in vitro evaluation of the efficiency of Artemisia judaica leaf extract on sporulation of Eimeria papillate oocysts and its cytotoxicity

[Avaliação in vitro da eficiência do extrato da folha de Artemisia judaica na esporulação de oocistos de Eimeria papillate e sua citotoxicidade]

ABSTRACT

Eimeria is the parasite that is responsible for eimeriosis in the gut of numerous domestic mammals. While treating eimeriosis, the use of medication and some effects of synthetic anticoccidials has led to the development of resistant parasites, necessitating the search for alternative treatments. The treatment of a wide variety of parasite diseases can be done with natural products that don't harm the environment. The goal of the current investigation was to determine how Artemisia judaica leaf extract (AJLE) affected the oocyst sporulation of Eimeria papillate strain. Also, reaching the ideal concentration will affect the parasite and limit infection. In vitro: Artemisia judaica leaf extract was applied at four different concentrations (50, 100, 200, and 300mg/mL), while 2.5% potassium dichromate solution served as the control. The sporulation of oocysts was significantly decreased by AJLE, reaching 12.6% at 300mg/mL, and the effect of inhibition on the oocyst sporulation percentages of E. papillata was observed in a dosage-dependent manner in comparison to the control group. Our results revealed that A. judaica has cytotoxic activity against breast and lung cancer cell line with a promising IC₅₀ of 480.3 and 359.2µg/mL, respectively compared to doxorubicin as a standard.

Keywords:
Artemisia judaica extract; Eimeria; oocysts; sporulation; in vitro; cytotoxic

RESUMO

Eimeria é o parasita responsável pela eimeriose no intestino de vários mamíferos domésticos. Durante o tratamento da eimeriose, o uso de medicamentos e alguns efeitos dos anticoccidianos sintéticos levaram ao desenvolvimento de parasitas resistentes, exigindo a busca de tratamentos alternativos. O tratamento de uma ampla variedade de doenças parasitárias pode ser feito com produtos naturais que não prejudicam o meio ambiente. O objetivo da presente pesquisa foi determinar como o extrato de folhas de Artemisia judaica (AJLE) afetou a esporulação de oocistos da cepa Eimeria papillate. Além disso, atingir a concentração ideal afetará o parasita e limitará a infecção. In vitro: O extrato da folha de Artemisia judaica foi aplicado em quatro concentrações diferentes (50, 100, 200 e 300 mg/mL), enquanto a solução de dicromato de potássio a 2,5% serviu como controle. A esporulação de oocistos foi significativamente reduzida pelo AJLE, chegando a 12,6% a 300mg/mL, e o efeito da inibição sobre as porcentagens de esporulação de oocistos de E. papillata foi observado de maneira dependente da dosagem em comparação com o grupo de controle. Nossos resultados revelaram que a A. judaica tem atividade citotóxica contra a linha celular de câncer de mama e de pulmão com um IC50 promissor de 480,3 e 359,2 µg/mL, respectivamente, em comparação com a doxorrubicina como padrão.

Palavras-chave:
extrato de Artemisia judaica; Eimeria; oocistos; esporulação; in vitro; citotóxico

INTRODUCTION

Coccidiosis is a dangerous intestinal parasitic disease caused by protozoan parasites of genusEimeria which affects the intestinal tract of a wide range of animals. Eimeria is the parasite responsible for causing Coccidiosis (Bangoura et al., 2018). Since it was discovered more than a century ago, it has been known to be one of the most prevalent parasites that impact animals’ intestinal tract and can lead to serious problems. Additionally, this disease has a significant negative impact on the worldwide economy (Chapman, 2014). From mild to severe, it can be pathogenic. It comes accompanied by weight loss, moderate to severe diarrhea, and mucusy or stools that are bloody. Dehydration and decreased animal reproduction may result from these side effects (Kvičerová et al., 2008). This leads to a higher chance of animal death.

The life cycle of Coccidia includes both asexual and sexual phases of reproduction. Additionally, it generates oocysts, which are resistant stages of parasites released into the environment to aid in the spread of disease (Graat et al., 1994). It is challenging to regulate Eimeria oocysts because of their relative resilience to environmental changes (Graat et al., 1994). For these parasites to be under control, it is necessary to stop the sporulation process (Mai et al., 2009). All alternative or natural medicine systems primarily rely on medicinal plants as their major source of components, and these plants are viewed as therapeutic means of getting powerful chemical compounds. When used on hosts who are afflicted with Eimeria, these compounds display organ-protective properties and kill parasites (Wunderlich et al., 2014).

