Evaluation of the in vitro and in vivo inhibitory effects of Artemisia herba-alba against the growth of piroplasm parasites

Objective: The effect of Artemisia herba-alba methanolic extract monotherapy and combination therapies on the in vitro growth of several Babesia and Theileria parasites in vitro and mice was investigated in this study. Materials and Methods: Fluorescence assay using SYBR Green I stain was used to evaluate the antibabesial efficacy inhibitory of A. herba-alba either in vitro or in vivo. Hematological parameters in the treated mice were analyzed using a Celltac MEK-6450 computerized hematology analyzer. Results: Artemisia herba-alba reduced the growth of Babesia bovis, Babesia bigemina, Babesia divergens, Theileria equi, and Babesia caballi in vitro in a dose-dependent manner. The in vitro inhibitory impact of A. herba-alba on B. divergens and B. caballi cultures was amplified when combined with either diminazene aceturate (DA). In B. microti-infected mice, a combination therapy consisting of A. herba-alba and a low DA dose inhibited B. microti growth significantly (p < 0.05) better than treatment with 25 mg kg−1 DA. Conclusions: These data show that A. herba-alba, when paired with a modest DA dose, could be a promising medicinal plant for babesiosis treatment.


Introduction
Babesia and Theileria are tick-borne parasites that infect animals' erythrocytes, causing enormous economic losses in the agricultural industry and worldwide trade [1,2]. Clinical indicators of this infection include fever, malaise, jaundice, hemoglobinuria, and death [3,4]. The infection is mainly caused by either Babesia bovis (B. bovis) and Babesia bigemina (B. bigemina) in cattle [3] or Theileria equi (T. equi) and B. caballi in horses [5]. Because the inhibitory effects of recently developed antibabesial drugs should be evaluated in laboratory animals before they are used in the field, and because there are no acceptable laboratory experimental animals for bovine and equine Babesia infections, a rodent Babesia model infected with B. microti or a gerbil infected with Babesia divergens is used for drug evaluation [6,7].
For many years, the standard therapies for babesiosis were diminazene aceturate (DA) and imidocarb dipropionate [8,9]. However, they have significant drawbacks, such as a long time to remove tissue, toxicity, and, in the case of DA, unavailability in certain regions [9]. Furthermore, new research has revealed that Babesia parasites may develop DA resistance [10,11]. As a result, finding more effective and safer antipiroplasm drugs has become a top objective.
Natural phytochemicals could be a potential alternative in this scenario. In the same vein, Artemisia herba-alba, commonly known as desert or white wormwood, is used in folk medicine to treat various diseases [12,13]. Several studies have reported the wide pharmacological activities of A. herba alba as antidiabetic, antimicrobial, antimalarial, acaricidal [12,14,15], anticancer, and antioxidant [16,17]. However, A. herba-alba extract's antibabesial efficacy is yet to be determined. As a result, in the current investigation, we evaluated the antipiroplasm of A. herba-alba against the growth of B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi in vitro, and B. microti in mice.

Ethical approval
The Animal Care and Use Committee at Obihiro University of Agriculture and Veterinary Medicine approved all of the study's experimental protocols (Approval No. 27-65). The trials followed the Fundamental Guidelines for the Proper Conduct of Animal Experiments and Related Activities at Academic Research Institutions published by the Ministry of Education (Culture, Sports, Science, and Technology, Japan).
The chemotherapeutic efficacy of A. herba-alba against B. bovis (Texas strain) [22], B. bigemina (Argentina strain) [23], B. divergens (German strain) [24], B. caballi [25], and T. equi (U.S. Department of Agriculture) [25] was investigated in the current study by a fluorescence assay using a nucleic acid stain SYBR Green I (Lonza, Rockland, ME) [8,22]. The concentrations of A. herba-alba utilized ranged from 0.025 to 30 mg/ml. The in vitro study used DA, a routinely used antibabesial medication, as a positive control agent with concentrations ranging from 0.1 to 10 µg/ml [22]. Cultures without the drug and cultures with only DMSO (0.3% for A. herba-alba) and DDW (0.02% for DA) served as negative experimental controls (Wako Pure Chemical Industries, Ltd., Osaka, Japan). RBCs infected with 1% parasitemia of bovine and equine Babesia/Theileria parasites were cultured in 96-well plates for 4 days without daily medium replacement, using 2.5% hematocrit (HCT) for B. bovis and B. bigemina parasites and 5% HCT for other Babesia and Theileria parasites, as previously established [22,24]. All screened parasites' in vitro regrowth after ceasing A. herba-alba therapy was monitored using a viability assay, as described earlier in our study [24].
The combination therapy of A. herba-alba and DA was tested against in vitro cultures of bovine Babesia and horse piroplasm parasites having the highest IC 50 values, for B. bovis and T. equi, as previously detailed [6,26]. All in vitro tests were carried out three times.

