Phytochemical Screening and Antiprotozoal Effects of the Methanolic Berberis Vulgaris and Acetonic Rhus Coriaria Extracts

Berberis vulgaris (B. vulgaris) and Rhus coriaria (R. coriaria) have been documented to have various pharmacologic activities. The current study assessed the in vitro as well as in vivo inhibitory efficacy of a methanolic extract of B. vulgaris (MEBV) and an acetone extract of R. coriaria (AERC) on six species of piroplasm parasites. The drug-exposure viability assay was tested on three different cell lines, namely mouse embryonic fibroblast (NIH/3T3), Madin-Darby bovine kidney (MDBK) and human foreskin fibroblast (HFF) cells. Qualitative phytochemical estimation revealed that both extracts containing alkaloid, tannin, saponins and terpenoids and significant amounts of flavonoids and polyphenols. The GC-MS analysis of MEBV and AERC revealed the existence of 27 and 20 phytochemical compounds, respectively. MEBV and AERC restricted the multiplication of Babesia (B.) bovis, B. bigemina, B. divergens, B. caballi, and Theileria (T.) equi at the half-maximal inhibitory concentration (IC50) of 0.84 ± 0.2, 0.81 ± 0.3, 4.1 ± 0.9, 0.35 ± 0.1 and 0.68 ± 0.1 µg/mL and 85.7 ± 3.1, 60 ± 8.5, 90 ± 3.7, 85.7 ± 2.1 and 78 ± 2.1 µg/mL, respectively. In the cytotoxicity assay, MEBV and AERC inhibited MDBK, NIH/3T3 and HFF cells with half-maximal effective concentrations (EC50) of 695.7 ± 24.9, 931 ± 44.9, >1500 µg/mL and 737.7 ± 17.4, >1500 and >1500 µg/mL, respectively. The experiments in mice showed that MEBV and AERC prohibited B. microti multiplication at 150 mg/kg by 66.7% and 70%, respectively. These results indicate the prospects of these extracts as drug candidates for piroplasmosis treatment following additional studies in some clinical cases.