A. judaica is broadly distributed in the desert and along the Mediterranean coast, as well as in certain countries in the Middle East, including Jordan, Algeria, Saudi Arabia, and the Sinai Peninsula in Egypt (Abu-Darwish et al., 2016 and Dob et al., 2006). Additionally, it is advised for treating skin conditions, weakened immune systems, gastrointestinal problems, diabetes, fungal infections, and arthritis, as well as for improving eyesight and capillary strength. Additionally, it can be utilized to protect against cardiovascular disease and lower the risk of atherosclerosis (Abu-Darwish et al., 2016). Additional biological actions by A. judaica included an anti-blastocystis response (Mokhtar et al., 2019), analgesic, anti-inflammatory and antipyretic (Batanouny et al., 1999). According to studies on the phytochemistry of A. judaica, it contains a variety of bioactive substances, including flavonoids (Saleh et al., 1987 and Moharram et al., 2021), sesquiterpene lactones (Khafagy et al., 1988), phenolic acids (Allam et al., 2019), sterols, triterpenes (Abd-Alla et al., 2014).

The current study set out to assess the pharmacological abilities of A. judaica leaves, as well as their impacts on sporulation and in vitro cytotoxicity properties.

MATERIALS AND METHODS

A. judaica leaves were gathered in Al Badiya-Tabuk, Saudi Arabia. The identification and confirmation of the plant material in the herbarium were made by a taxonomist from the King Saud University's Botany Department in Riyadh, Saudi Arabia. Between July and September 2023, this experiment was finished in the zoology department of King Saud University's College of Sciences. The procedure described by Manikandan et al. (2008) was used to prepare the 70% methanol extract of A. judaica leaves, with the following modifications: electric blenders (Senses, MG-503T, Korea) were used to powder the air-dried leaves of A. judaica. Leaf powder of A. judaica (100 g) was macerated in 70% methanol for 24 hours at 4 °C, then percolated 5-7 times to ensure complete extraction. After filtering, methanol was separated from the extract using a vacuum evaporator with low pressure and a temperature of 50 °C. Before being used, the crude extract was lyophilized and stored at -20°C.

Plant extract was evaluated on the optical spectrometer NICOLET 6700 (Thermo Scientific, Waltham, USA) FT-IR spectroscopy using the KBr pellet method with a range of 400-4000cm_-1 (Abu Hawsah et al., 2023).

The total phenolic content (TPC) of the leaf extract was calculated using the (Ainsworth and Gillespie, 2007) technique. 100µL of the Folin-Ciocalteu reagent and 300µL of sodium carbonate solution (20%) were added to 100 µL of the leaf extract. The sample was incubated for 30 minutes at room temperature and in the dark. The wavelength was measured using a UV-Visible spectrophotometer (SHIMADZU, UV-1800), and it was 765 nm. The total phenolic in the samples was determined using the following linear equation (y = 0.0021x + 0.0021 with R2 = 0.9995) based on a standard curve developed using various gallic acid concentrations (25-400 g/mL). The total phenolic content was represented as mg/g DW.

The total flavonoid content (TFC) of plant materials was calculated using the (Ordonez et al., 2006) technique. 0.5 mL of methanol extract was added to the same volume of a 2% AlCl3 water solution. After two hours at 25°C, the wavelength was determined to be 420 nm. The TFC was determined using an equation (y = 0.0172x + 0.0507 with R2 = 0.995) to fit a calibration curve made up of various quercetin standard values (50-0400 g/mL). The estimated TFC has been represented by quercetin (mg/g DW).

Lung (A549) and breast (MCF-7) cancer cell lines were routinely grown in DMEM medium (Gibco, USA) with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) (Gibco, USA The cells were incubated at 37°C in a humidified atmosphere with 5% CO₂.The ability of plant extract to cause cytotoxicity was assessed using an MTT assay. Briefly, cells were plated in a 96-well culture plate at a density of 5 x 104 cells per mL and left 24 hours to proliferate. When cells were treated to different doses of plant extract (500, 250, 125, 62.5, 31,125, and 15.625g/mL), doxorubicin was used as a positive control.

