In vitro antiparasitic activity of ethanolic leaves extract of Anethum graveolens

ABSTRACT Natural products are safe environmentally friendly agents and have no negative impact on the environment, they can be used to combat parasitic diseases. Helminthiasis and coccidiosis are parasitic diseases that harm both health and the economy. This research aimed to see how Anethum graveolens leaves extract (AGLE) worked as an anti-parasitic modulator during oocyst sporulation of an Eimeria papillata infection. FT-IR phytochemical analysis revealed the presence of eight compounds. The time required to induce paralysis and death in worms at the highest concentration (200 mg/mL) was 4.57±0.26 and 5.22±0.10 min, respectively. In an in vitro study, AGLE (300 mg/ml) inhibited sporulation by approximately 100% after 72 and 96 hr. AGLE (200, 100, and 50 mg/ml), amprolium, DettolTM, and phenol induced variable inhibition levels at 96 hr of 5.54%, 1.01%, 37.33%, 81.33%, and 89.33%, respectively. Our findings suggest that AGLE has potent anthelmintic and anticoccidial properties that could be further developed into a novel therapeutic agent.


INTRODUCTION
 Protozoa of the phylum Apicomplexa, such as Toxoplasma, Plasmodium, and Eimeria, are of great medical and veterinary importance as pathogens that cause various human and veterinary diseases worldwide (Habib et al., Arq. Bras. Med. Vet. Zootec., v.75, n.5, p.893-901, 2023 This disease causes significant economic losses worldwide (Chapman, 2014).It has an asexual and sexual reproduction cycle, and it produces resistant parasite stages known as oocysts that are released into the environment, facilitating the spread of infection.Because of their resistance to environmental factors, controlling Eimeria oocysts is difficult (Graat et al., 1994).Eimeria tenella is one of the most common and dangerous pathogens in the genus Eimeria.These parasitic infections cause animals' nutrient uptake to be disrupted, resulting in decreased body weight and increased susceptibility to secondary infections (López-Osorio et al., 2020).
Conventional methods for preventing and controlling coccidiosis include anti-coccidian drugs and live vaccines; however, these measures raise concerns about drug resistance, food security, production costs, and species cross-protection (Chapman, 1997;Sharman et al., 2010).Therefore, there is an urgent need to seek new ideas and perspectives on coccidiosis prevention and control.
Anethum graveolens (Dill), belonging to the family Apiaceae, is an aromatic plant native to the Mediterranean region that has been widely used as a seasoning in the preparation of various foods (Slupski et al., 2005).It is rich in flavonoids, polyphenols, essential minerals, antioxidants, and vital vitamins like riboflavin, folic acid, vitamin A, niacin, vitamin C, and bcarotene (Jana and Shekhawat, 2010).Numerous studies have demonstrated that A. graveolens acts against fungi (Kumarasingha et al., 2016;Vieira et al., 2019), bacteria (Kaur andArora, 2009), andprotozoa (Sahib et al., 2014).It has also been utilized in traditional herbal medicine as a diuretic effector and as a treatment for the gastrointestinal disorders (Hosseinzadeh et al., 2002).
Given these benefits, the present investigation aimed to evaluate the possible anticoccidial activity of A. graveolens leaves extract against Eimeria papillata as well as in vitro anthelmintic activity.

