Efficacy of Cyperus rotundus extract against cryptosporidiosis and toxoplasmosis in murine infections

Azza M Fahmy; Amal M Alshenawy; Eman A El-Wakil; Amany M Hegab

doi:10.4103/epj.epj_27_21

ORIGINAL ARTICLE

Year : 2021 | Volume : 20 | Issue : 3 | Page : 242-248

Efficacy of Cyperus rotundus extract against cryptosporidiosis and toxoplasmosis in murine infections

Azza M Fahmy¹, Amal M Alshenawy¹, Eman A El-Wakil², Amany M Hegab³
¹ Parasitology Lab, Department of Immunology and Drug Evaluation, Theodor Bilharz Research Institute, Giza, Egypt
² Department of Medicinal Chemistry, Theodor Bilharz Research Institute, Giza, Egypt
³ Developmental Pharmacology Department, National Organization for Drug Control and Research, Cairo, Egypt

Date of Submission	12-Jun-2021
Date of Decision	24-Jun-2021
Date of Acceptance	26-Jun-2021
Date of Web Publication	20-Sep-2021

Correspondence Address:
PhD Azza M Fahmy
Department of Parasitology and Immunology, Theodore Bilharz Research Institute, Cornish El-Nile-Warrak El-Hadar, PO Box 30, Imbaba, Giza 12411
Egypt

Source of Support: None, Conflict of Interest: None

Check

DOI: 10.4103/epj.epj_27_21

Abstract

Background Apicomplexa is a phylum of single-celled, obligate intracellular protozoan parasites that are among the most common morbidity-causing diseases worldwide. This phylum contains a variety of intestinal protozoa of medicinal and veterinary interest, such as Cryptosporidium and Toxoplasma. These parasites can be acquired orally, before infecting or infiltrating the intestinal epithelium. Nitazoxanide (NTZ) is the only FDA-approved medicinal therapy currently in use. The conventional pharmacological therapies for toxoplasmosis include pyrimethamine and sulfadiazine; nevertheless, they have major limitations. The use of medicinal plants for treatment and to reduce dependence on chemical drugs has become an important goal for therapeutic research.
Objective Intending to develop alternative therapeutic options to address these health problems, we examined the efficacy of an ethanol extract of Cyperus rotundus, which has been demonstrated to have antiparasitic and hepatoprotective effects against Cryptosporidium and Toxoplasma in mice, with the goal of developing alternative therapeutic options to treat these health problems.
Materials and methods A total sample of 72 male mice was used for the experiment, the animals were separated into two groups of 36 mice each: the first group was used to examine the activity of ethanol extract of C. rotundus against Cryptosporidium, and the second group was used to examine its activity against Toxoplasma. Each experimental model was divided into six subgroups of six mice each: the first group was noninfected nontreated, the second infected nontreated, third infected and treated with the standard drug, fourth and fifth infected and treated with C. rotundus at 250 and 500 mg/kg body weight, respectively, and the sixth infected and received a combination of half doses of both drugs [C. rotundus (250 mg/kg/day) and half dose of the standard drug (NTZ or Spiramycin)]. The parasitological parameters and reduced glutathione, super oxide dismutase, and malondialdehyde levels in the liver homogenates were used to determine the infections and medication impacts.
Results and conclusion The results showed a promising finding that ethanol Egyptian herbal extract of C. rotundus and its combination with the standard drugs NTZ and Spiramycin have a promising antiparasitic and hepatoprotective activity against murine cryptosporidiosis and toxoplasmosis, respectively. The combined therapies resulted in the highest effectiveness of standard medications.

Keywords: Cryptosporidium oocysts, Cryptosporidium trophozoite, Cyperus rotundus extract, hepatoprotective activity, Toxoplasma tachyzoites

How to cite this article:
Fahmy AM, Alshenawy AM, El-Wakil EA, Hegab AM. Efficacy of Cyperus rotundus extract against cryptosporidiosis and toxoplasmosis in murine infections. Egypt Pharmaceut J 2021;20:242-8

How to cite this URL:
Fahmy AM, Alshenawy AM, El-Wakil EA, Hegab AM. Efficacy of Cyperus rotundus extract against cryptosporidiosis and toxoplasmosis in murine infections. Egypt Pharmaceut J [serial online] 2021 [cited 2022 Aug 18];20:242-8. Available from: http://www.epj.eg.net/text.asp?2021/20/3/242/326239

Introduction

Cryptosporidium parvum and Toxoplasma gondii are pathogenic protozoan parasites belonging to the phylum Apicomplexa. Cryptosporidium was the second most prevalent cause of moderate-to-severe diarrhea in children under the age of 2 years [1] Furthermore, in immunocompromised persons, it might be a life-threatening infection [2]. There is no vaccine, and the sole FDA-approved medication, Nitazoxanide (NTZ), has been demonstrated to have effective limits in many patient groups known to suffer high-illness risk [3].