Each well received 10L of MTT solution (5mg/mL in PBS) after the initial 48 hours of incubation, which was followed by an additional 4 hours of incubation. After adding 100L of acidified isopropanol to each well, the formazan product was solubilized. The plate was then agitated for 10 minutes. A microplate reader (BioTek, USA) was used to measure the absorbance at 570nm.

Cell Viability = Mean absorbance [treated cells / untreated cells] × 100 Using OriginPro software, the IC₅₀ values (concentration of extract that caused 50% inhibition) were calculated from the dose-response curve of cell viability percentage.

Fresh fecal cells from infected mice were used to get the parasite. Oocysts were separated from collected feces using the flotation method, and they were then used in an in vitro experiment.

The non-sporulated oocysts (1×10⁵) were incubated in each group, which contained the following: 5 ml distilled H₂O (negative control), 5 ml potassium dichromate (K₂Cr₂O₇) 2.5% (positive control), and finally, 5 ml K₂Cr₂O₇ (2.5%) containing one of the following: AJLE (50, 100, 200, and 300mg/mL), 8.3mg amprolium (Veterinary Agriculture Products Company [VAPCO], Jordan), 109 μl dettol^TM, 25μL phenol, and formalin (5%). Using an Olympus compound microscope (Olympus Co., Tokyo, Japan), sporocysts were examined and monitored the oocysts' sporulation. All Petri dishes were incubated for 72 and 96 hrs at 25 to 29°C (Gadelhaq et al., 2018). The sporulation % and sporulation inhibition percentage were computed in accordance with (Daiba et al., 2022; Cedric et al., 2018), respectively.

$Sporulation\left(Sp\right)\%=\frac{Numberofsporulatedoocyts}{Totalnumberofoocysts}X100$

$Sporulation\left(Sp\right)inhibitionpercentage=\frac{Sp\%ofcontrol-Sp\%ofextract}{Sp\%ofcontrol}X100$

One-way analysis of variance (ANOVA) was used to examine the data in SigmaPlot® version 11.0 (Systat Software, Inc., Chicago, IL, USA). Differences between groups were considered significant at a p-value ≤ 0.01.

RESULTS

The examination of AJLE with FT-IR displayed chief groups at 3390.30cm^-1, 2934.71cm^-1, 1763.99cm^-1, 1606.14cm^-1, 1514.66cm^-1, 1451.34cm^-1, 1383.43cm^-1, 1266.30cm^-1, 1175.69cm^-1, 1118.10cm^-1, 1069.40cm^-1, 832.42cm^-1, 765.02cm^-1, 769.00cm^-1 and 612.83cm^-1 (Figure 1 and Table 1). N-H widening was shown by the group at 3390.30cm^-1 approving the presence of aliphatic primary amine. The band at 2934.71cm^-1 implied C-H stretching for the occurrence of alkane. C=O stretching to 1763.99cm^-1 confirms the existence of carboxylic acid. The group at 1606.14cm^-1 parallels to C=C stretching for the occurrence of unsaturated ketonea. The group at 1514.66cm^-1 confirms the occurrence of N-O nitro compound. The gang at 1451.34cm^-1 implied the presence of C-H alkene, The band at 1383.43cm^-1 confirms the occurrence of C-H alkene The band at 1266.30cm^-1 implies the presence of C-N aromatic ester, The band at 1175.69cm^-1 confirms the occurrence of C-O ester. The band at 1118.10cm^-1 confirms the presence of C-O secondary alcohol. The group at 1069.40cm^-1 confirms the occurrence of S=O sulfoxide. The group at 832.42cm^-1, confirms the presence of C=C alkene. The band at 765.02cm^-1, confirms the presence of C-H 1,2,3-trisubsituted. The band at 769.00cm^-1 confirms the attendance of C-H 1,2 disubstituted. and The group at 612.83cm^-1 confirms C-I halo compound (Table 1).

The proportion of sporulated oocysts was calculated for the control and experimental groups at 72 and 96 hours. In comparison, there were significant amounts of sporulated oocysts in the 2.5% potassium dichromate (control) sample. The E. papillata oocysts displayed high levels of inhibition after 72 hours of incubation with AJLE (300g/mL and phenol). The amounts of sporulation in oocysts incubated with AJLE (200, 100, 50mg/mL) was rather low (Figure 2). Furthermore, after 96 hours of incubation with AJLE (300 g/mL and phenol), low levels of sporulated oocysts, the E. papillata oocysts showed high levels of inhibition. Oocysts treated with AJLE (200, 100, 50 mg/mL) showed relatively little sporulation (Figure 3).