Introduction
Piroplasmosis is the exhausting ailment caused by hematotropic piroplasm parasites [1]. Up to date, numerous drugs have been used for the treatment of piroplasmosis infections [2,3]. For instance, clindamycin-quinine, pentamidine-cotrimoxazole and atovaquone (ATV) combination with either azithromycin or proguanil have been reported for zoonotic babesiosis treatment, yet some investigations reported Babesia (B.) gibsoni resistance towards ATV [4,5]. Additionally, diminazene aceturate (DMA), oxytetracycline and imidocarb dipropionate, the treatment choices for bovine and equine piroplasmosis showed limited efficacy and there have been reported of unwanted symptoms and the emergence of resistant parasites [6]. In general, advances in piroplasmosis treatment is vital for improving disease treatment and tick control [7,8]. Several antipiroplasmic molecules and drug targets were identified, presenting an alternative choice for disease control [4][5][6][9][10][11]. The extensive socio-economic and welfare effects of bovine and equine piroplasmosis on animals and human babesiosis on humans have sustained the demand for pharmaceutical advancements to develop novel drug candidates.
Herbal remedies have been attracting attention as a prospective alternative resource of therapy for diverse diseases across many nations [12,13]. In recent decades, medicinal plants have been gaining wider acceptance due to the perception that these plants being natural products have lesser side effects and improved efficacy than their synthetic counterparts [9,12]. Many plant species have been reported to have pharmacological activities attributable to their phytoconstituents such as glycosides, saponins, flavonoids, steroids, tannins, alkaloids, terpenes, etc. [11,14].
Berberis vulgaris L. Sp. Pl. 1: 330 (-331) (1753) (B. vulgaris), in the genus Berberis (family Berberidaceae), is the most widely known Berberis that is indigenous to semi-tropical areas in Africa, Asia, Europe, North America, and South America and it is used mainly as food [15]. Pharmacologically, B. vulgaris have been documented to have several therapeutic properties, including anti-inflammatory, sedative, antipyretic, antiemetic, antioxidant, anti-cholinergic, anti-arrhythmic, anti-leishmaniasis, antimicrobial, and anti-malarial [16][17][18]. The phytochemical examination showed the existence of several molecules such as phenolic compounds, tannins, sterols, vitamins (vitamins A and C), protein, carotenoid, anthocyanin, dextrose, malic acid, fructose, citric acid, pectin, tartaric acid, resin, elements (calcium, iron, and potassium, zinc, copper, and manganese), alkaloids and triterpenes [18]. Berberine and berbamine are the most valuable alkaloids exhibiting a variety of activities, such as hypoglycemia, anti-inflammatory, hypotensive, antioxidant, and hypolipidemic properties [19,20]. Previous studies documented the berberine significance in the treatment of schistosomiasis disease incomparable to the anti-schistosomiasis drug; praziquantel and they reported the beneficial effects of berberine on Schistosoma (S.) mansoni by inducing oxidative stress which may be the main cause of its antioxidant activity [21]. Additionally, berberine has the same effect in Plasmodium (P.) chabaudi through its anti-inflammatory effect as it acts by reducing the inflammatory response induced by the parasite and increasing the activities of alanine and aspartate transaminases [22,23]. Interestingly, recent reports documented the potent antiparasitic effect of B. vulgaris as well as berberine against Trichomonas vaginalis, Giardia lamblia, Entamoeba histolytica, and some Leishmania (L.) spp. For instance, Mahmoudvand et al. [20] revealed that B. vulgaris methanolic extract showed potent leishmanicidal activity against L. infantum and L. tropica (IC 50 = 4.83 µg/mL) in vitro. Additionally, Vennerstrom et al. [24] reported that berberine alone demonstrated a potential leishmanicidal effect with an IC 50 value of 7.1 µM and ranging from 2.1 to 26.6 µg/mL against L. donovani, L. tropica, and L. major promastigotes, respectively. The root bark extract of B. vulgaris was used to prepare a lotion formula which led to good suppression effects in mice with 90% recovery from cutaneous leishmaniasis [25,26].
Although these plants have been reported for various pharmacological values, there is no evidence on the antipiroplasmic activity of B. vulgaris and R. coriaria crude extracts. Thus, the current study examined the effectiveness of a methanolic extract of B. vulgaris (MEBV) and an acetone extract of R. coriaria (AERC) on the multiplication of Theileria (T.) equi, B. bigemina, B. bovis, B. caballi and B. divergens using the in vitro fluorescence assay and their chemotherapy prospects against B. microti-infected mice.

Plant Extraction, Phytochemical and Total Phenolic and Flavonoid Contents Evaluation of MEBV and AERC Extracts
The yield percentage of the MEBV and AERC were 7.31 and 8.06% w/w dry matter and dark in color. Preliminary examination of MEBV pointed to the existence of different phytoconstituents such as tannins, saponins, alkaloids, and terpenoids, while AERC contains saponins, alkaloids, and terpenoids that may be responsible for their pharmacological activities. Moreover, considerable amounts of polyphenols and flavonoid contents were observed in MEBV and AERC. Notably, MEBV (63.2 ± 4.2 mg of GAE/g DW) showed the highest total phenolic content followed by AERC (59.9 ± 9.4 mg of GAE/g DW). Moreover, AERC (42.9 ± 5.1 mg of CAE/g DW) had the highest total flavonoid content followed by MEBV (39. 2 ± 4.7 mg of CAE/g DW).

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
The GC-MS analysis of MEBV and AERC revealed the existence of 27 and 20 phytochemical compounds, respectively. The identified chemical composition of MEBV is shown in Table 1 and represented 27 compounds, while the identified chemical composition of AERC is shown in Table 2 and represented 20 compounds. The phytochemical compounds' identification was established on the basis of the peak area, and retention time. The active principles with their retention time (RT) and percentage of peak area (%) are expressed in Figures S1 and S2.

Growth-Inhibition Efficacy of MEBV and AERC In Vitro
The preliminary evaluation of MEBV and AERC was performed to detect their efficacy on the host red blood cells (RBCs)prior to the subculture of T. equi and B. bovis. The parasite proliferation did not significantly differ between RBCs treated with either MEBV or AERC and the untreated one for both species. For the growth-inhibitory effect, MEBV ( Figure 1) and AERC ( Figure 2) affected the multiplication of all treated species in a dose-related manner.   Table 3. For the reference antibabesial drugs, DMA, ATV and clofazimine (CLF) restricted B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi multiplication with IC 50 s shown in Table S2.