T. gondii is the causative protozoan agent of toxoplasmosis, a widespread illness that is found all over the world [4]. Toxoplasmosis is associated with behavioral and neurochemical alterations [5],[6],[7]. Toxoplasma uses a variety of survival mechanisms, including intracellular parasitism and immunological disruption, to evade the host’s immunological response, making vaccine development exceedingly challenging [8]. The usual medication therapies for toxoplasmosis include pyrimethamine and sulfadiazine; however, these medicines cannot eliminate T. gondii bradyzoites [9] and several failure cases have also been documented indicating the presence of drug-resistant strains [10].

Herbal extracts are now seen to be a promising source for the development of novel medications. These herbal extracts possess a wide range of bioactive components that have a particular physiological impact on the human body, such as tannin, flavonoids, alkaloids, and phenolic substances, and may serve as possible substitutes for many synthetic drugs [11]. Since ancient times, Egypt has been known for its herbal medicine. The trends in the use of traditional alternatives to pricey pharmaceuticals, either alone or as complements to the chemical pharmaceuticals in treatment protocols, were discovered [12].

Cyperus rotundus belongs to the Cyperaceae family and distributes all over the world [13], including Egypt [14],[15]. Antibacterial [16], antiviral [17], insecticidal [18], antiplasmodial [19], and antihelmintic [20] actions have been reported for C. rotundus, in addition to anti-inflammatory [21], antidiabetic [22], antidiarrheal [23], cytoprotective [14], antioxidant [24], cytotoxic and apoptotic [25], and antipyretic and analgesic activities [26].

Therefore, in an attempt to develop a new therapy from Egyptian herbal extract for C. parvum and T. gondii, the parasitological and hepatoprotective activities of ethanol extract of C. rotundus against C. parvum and T. gondii in mice were evaluated. In addition, combinations of the extract with the validated anti-apicomplexan drugs may offer a viable therapeutic strategy to be evaluated to boost antiparasitic effects, minimize costs, enhance treatment quality, and reduce medication toxicity.

Materials and methods

Ethical and regulatory guidelines

The experimental animals’ studies were conducted following internationally accepted guidelines, and the Ethical Committee for Animal Experimentation, Theodor Bilharz Research Institute, Egypt, while making efforts to abate animal pain.

Preparation of plant extract

Fresh C. rotundus samples were collected from Orman garden, Giza, Egypt. The rhizomes were identified by a botanical specialist and consultant at Orman botanic gardens. The plant was dried, powdered into a fine powder, and stored in secure containers, Theodor Bilharz Research Institute’s Medicinal Chemistry Department. Five-hundred grams of dry powder was extracted with 85% ethanol for 1 week. The extract was then filtered and concentrated under a vacuum using a rotatory evaporator (BUCHI, Flawil, Switzerland). The crude ethanol extract was then collected and dried for future use [27],[28].

Experimental animals and parasites

A total of 72 male albino mice, —5–6 weeks old, ∼20–22 g, were obtained from Schistosome biological supply program (Theodor Bilharz Research Institute, Cairo, Egypt). To exclude parasite infection, their feces were examined for 3 consecutive days before the commencement of the experiments as determined using the formol–ether concentration method [29] and the modified Ziehl–Neelsen technique [30]. They were kept in a room with temperature, lighting, and relative humidity controls. They were fed conventional food and given unlimited access to water. The animals were separated into two groups of 36 mice each: the first group was used to examine the activity of C. rotundus against Cryptosporidium, and the second group was used to examine the activity against Toxoplasma. The parasitological parameters and oxidative stress in the liver were used to determine infection and medication impact.

The activity of Cyperus rotundus extract against Cryptosporidium

Except for the control group, mice were infected intraesophageally with 1×10³ Cryptosporidium oocysts by gastric gavage [31]. The Cryptosporidium isolates utilized in this study originated from infected patients in Theodor Bilharz Research Institute Hospital. A modified formalin–ether-sedimentation procedure was used to concentrate the samples. To confirm the presence of oocysts, the stool samples were stained using a modified Ziehl–Neelsen staining method. Nanazoxid suspension (100 mg/5 ml) from Utopia Pharmaceuticals (Cairo, Egypt) was administered orally in a dosage of 200 mg/kg daily for 5 consecutive days to mice, based on prior studies [32].

