The waterborne pathogen Cryptosporidium sp. is widely recognized as having medical importance for both immunocompetent and immunocompromised patients. Around 905 waterborne and 25 foodborne outbreaks are reported by Cryptosporidium sp. worldwide (Baldursson and Karanis, 2011; Efstratiou et al., 2017; Ahmed and Karanis, 2018b).

The most common transmission routes are contaminated food or water consumption, zoonotic transmission, person-to-person contact, and the underestimated but critical route of airborne transmission (Ahmed et al., 2018; Ahmed and Karanis, 2020). At least 47 species (Ježková et al., 2021; Zahedi et al., 2021) and more than 120 genotypes of Cryptosporidium sp. have been identified (Ryan et al., 2021). Almost 20 species of them have been observed in humans, with Cryptosporidium hominis (C. hominis) and Cryptosporidium parvum (C. parvum) being the two most notable species (Innes et al., 2020).

In the first 2 years of life, Cryptosporidium sp. is the second most prevalent cause of diarrhea in developing countries (Kotloff et al., 2012). In seven areas of Sub-Saharan Africa and Asia, it is considered the fourth leading cause of diarrhea (Checkley et al., 2015).

With the highest estimated prevalence range of 21–50%, Cryptosporidium sp. is the third most often reported pathogenic protist in African nations. In Egypt, the prevalence of Cryptosporidium sp. in humans is between 11 and 20% (Ahmed et al., 2022). Egypt led the way in reporting more than one-third (36/122) of waterborne protozoa reports in Africa, with Cryptosporidium sp. likely being common in numerous Egyptian water supplies (Ahmed et al., 2018). Despite the availability of several diagnostic procedures for detecting Cryptosporidium sp. in feces, food, and water (Ahmed et al., 2018; Ahmed and Karanis, 2018a,b), diagnosis for Cryptosporidium sp. is not controlled in Egyptian laboratories (Ahmed et al., 2022) indicating that the prevalence above is greatly underestimated.

Nitazoxanide is the only drug that has been FDA-approved for treating Cryptosporidium sp. infection in people with healthy immune systems. However, the efficacy of nitazoxanide in immunocompromised patients is unclear (CDC, 2021; Schneider et al., 2021). The lack of continuous culturing, cumbersome screening procedures for chemotherapeutic treatments, and a scarcity of genetic manipulation tools limits drug and vaccine research against Cryptosporidium sp. (Karanis, 2018; Gururajan et al., 2021).

Severe cases frequently necessitate hospitalization in immunocompetent individuals and are more common in youngsters and the elderly (Shaposhnik et al., 2019; Collier et al., 2021). Immuno-compromised people, including transplant patients, HIV and cancer patients receiving chemotherapy, frequently have to temporarily halt their treatment regimens when they contract Cryptosporidium sp. infection (Sparks et al., 2015; Schneider et al., 2021; Ahmed et al., 2023). Significant economic losses and zoonotic relevance are associated with cryptosporidiosis, specifically C. parvum. Due to the disease’s harmfulness and the ease with which newborn calves might develop it, it is difficult to control (Zhang et al., 2018). Anti-cryptosporidials would undoubtedly benefit patients at high risk of Cryptosporidium infection and the veterinary industry.

Herbal formulations with anti-Cryptosporidium sp. activity have been identified in recent years. They proposed effective treatment against C. parvum in vitro (Jin et al., 2019; Mendonça et al., 2021). The biodiversity of plants makes them a commonly explored source of novel bioactive compounds, providing molecules with distinct structures, complex or simple, with colossal chemical variety (Morais et al., 2020). Natural products, particularly crude forms, have been used traditionally to treat various parasitic diseases, such as babesiosis, leishmaniasis, malaria, and trypanosomiasis (Ismail et al., 2020; Kingston and Cassera, 2022). Moreover, natural products have afforded the pharmaceutical industry potential antiparasitic drugs, such as the antimalarial drugs artemisinin from Artemisia annua and quinine from Cinchona succirubra (Kayser et al., 2002) in addition to licochalcone A, amphotericin B, and ivermectin (Kayser et al., 2003).