Oocyst sporulation was completely suppressed by formalin while AJLE (300 mg/mL) incubation at 72 and 96 hours was (6.7±1 and 21.1±1) respectively (Figures 4, 5). At 72 and 96 hours, respectively, there was a notable amount of in vitro oocyst sporulation in purified H₂O, measuring 24.2± 2 and 15.7± 2, respectively (Figures 4, 5. At 72 hr, AJLE 200, 100, 50mg/mL, amprolium, Dettol^TM, phenol, and formalin showed fluctuating levels of inhibition by 92.3%, 85.7%, 45.4%, 26.9%, 12.6%, 34.61%, and 76.92%, 92.30% and 100%, respectively (Figure 4). At 96 h, AJLE 200, 100, 50 mg/mL showed fluctuating points of inhibition by 15.7%, 77.6%, 20.4%, 3.9%, 1.3%, 37.33%, and 81.33%, 89.33% and 100%, respectively (Figure 5).

In Figure (6), displayed the concentration of phenolic and flavonoid components in AJLE and determined that the methanolic extract had a phenolic compound concentration of (144±0.7 1mg/mL). Additionally, the lowest flavonoid content concentration was (26±0.1 1mg/mL).

The methanolic excerpt of A. judaica L. was separated for its cytotoxicity against a sheet of cancer cells, specifically MCF-7 Breast cell and A549 lung cells, using the MTT assay, as seen in Table 2 and Figure 7. When compared to doxorubicin, the standard medication (IC₅₀ = 1.55µg/mL), the crude extract showed significant cytotoxic action towards the A549 lung cancer cell line, with a prospective IC₅₀ of 359.2µg/mL. Additionally, the crude excerpt displayed effective cytotoxic activity contrary to the MCF-7 Breast cancer cell line, with a hopeful IC₅₀ of 480.3µg/mL related to doxorubicin as the standard drug (IC₅₀ = 0.95µg/mL) (Table 2).

Figure 1
FTIR of AJLE displays the material's functional properties.

Table 1
FT-IR for A judaica leaves extract

Absorption (cm-1)	Transmittance (%)	Appearance	Group	Compound class
3390.30	11.13726	medium	N-H stretching	aliphatic primary amine
2934.71	4.068068	medium	C-H stretching	alkane
1763.99	3.403546	strong	C=O stretching	carboxylic acid
1606.14	4.278121	strong	C=C stretching	α,β-unsaturated ketone
1514.66	9.217903	strong	N-O stretching	nitro compound
1451.34	4.927714	medium	C-H bending	alkane
1383.43	4.108677	medium	C-H bending	alkane
1266.30	4.831751	strong	C-N stretching	aromatic ester
1175.69	6.214668	strong	C-O stretching	ester
1118.10	4.295302	strong	C-O stretching	secondary alcohol
1069.40	3.638029	strong	S=O stretching	sulfoxide
832.42	10.90045	medium	C=C bending	alkene
795.02	10.64206	strong	C-H bending	1,2,3-trisubstituted
769.00	9.613958	strong	C-H bending	1,2-disubstituted
612.83	8.722485	strong	C-I bending	halo compound

Figure 2
Anticoccidial effects of A. judaica on the percentage sporulation of E. papillata oocysts at 72 h. *Significance against (2.5%) potassium dichromate group (p ≤ 0.01).

Figure 3
Anticoccidial effects of A. judaica on the percentage sporulation of E. papillata oocysts at 96 h. *Significance against (2.5%) potassium dichromate group (p ≤ 0.01).

Figure 4
A. judaica anti-coccidial effects on the Inhibition of E. papillata oocysts sporulated (%) at 72 h. *Significance against (2.5%) potassium dichromate group (p ≤ 0.01).

Figure 5
A. judaica anti-coccidial effects on the Inhibition of E. papillata oocysts sporulated (%) at 96 h. *Significance against (2.5%) potassium dichromate group (p ≤ 0.01).

Figure 6
Flavonoids and total polyphenols in the leaves methanolic extract of the A. judaica plant.

Figure 7
Cytotoxicity (MTT) assay for tested A. judaica at different concentrations (µg/mL) against breast (MCF-7) cancer and Lung (A549) cell lines after 48 h of incubation.