Mice were divided into six subgroups of six mice each. The first subgroup was kept as negative control (neither infected nor treated). The second subgroup was infected but not treated. The third subgroup was infected and given 200 mg of NTZ. In the fourth and fifth subgroups, infected mice were given Cyperus extract at a dosage of 250 and 500 mg/kg body weight, respectively. The sixth subgroup was infected and treated with the combination of Cyperus and NTZ (half dose of both, 250 mg/kg Cyperus+100 mg/kg NTZ). Doses were administered daily for 5 consecutive days [32]. On the 14^th day after treatment, feces were collected from each mouse in the treated and control groups, and oocysts were counted/feces weight. Mice were sacrificed by rapid decapitation, and liver was collected for assessment of different oxidative stress-related biochemical parameters [glutathione (GSH), super oxide dismutase (SOD), and malondialdehyde (MDA)] using the spectrophotometer. Duodenal content was also examined for trophozoite count after scarification of mice, smeared on a slide and stained by modified Zeihl–Neelsen stain, and examined microscopically.

The activity of Cyperus rotundus extract against Toxoplasma

Tachyzoites from the virulent RH strain of T. gondii were maintained in Swiss albino mice by intraperitoneal passages at the laboratory, Department of Parasitology (Theodor Bilharz Research Institute), Egypt. Tachyzoites were extracted from the ascites’ fluid of infected mice on the fourth day of infection [33]. Debris and host cells were filtrated through a sheet of glass-wool fibers. The filtrate was washed three times and diluted with phosphate buffer saline, pH 7.4. After counting in a hemocytometer, suspensions were adjusted to 2×10² tachyzoites/ml with saline and 0.4-ml aliquots were injected intraperitoneally into mice as Grujić et al. [34] described.

All medications were administered on the first day of infection and continued for 5 days. Mice were divided into six subgroups, as described in the Cryptosporidium model: the first was noninfected, the second was infected but not treated, and the third was infected and treated with Spiramycin (Phraonia Pharmaceuticals, Cairo, Egypt, in the form of one-and-a-half milligrams per tablet). The Spiramycin tablet was ground and the dose per mouse was calculated and adjusted to dissolve in 100 μl of saline for oral administration at a dose of 200 mg/kg [34], the fourth and fifth groups were infected and treated with Cyperus extract at doses of 250 and 500 mg/kg body weight [22], respectively. The sixth subgroup was infected and treated with the combination of Cyperus and Spiramycin (half dose of both, 250 mg/kg Cyperus+100 mg/kg Spiramycin). On the 14^th day after infection, peritoneal fluid containing T. gondii tachyzoites was collected, and the mean number of tachyzoites was determined. The percentage reductions in the mean number of parasites in treated versus infected control mice were calculated as per the formula:

Reduction percent: %R=100 (C-T/C); C: the infected control subgroups, T: treated mice subgroups.

Biochemical analysis

The liver was homogenized in the appropriate buffer (1 g/10 ml) using a glass homogenizer. The homogenate was filtered and centrifuged for the analysis of antioxidant enzymes. Reduced GSH, SOD, and MDA levels in liver homogenates were determined using biodiagnostic assay kits according to the techniques of Beutler and Kelly [35], Marklund and Marklund [36], and Mihara and Uchiyama [37], respectively.

Statistical analysis

The Statistical Package for Graph Pad Prism application (San Diego, California, USA), version 6.0 for Windows, was used to analyze the obtained and confirmed data. To compare the different studied groups, quantitative data were presented as mean and SE and analyzed using the F test (analysis of variance) followed by Tukey’s multiple-comparison test. Differences were considered as significant at P value less than 0.05.

Results and discussion

C. parvum and T .gondii are significant parasites of both humans and animals globally, necessitating the development of novel and effective therapies [38]. Medicinal plants are regarded as a valuable source for the discovery of novel antiparasitic drug leads [39],[40],[41] for cryptosporidiosis [42],[43],[44] and toxoplasmosis [45],[46],[47],[48]. As a result, the current study was carried out to create novel anti-Cryptosporidium and anti-Toxoplasma treatments using our native medicinal plant C. rotundus. In this work, the anti-apicomplexan action of C. rotundus extract was markedly observed in mice in both monotherapy (500 mg/kg) and combination therapy against Cryptosporidium and Toxoplasma. A remarkable result to emerge from the data is that the most potent anti-Cryptosporidium and anti-Toxoplasma activity were recorded in the combination treatments of Cyperus with clinical drugs, NTZ and Spiramycin, respectively. The combined therapy resulted in the highest-percentage reduction in the number of Cryptosporidium trophozoites in intestinal contents; 72.6%, followed by NTZ treatment, which resulted in a decrease of 55.8% ([Table 1]). Stool analysis demonstrated a significant reduction in the number of Cryptosporidium oocysts in the group treated with Cyperus (500 mg/kg) and combination group. The combined NTZ-Cyperus-treated group had the largest-percent reduction in oocyst count (85.4%), followed by Cyperus (500 mg/kg) (76.3%) and finally Cyperus (250 mg/kg) group (65.2%). The group that received NTZ had the least reduction ([Table 2]). Peritoneal fluid examination of T. gondii-infected mice indicated that the combination Spiramycin–Cyperus treatment induced the highest-percent reduction in T. gondii tachyzoites count (66.2%), followed by Spiramycin (64.7%) and finally Cyperus (500 mg/kg) group (55.5%). The Cyperus (250 mg/kg) group had the lowest decrease (31.4%) ([Table 3]). To the best of our knowledge, this study is the first investigation to identify Cyperus as an anti-apicomplexan compound with potent efficacy against parasites representing all branches of the apicomplexan phylogeny.

