Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 57 issue 5 (may 2023) : 638-643

In vitro Effect of Arnebia densiflora and Artemisia annua Plant Extracts against Theileria annulata Schizonts

O. Girisgin1,*, M. Kürkcüoglu2, A.O. Girisgin3
1Karacabey Vocational School, Bursa Uludag University, Karacabey, Bursa-Turkey.
2Faculty of Pharmacy, Eastern Mediterranean University, Famagusta, North Cyprus via Mersin-10, Turkey.
3Department of Parasitology, Bursa Uludag University, Veterinary Faculty, Nilüfer, Bursa-Turkey.
Cite article:- Girisgin O., Kürkcüoglu M., Girisgin A.O. (2023). In vitro Effect of Arnebia densiflora and Artemisia annua Plant Extracts againstTheileria annulata Schizonts . Indian Journal of Animal Research. 57(5): 638-643. doi: 10.18805/IJAR.BF-1588.
Background: This study investigated the in vitro activities of naphthoquinones and artemisinin contained in the extracts obtained from Arnebia densiflora and Artemisia annua plants against peripheral blood mononuclear cells infected with Theileria annulata schizonts. For this purpose, the n-hexane extract of A. densiflora and the petroleum ether and methanol extracts of A. annua were obtained.

Methods: Non-infected and infected cells were cultivated and 2×106 cells were seeded in each well in 0.5 ml of RPMI 1640 medium. The n-hexane extract of A. densiflora was tested six times at concentrations of 125, 62.5, 15.625, 7.8125 and 3.9062 mg/ml. A. annua petroleum ether and methanol extracts were also tested six times at 125, 62.5, 31.25, 15.625, 7.8125, 3.9062, and 1.9531 mg/ml concentrations on PBMCs. A Mann-Whitney U test was used to determine differences between control and experimental groups.

Result: The A. densiflora extract killed both non-infected and infected cells at significant levels (P<0.05) compared to the control group at all tested concentrations. A. annua methanol extract had no adverse effect on non-infected cells and the effect on infected cells was insufficient, with the only observed significant effect at a concentration of 125 µg/ml. The A. annua petroleum ether extract had no adverse effect on non-infected cells and killed infected cells at significant levels (P<0.05) at concentrations of 15.625 µg/ml and above at 24 hours. At 48 hours, all infected cells were killed at a concentration of 62.5 µg/ml. In conclusion, A. annua petroleum ether extract can be used in future in vivo studies for Theileriosis at these effective concentrations.
Tropical theileriosis is a protozoan infection that causes high mortality and low productivity in imported high-grade cattle and crossbreeds, thereby negatively affecting economies (Agina et al., 2020).

Control of tropical theileriosis is achieved by i) vector control by application of acaricides, ii) drug administration against the disease agent and iii) use of attenuated macro schizont-infected cell culture vaccines for protection against the disease. Acaricide application has many disadvantages, such as high prices, residual drug waste in the meat and milk of cattle, contamination of the environment and resistance in ticks. Vaccines used for protection against the disease may be insufficient in controlling the disease due to inadequate amounts produced and difficulties in transportation and administration (Karagenc and Eren, 2002; Ghosh et al., 2006). Although antiprotozoal drugs such as halofuginone, parvaquone and buparvaquone are used in the treatment of cattle infected with Theileria annulata, treatment is very expensive and unsuccessful if not started early (Maharana et al., 2016; Nampoothiri, 2021).

Despite the availability of synthetic drugs whose dosage and stability can be easily adjusted, the use of herbal products as an alternative in the treatment of many diseases is becoming widespread in both developed and developing societies (Sofowora, 2013).

Various studies have been performed to determine the effects and possible toxic characteristics of herbal medicines which have been used traditionally for centuries (Efferth, 2017; Shahrajabian et al., 2020). Considering that there are more than nine thousand natural plant species in Turkey, there is great potential in terms of active herbal ingredients.

Naphthoquinones and artemisinin are molecules used in veterinary and human medicine against blood parasites (Sanford et al., 2016; Bhat et al., 2018; Wang et al., 2019). Naphthoquinones are used in parasitology for the treatment of some protozoan infections. It has been observed that naphthoquinone derivatives inhibit the mitochondrial electron transport in Theileria parva, causing the death of the parasite (Dobbeleare et al., 2000). In addition to these, naphthoquinones are used for the treatment of malaria, Toxoplasmosis and Pneumocystis pneumonia (Srivastava et al., 1999; Mone et al., 2021).

