Ameliorative Effects of Teucrium polium Leaves Ethanolic Extract on Liver Histopathology Induced by Eimeria papillata

Coccidiosis, caused by apicomplexan parasites of the genus Eimeria, is a well-recognized parasitic disease affecting the intestinal tract of wild and domestic animals (Sohaib and Jamil, 2017). The infection targets various intestinal sites and progresses rapidly (within 4-7 days), marked by parasite replication within host cells and significant damage to the intestinal mucosa. One such species, E. papillata, inhabits the jejunum of mice, leading to significant damage to the intestinal mucosa, inflammation and heightened oxidative conditions (Abdel-Tawab et al., 2020; Abdel-Tawab et al., 2024). While its primary impact is on the intestinal tract, it indirectly affects accessory organs like the liver. where is the liver exhibits an even more pronounced response, despite not being a direct target site of E. papillata (Dkhil et al., 2011a). Research indicates that Eimeria infection impact the liver and exhibits several signs of moderate inflammation, i.e., inflammatory cellular infiltrations around the central vein, dilatated blood sinusoids, an increase in vacuolated hepatocytes, hypertrophic Kupffer cells. Dkhil and Al-Quraishy (2012) reported metabolic disturbance and hepatic tissue damage induced by E. papillata infection. So, hepatic health is severely compromised due to metabolic deterioration and elevated levels of key hepatic parameters alongside hepatic tissue damage.
       
To combat coccidiosis and enhance poultry health, conventional strategies like administering coccidiostats in feed and preventive vaccinations are commonly employed. However, there's a growing concern over the emergence of drug-resistant oocysts and the presence of drug residues in broiler meat and its toxicity. Currently, a wide range of broad-spectrum anti-coccidial medications, including decoquinate, toltrazuril, amprolium, diclazuril and sulfonamide, are used to treat and prevent eimeriosis. These drugs have been developed toxicity and resistance as a result of frequent use. Nonetheless, efforts to find novel compounds with anti-Eimeria action should be made in light of growing concerns regarding parasite resistance, consumer health and the environmental safety of commercial medications (Wunderlich et al., 2014). Additionally, the issue of drug resistance has prompted numerous researchers to look for alternative approaches to treating coccidiosis.
       
In summary, a number of mechanisms are known by which coccidia become resistant to anticoccidial drugs. Resistance may involve altered permeability of the cell membrane so that the drug is no longer taken up or is rapidly pumped out of the cell (ionophores), use of an alternative biochemical pathway (clopidol, quinolones) and modification of the target sites in the coccidia parasites (amprolium).
       
Also, there are several processes that are understood to cause coccidial resistance to anticoccidial medications. Whereas, resistance can take several forms, such as changing the target locations in the coccidia parasites (amprolium), using an alternate biochemical pathway (clopidol, quinolones), or changing the permeability of the cell membrane so that the drug is either rapidly pumped out of the cell or no longer taken in (ionophores) (Abbas et al., 2011).
       
In response to these pressing global needs, there’s a heightened interest in safe and effective alternatives for controlling coccidiosis, including the use of plant extracts, essential oils and traditional medicinal products. Where it is considered medicinal plants serve as the primary source of alternative or natural medical systems, esteemed for their therapeutic properties. These approaches are valued for their efficacy in yielding effective chemical ingredients (Sidiropoulou et al., 2020 and Mosaddeghi et al., 2021)., showcasing organ-protective characteristics in Eimeria-infected hosts while targeting the parasites directly (Wunderlich et al., 2014).
       
T. polium, a perennial wild flowering plant that is widely distributed, has been traditionally utilized in treating various ailments (Akin et al., 2010; Çakýlcýoðlu et al., 2010; Couladis et al., 2003). Recent studies on this plant have unveiled its efficacy in treating a multitude of disorders, likely attributed to its flavonoids and sterols, known for their anti-inflammatory properties (Abdollahi et al., 2003). T. polium, fragrant in nature, contains a rich array of compounds, including tannin, terpenoid, saponin, flavonoid, glycoside-a, sterol, leucoanthocyanin, beta-caryophyllene, caryophyllene oxide, diterpenoid, asparagine and dietrin, some of which exhibit anti-inflammatory effects (Aburjai et al., 2006; Ansari et al., 2009; Oganesyan et al., 1991).
       