Table 1 The mean number and the percentage of reduction of Cryptosporidium trophozoites in intestinal content 14 days post-treatment

Click here to view

Table 2 The mean number and the percentage of reduction of Cryptosporidium oocyst in stool 14 days post-treatment

Click here to view

Table 3 The mean number and the percentage of reduction of Toxoplasma tachyzoites in peritoneal fluid 14 days post-treatment

Click here to view

The evident anti-apicomplexan action of C. rotundus extract discovered in the present study might be attributed to its functional bioactive components, such as alkaloids, flavonoids, saponins, tannins, and triterpenoids [27],[49],[50],[51]. Alkaloids disrupt the parasite’s amino acid metabolism and/or DNA synthesis of the parasite [52]. Some flavonoids have been shown to suppress C. parvum [53],[54] and T. gondii [55],[56] by damaging cell membranes and inhibiting DNA, RNA, and proteins synthesis, or inhibiting microorganisms’ reproduction [53],[54],[55],[56],[57]. Furthermore, saponins affect the permeability of parasite cell membranes and promote cytotoxic action [58], resulting in parasite degeneration [59]. Tannins have antiparasitic activity as they inhibit parasite metabolism [60],[61],[62]. Terpenoids have been shown to have anti-apicomplexan activity [63],[64], and their recognized actions include cell membrane instability, inhibition of key parasite enzymes with the resulting ultrastructural changes, and cell death [65].

Our findings support the notion that C. parvum and T. gondii cause oxidative stress in experimentally infected mice. This finding was approved by a significant rise in hepatic MDA levels in C. parvum and T. gondii-infected mice, as well as a significant drop in hepatic SOD and GSH levels. Previous findings related to C. parvum [66],[67],[68],[69] and T. gondii infections [70],[71],[72] are consistent with our findings. Another promising finding was that C. rotundus treatment improved oxidative damage generated by Cryptosporidium and Toxoplasma by increasing antioxidant contents (SOD and GSH) and reducing MDA levels in liver tissue ([Table 4] and [Table 5]). Our findings are in line with earlier studies that confirmed the antioxidant activity of C. rotundus extract in vitro and in vivo [14],[24],[27],[73],[74],[75],[76],[77]. C. rotundus was safe up to 2 g/kg body weight and did not cause toxicity to the host cells in vitro and in vivo [49],[78],[79],[80].

Table 4 Effect of Cyperus rotundus on oxidative stress parameters in liver of mice infected with Cryptosporidium 14 days post-treatment

Click here to view

Table 5 Effect of Cyperus rotundus on oxidative stress parameters in liver of mice infected with Toxoplasma 14 days post-treatment

Click here to view

The most striking result to emerge from the data is that combination treatments have antioxidant activity against Cryptosporidium and Toxoplasma that is superior to standard drugs and even improve to the point of approaching that of healthy control. The hepatoprotective activity of C. rotundus extract may be attributed to the presence of flavonoids, alkaloids, terpenoids, and phenols [24],[28],[50],[73]. Herbal medicines’ antioxidant capabilities are beneficial in decreasing the toxicity of hazardous substances [81] or other drugs [82].

It appears that combining medicinal plants that contain biological bioactive compounds with synthetic conventional pharmaceuticals might boost activities, lower the costs, enhance treatment quality, and lessen medication-adverse effects. The mechanism behind the observed combined effects of Cyperus and the synthetic medicines NTZ and Spiramycin in the current investigation remained unknown. It might be related to these drugs’ various synergistic modes of action.