MATERIALS AND METHODS
Anethum graveolens leaves were collected from the botanical gardens in Riyadh, Saudi Arabia.A taxonomist from the Botany Department (King Saud University, Riyadh, Saudi Arabia) identified and confirmed the plant material.The ethanolic extract of 70% of A. graveolens leaves (AGLE) was prepared using the method described by Manikandan et al. (2008), with some modifications as follows: air-dried leaves of A. graveolens were ground into a powder with an electric blender (Senses, MG-503T, Korea).Dried powder (100 g) of A. graveolens leaves was macerated in 70% ethanol for 24 hr at 4 ºC, followed by percolation 5-7 times until complete extraction.Following filtration, ethanol was isolated from extract using a vacuum evaporator at 50 ºC under reduced pressure.The crude extract was lyophilized and stored at -20 ºC until further use.
Fourier-transform infrared spectroscopy (FT-IR) NICOLET 6700 (Thermo Scientific, Waltham, USA) was used for the analysis of the plant extract through the KBr pellet method with a range of 4000 cm −1 to 400 cm −1 , following Al-Quraishy et al. (2020).
The phenolic contents of AGLE were determined according to Singleton et al. (1999), with some modifications.Briefly, 0.1 mL of Folin-Ciocalteu reagent, 1.5 mL of ultrapure water (Milli-Q), and 0.1 mL of AGLE (1 mg/mL) or gallic acid were mixed and left for 8 min, then, 0.3 mL 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 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 the extracts was calculated as gallic acid equivalent (mg/g DW), where (y) absorbance and (x) gallic acid equivalent concentration (mg/g).
The total flavonoids in AGLE were determined using the method of Ordonez et al. (2006).Briefly, 1.0 mL of 2% AlCl 3 water solution was mixed with 1.0 ml of AGLE (1 mg/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 = 0.0011x + 0.0928), where (y) is the absorbance and (x) is the quercetin equivalent concentration (mg/g), the flavonoids in the extracts were expressed as quercetin (mg/g DW).
The antioxidant activities of AGLE were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay (Liyana-Pathirana et al., 2005).Briefly, 1 ml of the extract was mixed with 1 ml of 0.135 mM 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 the samples and the control solutions (Ascorbic acid as positive control) was measured at 517 nm, and the percentage of DPPH scavenging activity of the extracts 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).This study was conducted using three doses of AGLE (50, 100, and 200 mg/ml) against the earthworm by following method of Ajaiyeoba et al. (2001).Five worms of roughly the same size were used per dose.As a control, worms in distilled water were used.The reference drug was mebendazole (10 mg/ml).The time it took to reach paralysis and death was measured in minutes (Dkhil, 2013).Before beginning the experiment, all the extracts and drug solutions were freshly prepared.
Small pieces of the earthworm body were removed and fixed in formalin (10%).Following fixation, specimens were dehydrated, embedded in wax, and then sectioned to 5 m thicknesses.For histological examinations, sections were stained with hematoxylin and eosin (Drury and Wallington, 1978).Sections were examined and photographed using a digital camera (DP 73) fitted on an Olympus B×61 microscope (Tokyo, Japan).
Five Swiss albino male 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 unsporulated oocysts (1×10 5 ) were incubated at 25-29 ºC for 72 and 96 hs in 5 mL 2.5% potassium dichromate solution K 2 Cr 2 O 7 (positive control), 5 ml distilled H 2 O (negative control) and finally in 5 ml K 2 Cr 2 O 7 containing one of the following: AGLE (300, 200, 100, and 50 mg/ml), 109 μl Dettol TM , 8.3 mg amprolium (Veterinary Agriculture Products Company [VAPCO], Jordan), 5% formalin and 25 μl phenol.The oocysts' sporulation was monitored by examining sporocysts under an Olympus compound microscope (Olympus Co., Tokyo, Japan).According to Thagfan et al. (2020), a total of 100 oocysts were counted in each control and treatment group to estimate the sporulation and inhibition (%) of E. papillata oocysts.
Data analysis was performed using SigmaPlot® version 11.0 (Systat Software, Inc., Chicago, IL, USA) and presented as mean ± SD with p-value ≤ 0.05.

RESULTS
The FT-IR evaluation indicated the presence of 8 expected compounds in AGLE.Some of these compounds have strong appearances including groups with N-O, S=O, C-O, and C-I stretching (Table 1, Figure 1).These expected compounds included nitro compound, sulfonyl chloride, primary alcohol, and halo compound.
Phenolic and flavonoid contents of AGLE were presented in Figure (2).From this Table, it appears that the concentration of phenolic compounds in the ethanolic extract (1mg/mL) was (32±0.7).while the lowest concentration of flavonoid content was observed in the extract of 23±0.1.Table (2) showed that the ethanolic extracts had the highest DPPH (65.4±0.9) at the concentration of 1000 μg/mL, while the lowest scavenging percentage (0) was at the concentration of 31.25μg/mL.The ethanolic extracts presented DPPH in a concentration-dependent manner, where the concentrations of the ethanolic extract exhibited good antioxidant properties.
The AGLE had anthelmintic activity against live adult E. fetida worms that was comparable to the conventional anthelmintic agent (mebendazole).Table (3) showed that time to paralysis and death were 4.57±0.26min and 5.22±0.10min, respectively, for the most effective dose of 200mg/mL.However, compared to the other AGLE concentrations the reference drug mebendazole (10mg/mL) had a lesser effect (Table 3).After receiving AGLE, the cuticle thickness and length of the segment for E. fetida was significantly reduced compared to the typical structure for those in water, and upmost layer was destructed in the treated group with mebendazole (Figure 3).Incubation with AGLE (300 mg/mL) and formalin at 72 and 96 hr inhibited oocysts sporulation by 100% (Table 4).Incubation with AGLE (200 mg/ml), at 72 and 96 hr, inhibited sporulation of E. papillata oocysts by 97.52% and 97.09% respectively.At 96 hr, AGLE (100 and 50 mg/mL), amprolium, Dettol TM , and phenol induced variable inhibition levels of 5.54%, 1.01%, 37.33%, 81.33%, and 89.33%, respectively (Table 4).