Asteraceae (Compositae) is considered one of the most prominent and widely distributed plant families (Panda and Luyten, 2018). In Egypt, this plant family comprises 14 genera, including the genus Artemisia (Mokhtar et al., 2019) which is represented by five species; A. monosperma Delile, A. vulgaris, A. verlotiorum, A. scoparia Waldst and A. judaica L. (Badr et al., 2012). Plants of this genus were reported to possess antioxidant, antimicrobial, and cytotoxic activities (Bora and Sharma, 2011; Pandey and Singh, 2017). These biological activities are linked to their phytoconstituents, of which terpenoids and phenolics predominate (Bora and Sharma, 2011; Pandey and Singh, 2017; Goda et al., 2021).

A. judaica (Asteraceae) is a perennial aromatic shrub, wildly distributed in Egypt, particularly in Sinai, Gabal Elba, Mediterranean, and Red Sea regions (Ahmed et al., 2017) and has been extensively employed in traditional medicine practices. Therefore, it is almost ubiquitous in Egyptian households. A. judaica has demonstrated potent wound healing, antiviral, antibacterial, antihelminth, and antiprotozoal properties in previous research (Mokhtar et al., 2019; Kshirsagar and Rao, 2021; EDA, 2022; Marakhova and Emam, 2022; Mohammed et al., 2022; Qanash et al., 2023). Due to its availability, affordability, and anti-protozoal activity, A. judaica is a prime candidate for testing against Cryptosporidium oocysts. This study, therefore, aims to investigate the cytotoxic activity of A. judaica L. ethanolic extract and its fractions against C. parvum using (a) in vitro experiments, (b) acting mechanism, (c) in vivo bioassay to demonstrate the infectivity of the treated oocysts.

Herbals have selective actions against parasites without reduction of host cell viability (Haynes and Krishna, 2004). Artemisia was reported worldwide as an effective treatment against several protozoa parasites, including Giardia duodenalis, Blastocystis sp., Leishmania major, and Plasmodium falciparum (Mokhtar et al., 2019; Abd-Elhamid et al., 2021; Gruessner and Weathers, 2021; Najm et al., 2021). Since ancient times in Egypt, natural products have been favored and widely used to cure parasitic infections and other diseases (Aboelsoud, 2010). The cost-effectiveness and absence of adverse effects of natural products made them favorable (Ahmed et al., 2019).

Artemisia judaica L. is indigenous to Sinai Peninsula (Bakr, 2014). A. judaica infusion treats many diseases in folk medicine. Moreover, its diverse pharmacological effects were evidenced (Goda et al., 2021). These bioactivities could be attributed to the plant’s phytochemicals. A. judaica accumulates volatile oil, sesquiterpene lactones, and phenolic compounds (Galal et al., 1974; Abdelgaleil et al., 2008; Janaćković et al., 2015). Recent phytochemical research by LC/MS/MS showed that A. judaica possessed a plethora of flavonoids, phenolic acids, and terpenoids (Goda et al., 2021).

Plant phenolics are a diverse, widely distributed group of phytochemicals. They comprise stilbenes, tannins, coumarins, phenolic acids and flavonoids (Abotaleb et al., 2019). Polyphenols are natural antioxidants in fruits and vegetables (Abotaleb et al., 2019; Zhang et al., 2022). The most predominant polyphenolic compounds in the plant kingdom are phenolic acids and flavonoids (Chen et al., 2022). In recent years, plant polyphenols have received increasing attention and drawn much interest due to their diverse biological activities (Zhang et al., 2022), including antiprotozoal activities (Phillipson and Wright, 1991). Therefore, In the current study, the total phenolics, flavonoids contents in A. judaica were quantified. The obtained results revealed that the plant was a rich source of phenolic and flavonoids (52.6 ± 3.1 mgGAE/g, 64.5 ± 3.1 mg QE/g, respectively).

On the other hand, terpenes and terpenoids are a large, chemically, and functionally diverse group of secondary metabolites, among which several members exhibited cytotoxic, antimicrobial, and antiparasitic effects (Ajikumar et al., 2008). Thus, in this study, the amount of such compounds in A. judaica was estimated spectrophotometrically, where linalool was employed as a standard. Our findings demonstrated that A. judaica contained a considerable amount of terpenoids (9.5 ± 1.1 mg Linol/g).