Conclusion

In conclusion, the study supports the effectiveness of the Egyptian herbal extract C. rotundus after being combined with NTZ and Spiramycin in controlling the murine cryptosporidiosis and toxoplasmosis, respectively, than synthetic drugs. In addition to its antiparasitic effectiveness, C. rotundus extract and combined therapy exhibit hepatoprotective activity with a superior effect of the combined therapy than the standard drugs and even improve to the extent of approximating that of healthy control. More research on these combinations is needed since the encouraging results warrant further investigations.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1.	Kotloff K, Nataro J, Blackwelder W, Nasrin D, Farag T, Panchalingam S, Wu YS. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet 2013; 6736:1–14.
2.	Nahrevanian H, Assmar M. Cryptosporidiosis in immunocompromised patients in the islamic republic of Iran. J Microbiol Immunol Infect 2008; 41:74–77.
3.	Ashigbie PG, Shepherd S, Steiner KL, Amadi B, Aziz N, Manjunatha UH et al. Use-case scenarios for an anti-Cryptosporidium therapeutic. Geary TG, ed. PLoS Negl Trop Dis 2021; 15:e0009057.
4.	Oliveira CB, Meurer YS, Andrade JM, Costa ME, Andrade MM, Silva LA et al. Pathogenicity and phenotypic sulfadiazine resistance of Toxoplasma gondii isolates obtained from livestock in northeastern Brazil. Mem Inst Oswaldo Cruz 2016; 111:391–398.
5.	Prandovszky E, Gaskell E, Martin H, Dubey JP, Webster JP, McConkey GA. The neurotropic parasite Toxoplasma gondii increases dopamine metabolism. PLoS ONE 2011; 6:e23866.
6.	Stock A-K., Dajkic D, Köhling HL, von Heinegg EH, Fiedler M, Beste C. Humans with latent toxoplasmosis display altered reward modulation of cognitive control. Sci Rep 2017; 7:10170.
7.	Meurer YDSR, Brito RMDM, da Silva VP, Andade JMDA, Linhares SSG, Pereira Junior A et al. Toxoplasma gondii infection damages the perineuronal nets in a murine model. Mem Inst Oswaldo Cruz 2020; 115:e200007.
8.	Han Y, Adeyemi OS, Kabir MHB, Kato K. Screening of compound libraries for inhibitors of Toxoplasma growth and invasion. Parasitol Res 2020; 119:1675–1681.
9.	Luft BJ, Remington JS. Toxoplasmic encephalitis. J Infect Dis 1988; 157:1–6.
10.	Montazeri M, Mehrzadi S, Sharif M, Sarvi S, Tanzifi A, Aghayan SA, Daryani A. Drug resistance in Toxoplasma gondii. Front Microbiol 2018; 9:2587.
11.	Djeussi DE, Noumedem JA, Seukep JA, Fankam AG, Voukeng IK, Tankeo SB et al. Antibacterial activities of selected edible plants extracts against multidrug-resistant Gram-negative bacteria. BMC Complement Altern Med 2013; 13:164.
12.	Mostafa NM, Singab AN. Prospective of herbal medicine in Egypt. Med Chem (Los Angeles) 2018; 08:04.
13.	Al-Snafi PD. A review on Cyperus rotundus. A potential medicinal plant. IOSR J Pharm 2016; 06:32–48.
14.	Hamed A. Antioxidant and cytoprotective properties of three Egyptian cyperus species using cell-free and cell-based assays. Pharm Crop 2012; 3:88–96.
15.	Samra RM, Soliman AF, Zaki AA, El-Gendy AN, Hassan MA, Zaghloul AM. Chemical composition, antiviral and cytotoxic activities of essential oil from Cyperus rotundus growing in egypt: evidence from chemometrics analysis. J Essent Oil Bear Plants 2020; 23:648–659.
16.	Nima Z, Jabier MS, Wagi RI, Hussain HAA. Extraction, identification and antibacterial activity of cyperus oil from Iraqi C. rotundus. Eng Technol 2008; 26:1156.
17.	Xu HB, Ma YB, Huang XY, Geng CA, Wang H, Zhao Y et al. Bioactivity-guided isolation of anti-hepatitis B virus active sesquiterpenoids from the traditional Chinese medicine: rhizomes of Cyperus rotundus. J Ethnopharmacol 2015; 171:131–140.
18.	Singh SP, Raghavendra K, Dash AP. Evaluation of hexane extract of tuber of root of Cyperus rotundus Linn (Cyperaceae) for repellency against mosquito vectors. J Parasitol Res 2009; 2009:1–5.
19.	Kaushik NK, Bagavan A, Rahuman AA, Mohanakrishnan D, Kamaraj C, Elango G et al. Antiplasmodial potential of selected medicinal plants from Eastern Ghats of South India. Exp Parasitol 2013; 134:26–32.