DISCUSSION
Coccidial and helminth infections constitute public health concerns worldwide.Plant extracts used as mendicants may help to overcome these obstacles.Also, plants have proven to be a promising alternative for controlling small ruminant nematodes.In addition to anthelmintic activity, it reduces the risk of chemical residues in animal products and the environment (Chagas et al., 2008).Carvone and limonene are the main chemical components of A. graveolens (Jana and Shekhawat, 2010).
The presence of the main chemical components may be responsible for these activities.
Meanwhile, other researchers have reported that carvone and limonene have antimicrobial properties (Palmeira et al., 2009).Limonene is found as anthelmintic activity in oils from different plants, such as Cymbopogon martini, Lippia sidoides, Eucalyptus staigeriana, and Mentha piperita (Katiki et al., 2011;Carvalho et al., 2012;Ribeiro et al., 2013).In the current in vitro study, a concentration of AGLE of 200 mg/ml generated significant anthelmintic activity comparable to the conventional anthelmintic agent, mebendazole.Many studies used earth worms as the model for the anthelmintic activity evaluation due to the physiological similarities between the E. fetida worms and some intestinal round worms that infect people (Abu Hawsah et al., 2023).The carvone and limonene found in AGLE may be the main components of the anthelmintic action observed in this study6 Stepek et al. (2005)  Flavonoids, alkaloids, tannins, and phenolic compounds are the most important plant bioactive compounds (Mehmood et al., 2015).
Several studies have shown that plant extracts containing phenolic compounds have inhibitory properties.Natural polyphenolic components derived from medicinal plants have been shown to inhibit E. tenella sporozoite cell invasion in vitro (Arlette et al., 2019).These researchers also noted that extracts with polyphenolic compounds may have the power to inhibit the enzymes necessary for the coccidian oocysts' sporulation process.It is thus possible that A. graveolens extract components exhibited anti-sporulation activity by interfering with the physiological processes necessary for sporulation, thereby inhibiting or inactivating the enzymes responsible for the sporulation process.The finding is supported by a study that linked Moringa oleifera's anticoccidial activity to its biological constituents, which include flavonoids and phenolic compounds with anti-inflammatory and antioxidant properties (Gadelhaq et al., 2018).
The results of this experiment demonstrated that AGLE has an in vitro anticoccidial effect on unsporulated oocysts of E. papillata in a concentration-dependent manner.This is consistent with the findings of Cedric et al. (2018), who reported the anticoccidial, antioxidant, and cytotoxicity activity of Psidium guajava extracts against four different species of Eimeria in a concentration-dependent manner.The effect of antioxidants on DPPH radicals is thought to be due to their ability to donate hydrogen.As a result, DPPH is commonly used as a substrate to assess antioxidant agents' antioxidative or free radical scavenging activity.

CONCLUSION
It could be concluded that AGLE has anticoccidial and anthelmintic efficacy, in vitro.
Further studies are recommended to include the in vivo effectiveness of AGLE and identify the pathway of its active compounds on parasite and host response.

Figure 1 .
Figure 1.FT-IR of Anethum graveolens leaves extract in ethanolic medium showing the functional characteristic of the material.

Table 4 .
In vitro, anti-coccidial effects of Anethum graveolent leaves extract on the sporulation percentage of Eimeria papillata oocysts Groups Time Sporulation of oocyst (%) Inhibition of sporulation (%) p-value Gadelhaq et al. (2018)onfirmed phenolic compounds' antioxidant role by referring to their redox properties, which are considered free radical scavengers.It is also shown that the commonly used disinfectant formalin (5%) is the most effective in inhibiting E. papillata oocyst sporulation, which agrees with Thagfan et al (2020).According toMai et al. (2009)andGadelhaq et al. (2018)Phenol and Dettol TM have been reported to inhibit sporulation by 89.33% and 81.33% respectively, and the oocyst wall is resistant to proteolysis and impermeable to water-soluble substances.