Plant antioxidants, among them polyphenolics (phenolic acids and flavonoids) and terpenoids exert their effect through various processes, including metal reduction, chelation, hydrogen transfer, electron transfer, and singlet oxygen quenching (Graßmann, 2005; Eltamany et al., 2020). In the current study, reactive oxygen species (ROS) scavenging mechanisms, the antioxidant potential of the ArEx, as well as ArT and ArPh, was evaluated simultaneously using four indicative assays (DPPH, ABT, FRAP and TAC). The current study findings demonstrated that ArPh exhibited the highest DPPH and ABTS^•+ radical quenching effects (IC₅₀ values of 47.27 ± 1.86 μg/mL and 66.89 ± 1.94 μg/mL, respectively) as well as the FRAP and TAC (2.97 ± 0.65 mMol Fe⁺²/g and 46.23 ± 3.15 mg GAE/g, respectively). The ArEx showed noticeable anti-free radical effects against DPPH and ABTS^•+ (IC₅₀ = 123.83 ± 2.71 μg/mL and 154.15 ± 4.92 μg/mL, respectively), FRAP (2.34 ± 0.42 mMol Fe⁺²/g) and TAC (40.91 ± 2.63 mg GAE/g) compared to ArT (233.24 ± 5.17 μg/mL, 481.92 ± 8.76 μg/mL, 1.38 ± 0.26 mMol Fe⁺²/g and 23.54 ± 1.98 mg GAE/g, respectively).

ROS boost infection as they regulate growth, proliferation, pathogenesis, and virulence of bacteria, viruses, and parasites at physiological levels as cellular signaling molecules (Goes et al., 2016; Szewczyk-Golec et al., 2021). Excessive generation of ROS can generate oxidative stress, resulting in cell damage and death. Consequently, cells include antioxidant networks to scavenge excess ROS (Poljsak et al., 2013). For instance, iron-dependent ROS signaling triggers the differentiation of virulent Leishmania amazonensis amastigotes (Mittra and Andrews, 2013). Blastocystis hominis virulence and infectivity were increased by oxidant-antioxidant homeostasis changes. Oxidative stress was also crucial to C. parvum infection in mice (Bhagat et al., 2017). Antioxidants slow infectious illnesses (Kaur et al., 2018). In vivo anti-Cryptosporidium actions were reported in olive and fig extracts, which increased glutathione reduced form, superoxide dismutase and catalase plasma levels compared to the infected control group (Abd El-Hamed et al., 2021).

The oocysts of Cryptosporidium sp. are known to be small (4–6 μm) and have a low infectious dose (1–10 oocysts); it is reported that they can survive in water for 6–12 months or longer and can produce epidemics even with the drinking of a treated water (Karanis, 2018; Omarova et al., 2018). The adverse effects and growing resistance to the available antiparasitic medicines used to treat C. parvum make it difficult for researchers to identify a natural herbal alternative that is nontoxic and easily accessible in sufficient numbers to replace the market-standard drugs (Abdelmaksoud et al., 2020). Therefore, Cryptosporidium sp. would be a parasite of particular interest in terms of its interaction with A. judaica.

Based on the preceding considerations and the demonstrated antioxidant activity of A. judaica crude extracts and fractions; specifically, the phenolic one, the ArEx and its fractions; ArPh and ArT were tested for their toxicity to C. parvum oocysts using the MTT assay. This colorimetric test estimates cellular metabolic activity as an indicator of cell viability and cytotoxicity. The ArPh demonstrated a potent toxic effect against C. parvum oocysts with an IC₅₀ value of (IC₅₀ = 31.6 μg / mL). MTT assay is the gold standard for cytotoxicity testing and promptly determines the viability of microorganisms following drug treatment (Iqbal and Keshavarz, 2017; Holzhausen et al., 2019). The potency of phenolic fraction in the present study is aligned with earlier studies that revealed the high concentration of phenolics was highly efficient in the treatment of quinine-resistant malaria (Ferreira et al., 2010; Fordjour and Adjimani, 2020; Mamede et al., 2020).