Likewise, artemisinin is a molecule used in human medicine against Plasmodium infections (Sanford et al., 2016; Wang et al., 2019), which has also been stated to be effective against some other protozoans and helminths (Kim et al., 2002; Mishina et al., 2007; Lam et al., 2018). The cytotoxic effects of artemisinin and its semi-synthetic derivatives have been investigated and it has been reported that these substances can be effective on cancer cells (Woerdenbag et al., 1993; Zhang et al., 2018) and also on Sars-CoV-2 virus, recently (Farmanpour-Kalalagh​ et al., 2022).

So far, in vitro and in vivo trials have limitedly been carried out with various extracts of different plants against T. annulata, T. parva and T. lestoquardi species. In in vitro experiments, Curcuma longa and Pavetta schumanniana plant extracts were tested against T. annulata, while different plants have been tested against T. parva and T. lestoquardi species and it has been determined that they have antitheilerial effects (Farah et al., 2012, 2013, 2014, 2015a).

This study aimed to determine the levels of artemisinin contained in the n-hexane extract of Arnebia densiflora and the petroleum ether and methanol extracts of Artemisia annua and their in-vitro activity against T. annulata schizonts to explore whether these extracts are promising for future in vivo trials.
Collection, drying and extraction of Arnebia densiflora with n-hexane
 
The A. densiflora (Ledeb. ex Nordm.) Ledeb. plant was collected from calcareous hills 10 km from the Eskişehir Alpu district of Turkey in June 2020. The roots of the collected plants were removed, dried in the open-air shade, mechanically powdered (drug) and extracted with n-hexane at room temperature in a Soxhlet extractor (Bozan, 1994).
 
Analysis of Arnebia densiflora extracts
 
Analysis of the extracts obtained from A. densiflora roots was performed by thin-layer chromatography (TLC)-densitometry and isolated naphthoquinones determined (Bozan et al., 1997).
 
Collection, drying and extraction of Artemisia annua with petroleum ethers and methanol
 
In order to evaluate the solubility of artemisinin, solvents with different polarities were applied to the plant samples. The above-ground parts of A. annua collected from open land in Bursa, Turkey were obtained in September 2020. Flowers and leaves were dried in the open-air shade and mechanically powdered. Extracts were obtained with petroleum ether and methanol maceration in an ultrasonic water bath (Christen and Veuthey, 2001).

Extracts obtained from A. densiflora and A. annua were stored in dark-coloured glass bottles at -20°C.
 
Gas chromatography analysis of extracts from Artemisia annua
 
Artemisinin in petroleum ether and methanol extracts of A. annua was separated according to retention time in a Chrompack capillary column and evaluated according to the relative percentage (Peng et al., 2006).
 
Assessment of anti-theilerial activity of plant extracts
 
Isolation of non-infected peripheral blood mononuclear cells (PBMCs) and those infected with Theileria annulata schizont
 
Non-infected and T. annulata schizont-infected PBMCs were used in the study. Non-infected PBMCs were isolated from the peripheral blood of a calf with Ficoll-Paque (Biochrom AG) and cultivated (Brown, 1987). The medium used in cultivation (RPMI 1640) contained 10% heat-inactivated neonatal calf serum (NCS), 2 mM L-glutamine, 100 µg/ml streptomycin and 100 U/ml of penicillin. Passages two and three of T. annulata from previously cultivated Osmanbükü-İbrahim Özkan isolate were used in the experiments.
 
Determination of in vitro efficacy of plant extracts
 
T. annulata schizont-infected and non-infected PBMCs were placed on separate plates and fresh medium was added to these cells as the first control group. As the second control group, 0.5% DMSO, used in solubilizing the extracts, was added to both cell groups at the highest concentration of 8 ml. This process was repeated six times for both control groups.

The effects of extracts on non-infected and T. annulata schizont-infected PBMCs were determined in 24-well plates. In one plate, 2 × 106 non-infected PBMCs were seeded to each well in 0.5 ml of medium (Wilkie et al., 1998). On the second plate, 1 × 105 T. annulata schizont-infected PBMCs were seeded to each well in 0.5 ml of medium.

The n-hexane extract of A. densiflora was tested six times at concentrations of 125, 62.5, 15.625, 7.8125 and 3.9062 µg/ml. A. annua petroleum ether and methanol extracts were tested six times at 125, 62.5, 31.25, 15.625, 7.8125, 3.9062 and 1.9531 mg/ml concentrations. The concentrations were determined by our preliminary trials and similar studies mentioned herein.

        The plates were incubated at 37°C and 5% CO2. Cell growth was checked under an inverted microscope at 24 and 48 hours. A cell suspension of 150 µl was taken from the wells for each dilution from which total cell numbers and live and dead cell proportions were determined by hemocytometry (Barker, 2004).