20.	Kasala S, Ramanjaneyulu K, Himabindhu J, Alluri R. Anthelmintic activity of Cyperus rotundus (L). J Pharmacogn Phytochem 2016; 5:407–409.
21.	Rocha FG, Brandenburg MDM, Pawloski PL, Soley BDS, Costa SCA et al. Preclinical study of the topical anti-inflammatory activity of Cyperus rotundus L. extract (Cyperaceae) in models of skin inflammation. J Ethnopharmacol 2020; 254:112709.
22.	Singh P, Khosa R, Mishra G, Jha K. Antidiabetic activity of ethanolic extract of Cyperus rotundus rhizomes in streptozotocin-induced diabetic mice. J Pharm Bioallied Sci 2015; 7:289.
23.	Uddin SJ, Mondal K, Shilpi JA, Rahman MT. Antidiarrhoeal activity of Cyperus rotundus. Fitoterapia 2006; 77:134–136.
24.	Kamala A, Middha S, Gopinath C, Sindhura H, Karigar C. In vitro antioxidant potentials of Cyperus rotundus L. rhizome extracts and their phytochemical analysis. Pharmacogn Mag 2018; 14:261.
25.	Mannarreddy P, Denis M, Munireddy D, Pandurangan R, Thangavelu KP, Venkatesan K. Cytotoxic effect of Cyperus rotundus rhizome extract on human cancer cell lines. Biomed Pharmacother 2017; 95:1375–1387.
26.	Rajamanickam M, Rajamanickam A. Analgesic and anti-inflammatory activity of the extracts from Cyperus rotundus Linn rhizomes. J Appl Pharm Sci 2016; 6:197–203.
27.	Safriani N, Erfiza NM, Arpi N. Antioxidant activities of Cyperus rotundus L. rhizome and Areca catechu L. seed. Int J Adv Sci Eng Inf Technol 2016; 6:285.
28.	Ahmed El-Wakil E, Morsi EA, Abel-Hady H. Phytochemical screening, antimicrobial evaluation and Gc-Ms analysis of Cyperus rotundus. World J Pharm Pharm Sci 2019; 8:129–139.
29.	Abdou AG, Harba NM, Afifi AF, Elnaidany NF. Assessment of Cryptosporidium parvum infection in immunocompetent and immunocompromised mice and its role in triggering intestinal dysplasia. Int J Infect Dis 2013; 17:e593–e600.
30.	Waldman E, Tzipori S, Forsyth JR. Separation of Cryptosporidium species oocysts from feces by using a percoll discontinuous density gradient. J Clin Microbiol 1986; 23:199–200.
31.	Gaafar MR. Effect of solar disinfection on viability of intestinal protozoa in drinking water. J Egypt Soc Parasitol 2007; 37:65-
32.	Li X, Brasseur P, Agnamey P, Lemeteil D, Favennec L, Ballet JJ, Rossignol JF. Long-lasting anticryptosporidial activity of nitazoxanide in an immunosuppressed rat model. Folia Parasitol (Praha) 2003; 50:19–22.
33.	Djurković-Djaković O, Nikolić A, Bobić B, Klun I, Aleksić A. Stage conversion of Toxoplasma gondii RH parasites in mice by treatment with atovaquone and pyrrolidine dithiocarbamate. Microbes Infect 2005; 7:49–54.
34.	Grujić J, Djurković-Djaković O, Nikolić A, Klun I, Bobić B. Effectiveness of spiramycin in murine models of acute and chronic toxoplasmosis. Int J Antimicrob Agents 2005; 25:226–230.
35.	Beutler E, Kelly BM. The effect of sodium nitrite on red cell GSH. Experientia 1963; 19:96–97.
36.	Marklund S, Marklund G. Involvement of the superoxide anoion radical in the auto-oxidation of pyrogallol and convenient assay for superoxde dismutase. Eur J Biochem 1974; 47:469–474.
37.	Mihara M, Uchiyama M. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 1978; 86:271–278.
38.	Abenoja J, Cotto-Rosario A, O’Connor R. Boromycin has potent anti-toxoplasma and anti-cryptosporidium activity. Antimicrob Agents Chemother 2021; 65:e01278–20.
39.	Wangchuk P, Giacomin PR, Pearson MS, Smout MJ, Loukas A. Identification of lead chemotherapeutic agents from medicinal plants against blood flukes and whipworms. Sci Rep 2016; 6:32101.
40.	Castaño Osorio JC, Giraldo García AM. Antiparasitic phytotherapy perspectives, scope and current development. Infect 2019; 23:189.
41.	Wink M. Medicinal plants: a source of anti-parasitic secondary metabolites. Molecules 2012; 17:12771–12791.
42.	Teichmann K, Kuliberda M, Schatzmayr G, Pacher T, Zitterl-Eglseer K, Joachim A, Hadacek F. In vitro inhibitory effects of plant-derived by-products against Cryptosporidium parvum. Parasite 2016; 23:2.
