidiosis (Remmal et al., 2011).Therefore, sporulation process disruption is a crucial area where this parasite can be managed (Mai et al., 2009).Many drugs are used as coccidiostats (such as toltrazuril, diclazuril, decoquinate, amprolium, and sulfonamide), and these can be either administered orally or through feed and water (Odden et al., 2019).However, because of the excessive use and misuse of drugs, have led to the emergence of drug-resistant strains of Eimeria species (Hema et al., 2015).As a result, developing new drugs from medicinal plants is a potentially sustainable alternative, because they have anti-bacterial and anti-parasitic properties (Cobaxin-Cardenas, 2018), less harmful, and have fewer side effects than conventional chemical agents (Wunderlich et al., 2014).
Our research is mainly focused on the in-vitro evaluation of the anticoccidial activity of Teucrium polium leaves extract (TPLE) against oocyst Eimeria papillata sporulation, in addition, to its anthelmintic activity.

MATERIALS AND METHODS
Leaves of Teucrium polium were collected from Tabuk, Saudi Arabia.A taxonomist at the Department of Botany and Micriobiology, College of Science, King Saud University (Saudi Arabia), confirmed the identification of the plant.The 150g of leaves were air-dried at 40°C, powdered, and then extracted with 50% ethanol for 24 hr at 4°C.According to Dkhil (2013), the resulting T. polium leaves extract (TPLE) was concentrated and dried in a rotary vacuum evaporator (Yamato RE300, Japan).
An excess of potassium bromide powder (1:99 wt%) was added to a tiny portion of TPLE, which was then processed to a homogeneous consistency before being finely ground and placed in a die for pellet formation.The instrument used for the study of Infrared (IR) is Thermo Scientific's optical spectrometer NICOLET 6700 Fourier-transform infrared spectroscopy (FT-IR).Maximum absorption was reported in the number of waves (cm -1 ).Spectra were registered from 4000 -400 cm −1 at 25°C.The total phenolic contents of TPLE were determined using the technique according to Singleton et al. (1999), with some modifications.Generate a standard curve, gallic acid solutions (25-150µg/mL) were used.Briefly, 0.1mL of Folin-Ciocalteu reagent, 1.5mL of ultrapure water (Milli-Q), and 0.1 mL of TPLE or gallic acid were mixed and left for 8 min, then, 0.3mL of sodium carbonate solution (20%) was added and mixed by a vortex in darkness for 2 hr, the mixture was incubated.A UV-visible spectrophotometer was used to measure the Arq.Bras.Med.Vet.Zootec., v.75, n.5, p.872-882, 2023 absorbance of the ensuing blue color at 765 nm.Using the equation based on the calibration curve (y = 0.005 − x − 0.0088), the total phenolic content of TPLE was calculated as gallic acid equivalent (mg/g DW), where (y) absorbance and (x) gallic acid equivalent concentration (mg/g).
The total flavonoids in TPLE were determined using a method reported by Ordoñez et al. (2006).Briefly, 1.0 mL of 2% AlCl 3 water solution was mixed with 1.0mL of leaves extract (1mg/mL).At 420 nm, absorbance was measured following an hour of incubation at room temperature.A quercetin solution (50-800 g/mL) was used to prepare the standard solution and create a standard curve (R2= 0.9996).Using the calibration curve equation (y = 0.0011 x + 0.0928), where (y) is the absorbance and (x) is the quercetin equivalent concentration (mg/g), the flavonoids in TPLE were expressed as quercetin (mg/g DW).
The antioxidant activities of TPLE were determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay (Liyana-Pathirana et al., 2005).Briefly, 1mL of TPLE was mixed with 1mL of 0.135mM DPPH at various concentrations (31.25-1000 g/mL).The mixture was held at room temperature in the dark for 40 min while being gently stirred.The absorbance of TPLE (sample) and the control solutions (Ascorbic acid as positive control) was measured at 517 nm, and the percentage of DPPH scavenging activity (%) of TPLE was calculated using the following equation: where:

Absorbance of DPPH + methanol (Abs control) Absorbance of the DPPH radical + sample (Abs sample)
A total of 25 earthworms, Eisenia fetida, were collected from agricultural lands and identified by a specialist in the College of Food and Agriculture Sciences (King Saud University).Five worms, approximately of equal size were placed in each Petri dish.Albendazole (10 mg/ml) was used as a positive control, and distilled H 2 O was used as a negative control.The extract from TPLE was prepared in distilled H 2 O at concentrations of 100, 50, and 25 mg/mL.Time for paralysis was recorded when no movement was observed except when shaken vigorously, while the time of death was recorded when the worms did not show any movement by vigorous shaking nor when dipped in warm water (50°C) (Parida et al., 2010).
The small parts of the earthworm body were fixed in 10% buffered neutral formalin, then processed for paraffin embedding, and 4 μm thick sections were stained with hematoxylin and eosin (H&E) (Drury and Wallington, 1973).Sections were examined and photographed using a digital camera (DP 73) fitted on an Olympus B×61 microscope (Tokyo, Japan).
Five Swiss albino mice were inoculated with 1×10 3 sporulated Eimeria papillata oocysts via oral gavage.On the fifth day of infection, feces were collected, and oocysts were separated by floatation technique and then used for in vitro study.
The phenolic and flavonoid contents in the TPLE were determined.Phenolics were found to be 78.89±0.73mg gallic acid/g of the sample.In addition, flavonoids were found to be 15.77±0.13mg quercetin/g of sample (Figure 2).
The antioxidant activities of TPLE were determined using free radical scavenging activity by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method.Overall, the scavenging activity against the DPPH radical increased with concentration increases in TPLE, peaking at 500 g/mL, and after that started to decline (Table 2).The results indicated that TPLE showed the highest percentage inhibition value of DPPH radical at 500µg/mL (70.9%).
In addition, the dermal layers of the worm cuticle in the control group showed no changes in histological sections stained with hematoxylin and eosin, but the vacuolation was significantly observed in the treated group with TPLE as well as the complete destruction of the upper layer with drug treatment (Figure 3).

DISCUSSION
Coccidiosis caused by Eimeria parasites affects various animal species and causes considerable economic damage through mortality and reduced weight gains and poor feed efficiencies (Abu Hawsah et al., 2023).Previous studies have attempted to determine a solution for this issue.
To avoid adverse effects on animal performance, there's a need to develop new agents with minimum side effects against coccidiosis.This study aimed to evaluate the anthelmintic and anticoccidial activities of T. polium.Potential anthelminthic medications include ones that harm the parasite but not the host, which causes paralysis of the parasite's musculature either by inhibiting the neuromuscular transition or energy generation enzymes (Andrews et al., 1980;Manger, 1991;Veerakumari and Munuswamy, 2000).Also, damage to the body wall allowing rejection or partial digestion by host's immune system (Andrews et al., 1980;Cortés et al., 2017).It has been proven that benzimidazole and mebendazole affect the energy metabolism of parasites (Kern, 2003).
Our results showed that TPLE (dose-dependent) had a significant effect on the oocyst sporulation of E. papillata, which is attributable to numerous bioactive phytochemical constituents studied by Silva et al. (2012), Ali et al. (2011), andFatima (2016).In vitro, both methanolic extracts of T. polium and T. chamaedrys showed time-and dose-dependent amoebicidal effects, which led to a decrease in the numbers of viable Acanthamoeba castellani trophozoites and cysts (Tepe et al., 2012).Regarding the effect of TPLE on the morphology of Eimeria oocysts, the deformity of oocysts was observed at a concentration of 300 mg/ml.

CONCLUSION
This study demonstrated that TPLE has anticoccidial and anthelmintic efficacy, in vitro.More research should be done to determine the in vivo effectiveness of TPLE.This will inform ongoing studies geared toward the development of TPLE as a novel drug that can be used to manage coccidian diseases that affect animals.

Figure 1 .
Figure 1.FTIR of TPLE in an ethanol medium showing the functional characteristic of the material.
are mean ± SD.All superscripts indicate significance at p ≤ 0.05, * compared to untreated (H 2 O), # compared to mebendazole.

Table 4 .
In vitro anti-coccidial effects of TPLE on the sporulation percentage of Eimeria papillata oocysts Gadelhaq et al. (2018), andAbu Hawsah et al. (2023)bd- Elrahman et al. (2022), which reported the effect of natural extracts of allicin and alcoholic garlic on E. tenella oocysts in chickens.Also, disinfectant formalin (5%) completely inhibited the sporulation of E. papillata, which agreed with Thagfan et al. (2020) and AbuHawsah et al. (2023)stated that this highly reactive chemical interacts with proteins in vitro and inhibits sporulation.In addition, dettol TM , and phenol have been reported to inhibit sporulation at 120 hr by 68.6%, and 46.6%, respectively, which is consistent withMai et al. (2009),Gadelhaq et al. (2018), andAbu Hawsah et al. (2023)that the oocyst wall is impermeable to water-soluble substances and resistant to proteolysis.