Multiple studies highlighted the hepatoprotective properties of T. polium extract. For instance, Kareem and Hamza (2022) demonstrated that the aqueous extract of T. polium mitigated liver tissue damage induced by the preservative sodium benzoate in male rabbits (Oryctolagus cuniculus). Similarly, Panovska et al., investigated the hepatoprotective effects of the ethyl acetate extract of T. polium L. against liver damage induced by carbon tetrachloride (CCl₄). They found that intraperitoneal injection of T. polium extract over 7 days led to the restoration of liver damage to a normal state (Kadifkova Panovska et al., 2007). Furthermore, liver morphology and histopathological findings corroborate the protective efficacy of this extract against liver damage induced by acetaminophen (Forouzandeh et al., 2013).
       
In a phytochemical investigation by Bahramikia and Yazdanparast(2012), extracts and isolated components from various parts of T. polium exhibited distinct biological effects, including antioxidant, antiseptic, anti-inflammatory, cytotoxic, antibacterial, antifungal properties and in vitro anthelminthic and anticoccidial activity (Al-Shaebi et al., 2023; Bahramikia and Yazdanparast, 2012).
       
The present study aimed to assess the anticoccidial activity and ameliorative effects of T. polium TPLEE extract against oxidative stress and histopathological changes in the liver of mice infected with E. papillata.

Between August 2023 and February 2024, this experiment was finished in the zoology department of King Saud University's College of Sciences. In this study, thirty-five adult Swiss albino male mice, aged between 8 to 10 weeks and weighing 30 to 35 g, were obtained from the animal house at the College of Science, King Saud University. The mice were housed in well-ventilated cages under specific pathogen-free conditions, with a controlled temperature of 23±5°C and a 12-hour light/12-hour dark cycle. Throughout the study, the animals were provided with standard pellet diets and tap water ad libitum. Before commencing the experiment, the mice were allowed a period of seven days for acclimatization.
       
In May 2022, T. polium leaves were gathered from Al-Badyah Tabuk, Saudi Arabia, situated at 27°45'59.5"N 36°31'48.8"E, approximately 80 km south of Tabuk. The plant’s identification was confirmed by a specialist at the herbarium of the Botany Department, College of Science, King Saud University, Riyadh, Saudi Arabia. Following the method outlined by Qabaha et al., (2021) with some modifications, T. polium leaf extract was prepared. The leaves were air-dried and subsequently ground into a powder. This powder was then subjected to a cold maceration extraction technique using an ethanol (50%) solvent system for a duration of 24 hours. The ethanolic extract was filtered, concentrated using a rotary evaporator under pressure and at a temperature of 50°C and then collected and stored in sealed bottles at -20°C.
       
This study used the coccidian parasite, E. papillata, as a model parasite. Eimeria parasite oocysts were passaged in laboratory mice (Mus musculus). Unsporulated oocysts were collected from faeces and allowed to sporulate in a solution containing 2.5% (w/v) potassium dichromate. They were then washed in a phosphate buffer solution to prepare them for the remainder of the experiment. A preliminary study was done to determine the most effective dose of TPLEE (50, 150 and 250 mg/kg). Seven groups of five mice each were used (Table 1), as follows: 
 

       
Groups (3-7) were orally inoculated with 100 ml of saline containing about 103 sporulated E. papillata oocysts. After 60 minutes of infection, groups 4,5, 6 and 7 were treated for five succeeding days with the mentioned doses of TPLEE and amprolium, respectively (Table 1).
       
On the 5^th day post-infection (p.i.), faecal pellets from each mouse in the 4 to 7 groups were collected. The total number of shed oocysts was determined following the method described by Schito et al., (1996). Additionally, the suppression (%) of oocyst shedding was calculated using the formula:
   
Subsequently, all mice were euthanized and their liver was harvested and preserved for use in the subsequent stages of the experiment.
       
Small pieces of liver tissue were extracted and immediately preserved in 10% neutral buffered formalin before being embedded in paraffin. Tissue sections, 5 mm thick, were then stained with hematoxylin and eosin to detect histopathological alteration. To show total carbs by the histochemical analysis, slices were stained using periodic Acid-Schiff’s procedure (Hotchkiss, 1948).
       