43.	Obiad HM, Al-alousi TI, Al-jboori AH. The in vivo effect of some medicinal plant extracts on Cryptosporidium parasite . J Univ Anbar Pure Sci 2012; 6:1–12.
44.	Khater M, El-Sayed S, Yousof HA, Mahmoud S, El-Dib N, El-Badry A. Anti-Cryptosporidium efficacy of Olea europaea and Actinidia deliciosa in a neonatal mouse model. Kasr Al Ainy Med J 2017; 23:32. [Full text]
45.	Cheraghipour K, Masoori L, Ezzatpour B, Roozbehani M, Sheikhian A, Malekara V et al. The experimental role of medicinal plants in treatment of toxoplasma gondii infection: a systematic review. Acta Parasitol 2020; 66:303–328.
46.	Youn HJ, Lakritz J, Kim DY, Rottinghaus GE, Marsh AE. Anti-protozoal efficacy of medicinal herb extracts against Toxoplasma gondii and Neospora caninum. Vet Parasitol 2003; 116:7–14.
47.	Wright CW, Anderson MM, Allen D, Phillipson JD, Kirby GC, Warhurst DC, Chang HR. Quassinoids exhibit greater selectivity against plasmodium falciparum than against Entamoeba Histolytica, Giardia Intestinalis or Toxoplasma gondii in vitro. J Eukaryot Microbiol 1993; 40:244–246.
48.	Choi KM, Gang J, Yun J. Anti-Toxoplasma gondii RH strain activity of herbal extracts used in traditional medicine. Int J Antimicrob Agents 2008; 32:360–362.
49.	Jebasingh D, Jackson DD, Venkataraman S, Emerald BS. Physiochemical and toxicological studies of the medicinal: plant Cyperus rotundus L (Cyperaceae). Int J Appl Res Nat Prod 2012; 5:1–8.
50.	Lydia J, Sundarsanam D. Phytoconstituents of Cyperus rotundus L. that attribute to its medicinal value and antioxidant property. Int J Pharma Sci Res 2012; 3:3304–3308.
51.	Jeong SJ, Miyamoto T, Inagaki M, Kim YC, Higuchi R. Rotundines A−C, three novel sesquiterpene alkaloids from Cyperus rotundus. J Nat Prod 2000; 63:673–675.
52.	Al-Shaibani IRM, Phulan MS, Shiekh M. Anthelmintic activity of Fumaria parviflora (Fumariaceae) against gastrointestinal nematodes of sheep. Int J Agri Biol 2009; 11:431–436.
53.	Mead JR, McNair N. Antiparasitic activity of flavonoids and isoflavones against Cryptosporidium parvum and Encephalitozoon intestinalis. FEMS Microbiol Lett 2006; 259:153–157.
54.	Forney JR, DeWald DB, Yang S, Speer CA, Healey MC. A role for host phosphoinositide 3-kinase and cytoskeletal remodeling during Cryptosporidium parvum infection. Infect Immun 1999; 67:844–852.
55.	Al Nasr I, Ahmed F, Pullishery F, El-Ashram S, Ramaiah VV. Toxoplasmosis and anti-Toxoplasma effects of medicinal plant extracts-a mini-review. Asian Pac J Trop Med 2016; 9:730–734.
56.	MacLaren A, Attias M, de Souza W. Aspects of the early moments of interaction between tachyzoites of Toxoplasma gondii with neutrophils. Vet Parasitol 2004; 125:301–312.
57.	Dzoyem JP, Hamamoto H, Ngameni B, Ngadjui BT, Sekimizu K. Antimicrobial action mechanism of flavonoids from Dorstenia species. Drug Discov Ther 2013; 7:66–72.
58.	Arabski M, Węgierek-Ciuk A, Czerwonka G, Lankoff A, Kaca W. Effects of saponins against clinical E. coli strains and eukaryotic cell line. J Biomed Biotechnol 2012; 2012:1–6.
59.	Wang C, Luo J, Amer S, Guo Y, Hu Y, Lu Y et al. Multivalent DNA vaccine induces protective immune responses and enhanced resistance against Cryptosporidium parvum infection. Vaccine 2010; 29:323–328.
60.	Cejas E, Pinto S, Prosdocimo F, Batalle M, Barrios H, Tellez G, Franceschi MD. Evaluation of quebracho red wood (Schinopsis lorentzii) polyphenolic vegetable extract for the reduction of coccidiosis in broiler chicks. Int J Poult Sci 2011; 10:344–349.
61.	Hoste H, Jackson F, Athanasiadou S, Thamsborg SM, Hoskin SO. The effects of tannin-rich plants on parasitic nematodes in ruminants. Trends Parasitol 2006; 22:253–261.
62.	Hoste H, Torres-Acosta JFJ, Sandoval-Castro CA, Mueller-Harvey I, Sotiraki S, Louvandini H et al. Tannin containing legumes as a model for nutraceuticals against digestive parasites in livestock. Vet Parasitol 2015; 212:5–17.
63.	Ho WE, Peh HY, Chan TK, Wong WSF. Artemisinins: pharmacological actions beyond anti-malarial. Pharmacol Ther 2014; 142:126–139.