Since the phenolic fraction of A. judaica was the most effective treatment based on the MTT assay, it was chosen to evaluate the effect of ArPh on C. parvum oocysts using parasitological experiments. The current investigation indicated that the harmful activity of ArPh against C. parvum oocysts is dose-dependent (Table 4). The influence significantly affected the count and the oocysts’s morphological characteristics. Another study proved that six polyphenolic compounds displayed anti - C. parvum and anti - Encephalitozoon intestinalis action, indicating that these compounds may be employed alone or in conjunction with other moderately active drugs to enhance efficacy (Mead and McNair, 2006). Against Trypanosoma brucei and Toxoplasma gondii cultures, phenolic compounds displayed strong action (Sun et al., 2016; Jafari et al., 2021). Extracts of the Artemisia herba-alba plant included a variety of phenolic compounds, which had antibacterial and antioxidant effects, and hence slowed the growth of both gram-positive and gram-negative bacteria (Mohammed et al., 2021).

In the current study, oocysts stained with TB and mZN exhibited microscopically observable morphological alterations produced by ArPh. The ArPh-TO were lysed, resulting in the loss of oocysts membrane and the expulsion of their contents (Figure 5). ArPh induced morphological alterations in C. parvum oocysts due to a cascade of programmed cell death, during which the oocysts membrane became deformed and created pores inside the cell membrane, resulting in cell lysis and death of the parasite. In another study that examined the effect of chitosan nanoparticles, a similar cascade to that of chitosan-treated C. parvum oocysts was seen, wherein the authors demonstrated that recorded changes altered the oocysts’ size, shape, interior content, and degree of staining (Ahmed et al., 2019). Additional research on other parasites confirmed the same fact. A. annua seeds and leaves have been observed to induce cell death in Leishmania donovani promastigotes (Islamuddin et al., 2014; Antwi et al., 2019). Infusions of A. afra at low concentrations had strong inhibitory effects on all Plasmodium species and stages examined (Gruessner and Weathers, 2021; Ashraf et al., 2022). Artemisinin was also observed to induce ultrastructural activity and damage the mitochondria of Toxoplasma gondii (Rosenberg et al., 2019).

In the current study, the exhibited ArPh activity against C. parvum could be linked to the previously reported flavonoids and phenolic acids in A. judaica (Goda et al., 2021). Quercetin and apigenin displayed an antiparasitic effect against Enterocytozoon intestinalis while naringenin and genistein were effective against C. pavum (Mead and McNair, 2006). Different phenolic components of A. judaica displayed substantial antileishmanial action via distinct pathways against diverse strains of Leishmania. Quercetin, fisitin, and luteolin inhibited darginase, a key enzyme in Leishmania (Manjolin et al., 2013). On the other hand, caffeic acid promoted amastigote apoptosis by TNF-α/ROS/NO production and reduced iron availability (Bortoleti et al., 2019). Apigenin was leishmanicidal against L. amazonensis-infected macrophages via the generation of ROS (Fonseca-Silva et al., 2016). It was demonstrated that p-hydroxybenzoic acid had an antiparasitic impact on Entamoeba histolytica trophozoites (Degerli and Tepe, 2015). The exact mechanism adopted by the ArPh in the damage of C. parvum remains of utmost interest.

The comet assay analyzed several types of DNA damage and repair in individual cells, including invertebrate and plant cells (Olive, 1999). This approach has the advantages of requiring a small number of cells, being highly sensitive to the detection of nuclear DNA damage and applying it to all eukaryotic cell types (Olive, 1999). Consequently, it was utilized to examine the apoptotic mechanism in ArPh-TO. In the present study, the comet tail moment indicated nuclear DNA of C. parvum produced by ArPh exposure increased in comparison to control. The tail length reflected the distance the DNA traveled from the oocyst cell and moved to its most distant location (Figure 6). This finding strongly implies that as the ArPh dose is increased, nuclear DNA fragmentation of oocysts increases.