The liver homogenate was prepared following the protocol outlined in Dkhil et al., (2012). The glutathione (GSH) level in the liver homogenate was determined according to Ellman (1959). During this process, GSH is decreased to produce a yellow molecule using Ellman’s reagent (5,52 dithiobis (2-nitrobenzoic acid). When chromogen absorbance is measured at 405 nm, it is directly correlated with GSH content. Similarly, the amount of lipid peroxidation in the liver homogenate was determined by heating the liver homogenate to a boiling point for thirty minutes in a water bath and adding one milliliter of each of trichloroacetic acid (10%) and thiobarbituric acid (TBA) (0.67%). By measuring the absorbance at 535 nm, thiobarbituric acid-reactive molecules were identified and malondialdehyde (MDA) equivalents were used as a gauge Ohkawa et al., (1979). The NO assay on the liver homogenate was conducted in accordance with the protocol outlined by Berkels et al., (2004). When N-(1-naphthyl) ethylenediamine was mixed with sulfanilamide, it was oxidized by the nitrous acid that was created in an acidic medium with nitrite. This resulted in the appearance of a vivid reddish-purple azo dye. The resultant color was measured at 540 nm. The homogenate’s superoxide dismutase (SOD) activity was measured using the Nishikimi et al., (1972) method. The success of the experiment depends on the enzyme’s capacity to stop the nitroblue tetrazolium dye from being reduced by phenazine methosulphate, which is visible at 560 nm.
       
SPSS (version 20) statistical program (SPSS Inc., Chicago, IL, USA) was used to analyze our data by one-way analysis of variance (ANOVA) to statistical comparisons among the groups. Results were expressed as mean ± SE and values of p£0.05 were considered statistically significant.

Based on a preliminary study, treatment of infected mice with various doses of TPLEE demonstrated a notable suppression of oocyst output. Particularly, treatment with 150 mg/kg TPLEE exhibited a suppression percentage of 91.2±4.1, closely resembling the efficacy of the reference drug amprolium (93.2±3.9). However, the other two doses showed a significant decrease in efficacy (Table 2). Consequently, for subsequent investigations, the dose of 150 mg/kg was selected.
 

       
Experimental infection of mice with E. papillata oocysts led to the development of different parasite stages in the epithelial cells of the jejunum (Table 3). The treatment with 150 mg/kg TPLEE resulted in a significant reduction in the number of parasitic stages.
 

       
Microscopic analysis of eosin/hematoxylin-stained liver sections did not show any evidence of parasites (Fig 1). However, the liver exhibited moderate pathological alterations, including inflammatory injury, vacuolated hepatocytes and hyperplasia of Kupffer cells, when compared to the livers of non-infected mice.
 

 
In addition, staining of the liver tissue of mice infected with the parasite with periodic acid-Schiff revealed a reduced amount of carbohydrates compared to the control group (Fig 2).
 

 
Infections with E. papilata resulted in a statistically significant (P<0.05) increase in NO and MDA levels in the liver of mice. TPLEE (150 mg/kg) treatment lowered the E. papillata-induced increase in the levels of both NO and MDA significantly (Fig 3 and 4).
 

 

       
Notably, the levels of GSH and SOD showed a significant (P<0.01) and (P<0.001) decrease as a result of infection with the parasite, reaching (143 ±14) and (67±10), respectively, compared to the control group, while treatment with the plant extract (150 mg/kg) led to a significant increase (P<0.05), reaching (175±10) and (96±9), respectively, compared to the infected and untreated group (Fig 5 and 6).
 

 

       
The liver serves as the initial site of interaction with the intestine via the portal vein. E. papillata typically targets the intestine, particularly the jejunum and normally does not invade the liver, though other lymphoid organs like the spleen and lymph nodes can be affected by related parasites such as E. coecicola (Renaux et al., 2001). Our study demonstrates that TPLEE effectively targets Eimeria parasites in the host. TPLEE exhibits significant anti-coccidial efficacy by markedly suppressing the percentage of oocysts and reducing the total number of parasite stages in the jejunum.
       
TPLEE’s anticoccidial activity has been documented in coccidiosis. where is this effectiveness is likely due to its saponin component, known for its anti-coccidian properties. Saponins interact with cholesterol on the parasite cell membrane, impeding protozoan growth and ultimately leading to parasitic death (Al-Shaebi et al., 2023 and Al Sulaibi et al., 2020).
       
In the current study, E. papillata infections induce a mild inflammatory response in the hepatic tissue of mice, characterized by an increase in Kupffer cells and the abundance of leukocytes, particularly lymphocytes, as a typical tissue response to injury. Moreover, it’s known that microbial and food antigens from the intestine can reach the liver through the hepatic portal system, triggering inflammatory and immune responses (Nagura and Sumi, 1988). The inflammation triggered by E. papillata prompts the activation and proliferation of numerous Kupffer cells (KCs). KCs, specialized macrophages located in the liver and integral to the mononuclear phagocyte system, demonstrate remarkable adaptability. They can alter both their morphology and function in response to shifts in the liver¢s microenvironment (Tacke and Zimmermann, 2014; Wynn and Vannella, 2016).
       