Cytotoxicity (LC50) tests were conducted on the data of DMSO and all experimental groups.
 
Statistical analysis
 
Statistical analysis was performed using SPSS 11.5 software. The arithmetic means and standard errors of the per cent proportions of dead PBMCs of the control and experimental groups were calculated at 24 and 48 hours. For comparison, a Mann-Whitney U test was used. Statistical significance was accepted as P<0.05.
As a result of the extraction of A. densiflora with n-hexane, 4.12 g of dark purple-coloured extract from 90.5 g of the drug was obtained. A. annua extraction from 100 g of drug with petroleum ether yielded 2.28 green-coloured extract, and with methanol 5.56 g of dark green-coloured extract.

Naphthoquinones isolated from the extract obtained from A. densiflora roots by TLC-densitometry were teracrylalkannin, β,β-dimethylacrylalkannin, a-methyl-n-butylalkannin, and isovalerylalkannin. Results of the gas chromatographic analysis of the amount of artemisinin in the petroleum ether and methanol extracts of A. annua are shown in Table 1.

Table 1: Gas chromatography analysis of artemisinin extracted from A. annua.



The A. densiflora extract had a negative effect on non-infected PBMCs at all concentrations tested (Table 2). At 24 and 48 hours, all tested concentrations of A. densiflora extract on infected PBMCs were found to be statistically different compared to the control group (Table 2).

Table 2: Dead cell ratios from efficacy trials of A. densiflora n-hexane extract against non-infected and infected PBMCs (n=6).



These results show that A. densiflora extract was effective against infected PBMCs even at the smallest concentration tested. At concentrations of 62.5 µg/ml and above, all infected cells died at 24 hours, whereas the mortality rate in the control group was very low. Cytotoxicity (LC50) values are about 6.63 µg/ml for infected cells and about 66.95 µg/ml for non-infected cells at 24 hours.

In the efficacy trials of A. annua petroleum ether extract on non-infected PBMCs, no statistically significant results were found between the control group and the experimental group in terms of dead cell ratios observed at 24 hours. At the 48th hour of the experiment, only the results obtained from the concentration of 125 µg/ml were found to be statistically significant (Table 3). This finding revealed that A. annua petroleum ether extract should be tested at concentrations lower than 125 µg/ml, as higher concentrations may damage non-infected cells. Cytotoxicity (LC50) values are about 23.43 µg/ml for infected cells and about 125 µg/ml for non-infected cells at 24 hours.

Table 3: Dead cell ratios from efficacy trials of A. annua petroleum ether extract on non-infected and infected PBMCs (n=6).



In the efficacy trials of A. annua petroleum ether extract against infected PBMCs, the results obtained with 125, 62.5, 31.25 and 15.625 µg/ml concentrations at 24 hours and 62.5 and 31.25 µg/ml at 48 hours were found to be significantly different compared to the control group. There was no statistically significant difference between the other concentrations and the control group at 24 or 48 hours (Table 3). These results showed that A. annua petroleum ether extract killed all infected PBMCs at concentrations of 62.5 µg/ml and above at 48 hours, whereas the mortality rate in the control group was low.

In the efficacy trials of A. annua methanol extract against non-infected PBMCs, there were no statistically significant differences between the control group and experimental group in terms of death rates observed at 24 and 48 hours (Table 4).

Table 4: Dead cell ratios from efficacy trials of A. annua methanol extract against non-infected and infected PBMCs (n=6).



The results obtained from the efficacy trials of A. annua methanol extract against infected PBMCs at concentrations of 125 µg/ml and 62.5 µg/ml at 24 hours and 125 µg/ml at 48 hours were statistically different from the control group. At 24 and 48 hours, no statistically significant results were found between the other experimental groups and the control group (Table 4). This finding revealed that A. annua methanol extract was not effective against infected PBMCs at concentrations <62.5 µg/ml. Cytotoxicity (LC50) values are about 206.61 µg/ml for infected cells and about 226.44 µg/ml for non-infected cells at 24 hours.

0.5% DMSO used for dissolving the extracts, at the highest concentration of 8 µl, did not show any statistically negative effects (Table 5). A cytotoxicity test (LC50) was conducted on the DMSO used in the lab and values were about 30 µg/ml for infected cells and about 17.22 µg/ml for non-infected cells at 24 hours.

Table 5: Dead cell ratios from efficacy trials of 0.5% DMSO against non-infected and infected PBMCs (n=6).