In vivo chemotherapeutic effect of A. herba-alba
The A. herba-alba in vivo inhibition assay for B. microti (Munich strain) [27] in 25 female BALB/c mice (CLEA Japan, Tokyo, Japan) was performed twice using a fluorescence assay [28]. Five groups of mice (five animals per group) were employed. Simultaneously, with the drug inhibitory effect evaluation, 10 µl of blood was drawn from each mouse's tail every 4 days to examine hematological parameters using a Celltac MEK-6450 computerized hematology analyzer (Nihon Kohden Corporation, Tokyo, Japan).

Statistical analysis
A one-way analysis of variance test was used in GraphPad Prism to discover significant differences between the analyzed groups (GraphPad Software, Inc., San Diego, CA). Statistical significance was defined as p-value less than 0.05.

Artemisia herba-alba effectively suppressed the in vitro growth of piroplasm
According to the computed IC 50 s, A. herba-alba has the most significant impact on the growth of T. equi and B. bigemina, followed by B. bovis (Table 1). 0.025 mg/ml A. herba-alba effectively suppressed the development of B. bigemina and B. bovis in vitro (p < 0.05) (Fig. 1). 0.10 mg/ ml A. herba-alba was found to be effective in inhibiting the growth of T. equi (Fig. 1). Furthermore, 0.50 mg/ml A. herba-alba treatments significantly reduced the development of B. divergens and B. caballi (p < 0.05) (Fig. 1).
Theileria equi and B. bigemina in vitro were suppressed at doses of 0.5 and 1 mg/ml, respectively, in the following viability test ( Table 2). The parasite regrowth was suppressed in vitro when B. bovis was given 5 mg/ml A. herba-alba (Table 2). With 10 mg/ml A. herba-alba, B. caballi regrowth began to be reduced ( Table 2). The lack of a significant difference (p > 0.05) between the DMSO-treated positive control well and the untreated wells shows that the diluent did not affect the efficacy of the A. herba-alba methanolic extract. Furthermore, compared to nontreated erythrocytes, pretreatment of erythrocytes with a high dose of A. herba-alba methanolic extract at 25 mg/ml did not affect parasite growth pattern or erythrocyte morphology (data not shown).  Table 3). Such findings validated A. herba-alba's potential anti-B. divergens effect, especially when given in large doses combined with the regularly used antibabesial medication, DA.