Previous studies demonstrated that the liver does respond strongly to E. papillata infections. Structural signs of moderate inflammation in the liver coincide with oxidative tissue damage (Dkhil et al., 2011b). Another species of Eimeria, E. coecicola, which targets the appendix part of the intestine in rabbits, also induces liver injury. Many rabbits exhibit several signs of moderate inflammation, including inflammatory cellular infiltrations around the central vein, dilated blood sinusoids, increased vacuolated hepatocytes and hypertrophic Kupffer cells (Al-Quraishy et al., 2012). TPLEE showed a hepatoprotective effect as it decreased signs of liver injury and restored the number of Kupffer cells. The ameliorative effects are attributed to its flavonoids and sterols, known for their anti-inflammatory properties (Çakýlcýoðlu et al., 2010).
       
The breakdown of liver glycogen, the buildup of triglycerides in the liver and the mobilization of adipose tissue are among the metabolic consequences of inflammation brought on by an E. papillata infection. More precisely, cytokines stimulate lipolysis directly, reduce feed intake and worsen insulin sensitivity to facilitate the breakdown of fat storage (Kushibiki et al., 2003). The low hepatocyte affinity for the periodic acid Schiff reaction (Fig 2) was indicative of this.
       
Our findings indicate that oxidative liver damage in mice infected with E. papillata infection is linked to a reduction in antioxidant enzymes like GSH and SOD and this is consistent with (Dkhil et al., 2012: Thagfan et al., 2023 and Al-Sayed et al., 2022), where is during an Eimeria infection, these enzymes are essential for shielding the animal body from free radical damage. When these enzyme levels drop, DNA and cellular membranes are harmed, lipid peroxidation is increased, protein oxidation is increased, intracellular stability is altered and cell death is induced. This irreversible damage is the result of an increase in reactive oxygen species.
       
In addition, the pathophysiology of intestinal coccidiosis was closely associated with nitric oxide (NO) and malondialdehyde (MDA), which were generated as a part of the host’s cellular immune response to the Eimeria infection and this is consistent with Dkhil et al., (2012). Using TPLEE to treat E. papillata-infected mice resulted in a decrease in oxidative stress in the infected liver, which was corroborated by an increase in SOD and GSH levels and a reduction in MDA and NO. These results support the hypothesis presented by Ljubuncic et al., (2006) that T. polium is rich in flavonoids, which have anti-free radical properties and lead to protection against oxidative stress. Also, our current study’s findings are consistent with a study by Alatawi et al., (2024) that found that TPE was effective as a natural antioxidant for reducing oxidative stress, enhancing antioxidant systems and stopping apoptosis in order to lessen nicotine’s effects on hepatic biochemical and histological changes. Some studies have reported that medicinal plants treat liver damage in mice resulting from infection with E. papillata, such as garlic (Dkhil et al., 2011a); Azadirachta indica (Dkhil et al., 2012); and Phoenix dactylifera (Metwaly et al., 2012. According to Saleh et al., (2023) research, Artemisia monosperma leaves are a good natural source of strong antioxidants and medications that treat anthelminthiasis. which encourages the performance of multiple in vivo studies in quest of a successful cure. This makes these medicinal plants promising in curbing the damage caused by parasites.
       
Also, the study by Saleh et al., (2024) shown that TPLE has enhanced the behavioral performance of mice, which encourages the undertaking of several studies on the substances present in plants.
       
Our findings have proved that TPLE, at a dose of (150 mg/kg body weight), can protect mice against E. papillate infection. Moreover, the effect of TPLE comes from an increase in the intestinal levels of SOD and GSH, along with a decrease in NO and MDA. TPLE, once incorporated into an animal’s regular diet, protects host tissue against impairments caused by a variety of pathogenic diseases. And although high doses of the plant had an effect, the most effective dose was 150 mg/kg.

The current study demonstrated that TPLEE exerted antioxidant properties and noteworthy anticoccidial activity against injuries induced by E. papillata in mice. The hepatoprotective effect was exerted mainly ability and enhancing the antioxidant defense system because of the high content of flavonoids and phenolics in the plant extract. improved histological structure of the liver were further by TPLEE treatment from injuries induced by E. papillata.

This work was supported by the Researchers Supporting Project (RSP2024R3) at King Saud University (Riyadh, Saudi Arabia).
 
Institutional review board statement
 
The animal study protocol was approved by the Institutional Review Board (or Ethics Committee) of Kingdom of Saudi Arabia (Ethics Committee, King Saud University, Ethics Agreement ID: KSU-SE-23-56).

The authors declare that there are no conflicts of interest.