In today’s world, there is growing interest in treatment trials using plant extracts. Herbal materials, which can be used as models for many synthetic drugs, can enlighten us as to the structures of the active ingredients they contain and as such are indispensable elements of modern medicine. In addition, when used well, they create great economic potential for countries (Salmerón-Manzano, 2020).

Bovine Theileriosis can be diagnosed in a variety of ways, both early and late phases (Al-Hosary et al., 2020). So the treatment in time is important to protect against more economical losses.

The plant extract A. densiflora is rich in naphthoquinones, but there is no study on its effectiveness on Theileriosis. Despite A. annua having a rich artemisinin content, there are few studies on the effectiveness of this plant extract on Theileriosis. For example, a semi-synthetic artemisinin derivative, arteether, has been applied to the Theileria-infected cattle in vivo. Intramuscular injection of arteether at 5 mg/kg body weight for three consecutive days revealed a per cent efficacy of 66.66% (Khawale et al., 2019). Besides that, another synthetic artemisinin-based drug derived from A. annua, Arthemeter 80, has effectiveness against T. lestoquardi 48 h after exposure in-vitro was 0%, 14%, 30% and 45% at concentrations of 0.01, 0.1, 1.0 and 10 mg/L, respectively (Farah et al., 2015b). Ethanolic extract of A. absinthium leaves has also been tested against Theileria equi in vitro. The extract exhibited an EC50 value of 1371.5±17.3 μg/mL against the human foreskin fibroblast (HFF) cell line (Batiha et al., 2019). Although our study was not in-vivo, the findings of this research are important as it is one of the first studies in this field and conducting in vitro studies is essential in terms of helping to guide and plan future in vivo studies. Furthermore, Arnebia or Artemisia spp. plant trials above were conducted on the species Theileria lestoquardi or T. equi. However, T. annulata schizonts were used in our study for the first time in terms of those two plants.

The n-hexane extract prepared from the roots of the A. densiflora plant collected from the Eskişehir region of Turkey was preferred because it is rich in naphthoquinones (Bozan, 1994). As a result of the study, it was determined that the extract had a negative effect on both non-infected and infected cells. This finding indicates that the extract is not suitable for in vivo trials at the concentrations tested. An argument for not experimenting with lower concentrations can be that although the effect level at the lowest concentration was statistically different, it was close to the average mortality rate in the control groups. It is thought that even if the possible efficacy of lower concentrations on infected cells lasts, the adverse effect on non-infected cells may continue (Table 2).

The efficiency of A. annua methanol extract on infected cells-even at low levels- could be observed at a concentration of 125 µg/ml. Higher concentrations must be tried to get strong efficacy with non-infected PBMCs. These findings are consistent with the proportion of artemisinin contained in the extract. As a result, since the amount of artemisinin in the concentrations tested remained at very low levels, it could not show a sufficient effect. Thus, it is thought that this extract should be tried in higher concentrations.

More effective results were obtained from the petroleum ether extract of A. annua. At the tested concentrations of this extract, no adverse effects were observed in non-infected cells at 24 hours. However, it was determined that infected cells died at a concentration of 15.625 µg/ml compared to controls (P<0.01). At a concentration of 125 µg/ml, there was no adverse effect on non-infected cells, while all infected cells died, which is the optimal desired result. At 48 hours, the infected cells exposed to a concentration of 62.5 µg/ml died and the non-infected cells were not damaged (P<0.001). At a concentration of 125 µg/ml, there was a partial loss of non-infected cells at 48 hours.

Some data appear contradictory in tables 2, 3, 4 and 5 which show the dead cell ratios are more in some non-infected cells after a particular dose, in control groups and in the DMSO group as compared to infected cells. However, a high mitotic activity that occurs in parasite-infected cells possibly causes this conclusion. This situation is also reported by some researchers working on the cell cultivation of Theileria spp. (von Schubert et al., 2010).
Based on these findings, in vivo trials should be conducted at concentrations of 15.625-62.5 µg/ml which is important because it indicates the starting point for future trials. According to the results of this experiment, the petroleum ether extract of A. annua could have promising effects against T. annulata infection in vivo.
We would like to thank Prof. Dr. Şevki Ziya COŞKUN for proposing work on this research topic and supporting us throughout our studies; Prof. Dr. Tülin KARAGENÇ for allowing the use of laboratory facilities and providing assistance, advice, support, and materials for cell culture experiments; the members of Anadolu University Faculty of Pharmacy, Department of Pharmacognosy, and the deceased Prof. Dr. Hulusi MALYER who helped in the stages of collecting the plants and obtaining the extracts.
None

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