64.	Anacleto-Santos J, López-Camacho P, Mondragón-Flores R, Vega-Ávila E, Islas GB, Mondragón-Castelán M et al. Anti-toxoplasma, antioxidant and cytotoxic activities of Pleopeltis crassinervata (Fée) T. Moore hexane fraction. Saudi J Biol Sci 2020; 27:812–819.
65.	Isah MB, Tajuddeen N, Umar MI, Alhafiz ZA, Mohammed A, Ibrahim MA. Terpenoids as emerging therapeutic agents: cellular targets and mechanisms of action against protozoan parasites. Vol 59. 1st ed. Amsterdam: Elsevier B.V; 2018.
66.	Elmahallawy EK, Elshopakey GE, Saleh AA, Agil A, El-Morsey A, EL-Shewehy DMM et al. S-methylcysteine (SMC) ameliorates intestinal, hepatic, and splenic damage induced by Cryptosporidium parvum infection via targeting inflammatory modulators and oxidative stress in Swiss albino mice. Biomedicines 2020; 8:423.
67.	Sood S, Yadav A, Katoch R, Bhagat M, Sharma A, Sharma S et al. Oxidative stress and clinico-pathological alterations induced by Cryptosporidium parvum infection in a rat model. Indian J Anim Res 2018; 53:1–5.
68.	Bhagat M, Sood S, Yadav A, Verma P, Manzoor N, Chakraborty D et al. Alterations in oxidative stress parameters and its associated correlation with clinical disease on experimental Cryptosporidium parvum infection in Swiss albino mice. J Parasit Dis 2017; 41:707–712.
69.	Wang C, Wu Y, Qin J, Sun H, He H. Induced susceptibility of host is associated with an impaired antioxidant system following infection with Cryptosporidium parvum in se-deficient mice. PLoS ONE 2009; 4:2.
70.	Dincel GC, Atmaca HT. Role of oxidative stress in the pathophysiology of Toxoplasma gondii infection. Int J Immunopathol Pharmacol 2016; 29:226–240.
71.	Al-Kennany ER, Al-Badrany SM. Pathological study on the capability of Toxoplasma gondii to induce oxidative stress and initiation a primary lesion of atherosclerosis experimentally in broiler chickens. J Anim Vet Adv 2007; 6:938–942.
72.	Hoseiny A, Nazarlu Z, Matini M, Bahmanzadeh M, Foroughi-Parvar F. Toxoplasma gondii: a possible inducer of oxidative stress in re-productive system of male rats. Iran J Parasitol 2020; 15:521–529.
73.	Yazdanparast R, Ardestani A. In vitro antioxidant and free radical scavenging activity of Cyperus rotundus. J Med Food 2007; 10:667–674.
74.	Huang B, Liu J, Fu S, Zhang Y, Li Y, He D et al. α-cyperone attenuates H2O2-induced oxidative stress and apoptosis in SH-SY5Y Cells via activation of Nrf2. Front Pharmacol 2020; 11.
75.	Kilani S, Sghaier MB, Limem I, Bouhlel I, Boubaker J, Bhouri W et al. In vitro evaluation of antibacterial, antioxidant, cytotoxic and apoptotic activities of the tubers infusion and extracts of Cyperus rotundus. Bioresour Technol 2008; 99:9004–9008.
76.	Kilani-Jaziri S, Bhouri W, Skandrani I, Limem I, Chekir-Ghedira L, Ghedira K. Phytochemical, antimicrobial, antioxidant and antigenotoxic potentials of Cyperus rotundus extracts. South Afr J Bot 2011; 77:767–776.
77.	Haseeb Kha A, Jabbar Abd A, Bahaa Sahi H, Adnan Fawz H. Anti-angiogenic and antioxidant activity of Iraqi Cyperus rotundus ethanol extract. Int J Pharmacol 2018; 14:546–552.
78.	Soumaya KJ, Dhekra M, Fadwa C, Zied G, Ilef L, Kamel G, Leila CG. Pharmacological, antioxidant, genotoxic studies and modulation of rat splenocyte functions by Cyperus rotundus extracts. BMC Complement Altern Med 2013; 13:28.
79.	Khojaste M, Yazdanian M, Tahmasebi E, Shokri M, Houshmand B, Shahbazi R. Cell toxicity and inhibitory effects of Cyperus rotundus extract on Streptococcus mutans, Aggregatibacter actinomycetemcomitans and Candida albicans. Eur J Transl Myol 2018; 28:4.
80.	Parvez MK, Al-Dosari MS, Arbab AH, Niyazi S. The in vitro and in vivo anti-hepatotoxic, anti-hepatitis B virus and hepatic CYP450 modulating potential of Cyperus rotundus. Saudi Pharm J 2019; 27:558–564.
81.	Mardani S, Nasri P, Tavakoli M. Contrast induced nephropathy; recent findings. J Nephropharmacol 2013; 2:27–30.
82.	Rafieian-Kopaei M, Baradaran A, Rafieian M. Oxidative stress and the paradoxical effects of antioxidants. J Res Med Sci 2013; 18:629.