Artemisia herba-alba suppressed the growth of B. microti in mice
A. herba-alba was tested in mice for its ability to suppress B. microti in vivo. Within the presence of 500 mg kg −1 A. herba-alba monotherapy, the greatest fluorescence values  within the A. herba-alba-treated groups reached a mean of 1702 at 12 days p.i. (Fig. 2). At 12 days p.i., 100 mg kg −1 A. herba-alba with 15 mg kg −1 DA demonstrated 991.15 mean fluorescence levels (Fig. 2). The peak fluorescence values in the positive control group, on the other hand, were 2033.65 at 12 days p.i (Fig. 2). Notably, when A. herba-alba was given at a low dose of DA, the suppression within the fluorescence values in mice was virtually identical to that shown in mice given 25 mg kg −1 DA at peak parasitemia days (Fig. 2). At 10 and 12 days p.i., oral injections of 100 mg kg −1 A. herba-alba in combination with a subcutaneous dose of 15 mg kg −1 DA inhibited parasite growth by 31.57% and 51.26%, respectively, compared to 54.64% and 73.24% inhibitions in the presence of 25 mg kg −1 DA (Fig. 2). The use of A. herba-alba in combination with a low DA dose normalized the hematological variables compared to those treated with 25 mg kg −1 DA (Fig. 3). These findings indicated A. herba alba's promising antibabesial activity when combined with a low DA dose. Such a regimen may aid in overcoming the toxic effects of high doses of the regularly used antibabesial medication, DA, and the parasite resistance resulting from this agent's prolonged use.   [18]. Similarly, the IC 50 for B. bovis in A. herba-alba was lower than those reported after in vitro treatment with turmeric (Curcuma longa) [29]. The efficacy of A. herba-alba as an agent with antimalarial activity [12,15] may explain the antibabesial efficacy of this herbal therapy because Plasmodium and Babesia parasites have striking biological similarities. Taken together, the antioxidant effect of A. herba-alba [17] may explain the antibabesial efficacy of this medicinal plant owing to the infection by Babesia is usually associated with increased levels of free radicals and oxidative stress markers [30], which is harmful to the infected host.
In the current investigation, very high concentrations of A. herba-alba exhibited no effect on bovine or horse RBCs. Additionally, A. herba-alba has been safely consumed for centuries without adverse effects. Previous studies reported the safe use of A. herba-alba in rats at >2 gm/ kg [12,31]. Such findings were confirmed via histopathological analysis of animal organs [31]. Interestingly, the 50% lethal dose (LD 50 ) value of A. herba-alba in mice was greater than 5,000 mg/kg [32].
The in vitro inhibitory activity of A. herba-alba and its safety have prompted us to study the inhibitory effect of A. herba-alba when taken alone or in combination with DA in mice. In our investigation, the in vitro inhibitory effect of A. herba-alba, combined with DA, against the growth of B. divergens and B. caballi was strengthened. These results are similar to the in vitro inhibitory effects of myrrh oil/DA [21], allicin/DA [33], and thymoquinone/DA combinations [27]. In an in vivo study, the inhibition of B. microti growth caused by A. herba-alba/DA is nearly similar to 56.35% Figure 2. Anti-B. microti of Artemisia herba-alba. All the animals received 1 × 10 7 B. microti RBCs intraperitoneally. When parasitemia in the infected mice reached about 1%, the treatment began and lasted for 5 days. In the control group, mice were given I/P doses of DMSO in phosphate buffer saline (0.02%). DA and Artemisia herba-alba were used in subcutaneous and oral doses, respectively. In the combination therapy, the drugs were given at the same time as the inoculation. The mean and standard deviation of five mice per experimental group are represented by each value. # denotes significant differences (p < 0.05) between Artemisia herba-alba/DA or DA monotherapy-treated groups and the control group. and 53.25% inhibition rates for 85 mg kg −1 PYR combined with 10 mg kg −1 DA, respectively [28]. Although the present study evaluated the inhibitory effect of A. herba-alba when used as monotherapy or in combination therapy against the growth of B. microti in mice, further studies are required to determine the LD 50 of this herbal extract in cattle before its application under field conditions.

Conclusion
In conclusion, B. bigemina and T. equi were the most sensitive Babesia species to Artemisia herba-alba's in vitro inhibitory action, followed by B. bovis. A. herba-alba was co-administrated with DA, a synergistic interaction against the in vitro growth of B. divergens was observed. The emitted fluorescence signal in the blood of mice treated with a combination therapy containing lower doses of A. herba-alba and DA was significantly reduced. Furthermore, a combination of A. herba-alba/DA therapy was used to correct hematological variables and treat hemolytic anemia caused by babesiosis. By overcoming the toxicity and resistance associated with long-term use of the antibabesial drug DA, A. herba-alba may be beneficial in treating animal piroplasmosis.