The nuclear DNA fragmentation of ArPh-TO might be related to both parasite-specific and ArPh-specific mechanisms. While C. parvum lacks genes for typical molecular therapeutic targets seen in other protozoan parasites, it has numerous genes encoding unique plant-like and bacterial-like enzymes that catalyze potentially critical biosynthetic and metabolic pathways (Abrahamsen et al., 2004). C. parvum lacks both the tricarboxylic acid cycle and the oxidative phosphorylation steps necessary for generating metabolic energy (ATP). As a result, the parasite solely depends on anaerobic respiration (the glycolytic pathway) to generate ATP, which is essential for the parasite’s survival and the pathogenesis it causes in the host (Abrahamsen et al., 2004). Previous research has demonstrated the significance of glycolytic enzymes (pyruvate kinase) as possible therapeutic targets for the treatment of cryptosporidiosis (Eltahan et al., 2018, 2019; Khan et al., 2022). An earlier study has reported that luteolin possessed anti-leishmanial activity exerted by the induction of apoptosis through the activation of topoisomerase II-mediated kinetoplast DNA cleavage (Mittra et al., 2000). Bioflavonoids induced apoptosis and DNA damage to Leishmania donovani amastigotes and promastigotes (Mehwish et al., 2021). Ferulic acid-induced cellular disturbances in adult worms characterized by in situ DNA fragmentation, nucleosomal DNA laddering, and chromatin condensation (Saini et al., 2012).

Through the previous results we can infer that apoptosis is the mechanism of action of ArPh-oocysts toxicity as indicated by the following: i. Trypan blue staining, which was used as a basic protocol to indicate early and late apoptosis (Zhivotosky and Orrenius, 2001); ii. Comet assay, which demonstrated DNA fragmentation of oocysts, an important biochemical hallmark of apoptosis (Zhang and Xu, 2000); and iii. High ROS–mediated DNA fragmentation apoptotic properties of ArPh (Higuchi, 2003).

The animal model remains the gold standard for assessing the infectivity of oocysts (Robertson and Gjerde, 2007). Mice were fed ArPh-TO after 48 h to evaluate whether the in vitro impact of ArPh could reach infectivity in vivo. In the current study, when compared to mice that were fed with an untreated C. parvum oocyst infection, ArPh-TO did not induce infection in mice at 200 μg/mL (the minimum fatal dose) of ArPh in vitro. Similar reported data were obtained from in vivo experiments with Cryptosporidium sp. at 20 mg/mL, A. spicigera possessed significant anti-Cryptosporidium efficacy in mice (Shahbazi et al., 2021). The methanol extract of Asafoetida reduced Cryptosporidium sp. infection in experimentally infected mice and improved the histological alterations of small intestinal villi (Abdelmaksoud et al., 2020). The ethanolic extract of olive (Olea europaea) pomace, after oil pressing and phenol recovery, reduced C. parvum development in a reproducible manner. Curcuma longa L. extract significantly impacted Cryptosporidium oocyst shedding in in vivo research (Khater et al., 2017). In contrast, neither water nor ethanol extracts of propolis could eliminate the infection, but they did lower oocysts shedding (Soufy et al., 2017). The promising antioxidant activity of phenolic fraction of A. judaica demonstrated in the current investigation may be the responsible for the negative results of the infectivity assay, since the C. parvum infection is ROS-dependent (Bhagat et al., 2017). However, the role of plant extracts and phytochemicals against Cryptosporidium oocysts is still debatable (Almoradie et al., 2018; Ullah et al., 2020). Further clinical trials would also be encouraging in future studies.

This study demonstrates that the phenolic fraction of A. judaica L. can be a potentially medication effective against C. parvum oocysts by activating nuclear DNA fragmentation and destroying its morphological characteristics and the number of viable oocysts.

The benefits of using ArPh are its simple application method and its high level of safety as a herb. A. judaica phenolic part can be utilized to treat water systems and evident Cryptosporidium sp. contamination and thereby avert multiple waterborne epidemics. It can also be added to food sources to avert foodborne cryptosporidiosis or formulated into a medicine to treat humans and animals, and particularly in farm cows, to prevent cryptosporidiosis-related industrial losses.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

The animal study was reviewed and approved by Suez Canal University's Research and Ethics Review Committee (Approval number: 5603, October 2022).

SA and PK contributed to conception and design of the study. SA, EE, SE, MN, and AM organized the database. SA, EE, SE, and MN performed the statistical analysis. SA wrote the first draft of the manuscript. EE, SE, and MN wrote sections of the manuscript. SA, EE, MN, and AM funded resources of the experiment. SE funding of publication. All authors contributed to manuscript revision, read, and approved the submitted version.

This research work was funded by Institutional Fund Projects under grant no. (IFPIP:1929-166-1443). The authors gratefully acknowledge technical and financial support provided by the Ministry of Education and King Abdulaziz University, DSR, Jeddah, Saudi Arabia.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.