Table 2
LC50 of the studied A. judaica plant extract

	Cell lines and IC50 (µg/ml)
	A549 Lung	MCF-7 Breast
Extract 3	359.2 ± 4.1	480.3 ± 4.5
Doxorubicin	1.55 ± 0.05	0.95 ± 0.01

DISCUSSION

Several research investigations have investigated the usage of plant extracts to create novel medications to treat coccidia. This is due to the difficulties that currently available conventional medications confront, such as the increase of drug-resistant coccidial species (Habibi et al., 2016). Measurements of the numeral and proportion of damaged Eimeria oocysts and sporulation inhibition were used to assess the anticoccidial effects of A. judaica extracts in vitro. Numerous animal species are affected by coccidiosis caused by Eimeria parasites, which has a significant negative economic impact due to mortality, lower weight growth, and deprived feed efficiency (Abu Hawsah et al., 2023).

There are numerous research that mention eimeriosis medication resistance (Chapman, 2014). Because natural compounds are more potent, less poisonous, and have fewer side effects than traditional chemical agents, emphasis is being directed to their use as antiparasitic agents (Wunderlich et al., 2014).

There were many recognized chemicals found in the A. judaica extract (aerial portions) according to FT-IR analysis. Moreover, the phenolic and flavonoid content were abundant in the aerial part of A. judaica (Qanash et al., 2023).

Plant bioactive chemicals that are most significant are flavonoids and phenolic compounds (Mehmood et al., 2015). Our findings showed that phenolic chemicals and flavonoids are present in the A. judaica extract. Thus, it has been demonstrated by numerous investigations that plant extracts holding phenolic mixtures have inhibitory effects.

Recent years have seen an increase in the use of plant extracts for the prevention and management of transferable diseases in fowl animals, such as coccidiosis. Numerous studies demonstrate the anticoccidial capabilities of the methanolic grape plant extract (Pieri et al., 2014). The daisy family Asteraceae includes the numerous, diversified genus Artemisia, which contains 200-400 species of plants.

The results of our study indicate that AJLE had a noteworthy impact on the oocyst sporulation of E. papillata. This effect can be ascribed to a range of bioactive phytochemical components that were investigated by (Ali and Shah, 2011; Fatima, 2016; Al-Shaebi et al., 2023). Also, In vitro studies, Arlette et al. (2019) have demonstrated that the natural polyphenolic components obtained from medicinal plants can block the invasion of E. tenella sporozoite cells.

According to the results of our research, AJLE at a dosage of 300mg/ml had an impact on oocyst sporulation. Several bioactive phytochemical substances may be responsible for this action. Similar findings were made by (Al-Shaebi et al., 2023), who reported that Teucrium polium leave extract had anticoccidial activity. Additionally, our findings were in line with those of (Ali and Shah, 2011).

The sporulation of E. papillata was totally prevented by formalin (5%) as reported by ABU Abu Hawsah et al., (2023), who also reported that this extremely reactive substance interacts with proteins in vitro and prevents sporulation. Furthermore, (Gadelhaq et al., 2018; Abu Hawsah et al., 2023) stated that dettol^TM, and phenol have been stated to prevent sporulation by different degrees.

A considerable drop in the production of E. papillata oocysts in the faeces and oocysts of infected mice after treatment with AJLE demonstrated anti-coccidiosis efficacy. These findings are in accordance with those of other research that looked at Calotropis procera leaf extract together with Azadirachta indica (Dkhil et al., 2013), Punica granatum plant (Amer et al., 2015) and grape extract (Abbas et al., 2020) as potential sources of anticoccidial chemicals.

The A. judaica extract displayed anticancer, antioxidant, anti-inflammatory, and antibacterial effects, according to (Song et al., 2022) and (Imam et al., 2022). Our findings supported those of previous studies that looked at the herb Artemisia as a potential cancer treatment plant.

CONCLUSIONS

A. judaica is a rich source of phenolic and flavonoid components with potential medicinal benefits, including anticancer and anticoccidial activity. The development of AJLE as a novel medicine to treat animal coccidian infections will be aided by this information, according to ongoing studies.

ACKNOWLEDGMENTS

This work was supported by the Researchers Supporting Project (RSP2023R3) at King Saud University (Riyadh, Saudi Arabia).

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Publication Dates

• Publication in this collection
  27 Jan 2025
• Date of issue
  Jan-Feb 2025

History

• Received
  02 Nov 2023
• Accepted
  10 Jan 2024