Parasite Viability and Morphological Changes After Treatment With MEBV and AERC
In the presence of 4 × IC 50 (16.4 and 3.24 µg/mL) MEBV, the regrowth of B. divergens and B. bigemina was completely suppressed, while T. equi, B. caballi, and B. bovis regrew even at 4 × IC 50 . AERC at 4 × IC 50 (360, 342.8 and 312 µg/mL) completely restricted T. equi, B. caballi and B. divergens regrowth, while B. bovis and B. bigemina were able to regrow even at 4 × IC 50 . Moreover, MEBV-and AERC-treated T. equi micrographs are shown in Figure 3. At 24 h, all captured micrographs revealed spindle shapes dividing B. bigemina, B. bovis, and B. caballi parasites in contrast with the normal piriform shape of the untreated one, while dot-shaped residues of dead parasites were observed within the erythrocytes at 72 h. At 24 h, T. equi treated with MEBV and AERC were smaller and pyknotic when compared with the normal oval form of the untreated one. In subsequent micrographs taken at 72 h, the dying parasites appeared dot-shaped ( Figure 3).

Toxicity of MEBV and AERC on Normal Cells
The cytotoxicity assay of MEBV and AERC was estimated on HFF, NIH/3T3, and MDBK cell lines to detect their effect on the host cell viability. MEBV and AERC showed inhibition on MDBK and NIH/3T3 cell lines with EC 50 shown in Table 3. Moreover, both extracts did not reduce the HFF cell viability at 1500 µg/mL. For the reference antibabesial drugs, neither DMA nor ATV (final concentration 100 µM) restricted the viability of HFF, NIH/3T3, or MDBK cell lines, while CLF in EC 50 of 34 ± 3.4 µM reduced the MDBK cell viability (Table S2). The highest selective index values (ratio of the EC 50 on the cell lines to the IC 50 on the parasites) for MEBV and AERC were 1023.1 and 13.2 times toward T. equi and B. bigemina, respectively, whilst the lowest of MEBV and AERC was achieved on B. divergens with a selectivity index of 169.7 and on B. divergens with a selectivity index of 8.2, respectively (Table 3).

In Vitro Potential of The Combination of MEBV and AERC With Other Drugs (ATV, DMA, and CLF)
MEBV-and AERC-DMA treatment was additive toward all tested species except toward B. caballi it was synergetic. The combination treatments of MEBV and AERC with ATV or CLF were synergetic and additive toward all tested species (Table 4).

In Vivo Chemotherapeutic Efficacy of MEBV and AERC
To examine the chemotherapeutic effects of MEBV and AERC in vivo, female BALB/c mice were affected by B. microti and the two drugs were administered for five days after the infection reach 1% parasitemia. On the eighth day post-infection (p.i.), the DDW control group showed rapid parasitemia growth reached 62.6% and the parasitemia reduced slowly on the subsequent days. The peak parasitemia level in all treated groups was 20.9%, 18.8% and 5% in MEBV (150 mg/kg BW), AERC (150 mg/kg BW) and DMA (25 mg/kg BW), respectively (Figure 4).

Discussion
In the current study, MEBV and AERC were examined for the existence of various biologically active compounds and the primary screening emphasized that both extracts containing terpenoids, alkaloids, flavonoids, and tannins. The qualitative examination revealed the presence of significant amounts of polyphenols and flavonoids. Notably, this finding conforms to the report by Gabr et al. [33] and Sequeda-Castañeda et al. [34] who revealed the existence of these active constituents in both extracts. It has been shown that all these secondary metabolites have many therapeutic properties and are known to be pharmacologically active components. Among these constituents, some flavonoids, polyphenols, and alkaloids have been reported to possess an antiparasitic effect against several parasites including, Plasmodium, Leishmania, Trypanosoma, Schistosoma and Trichomonas Vaginalis [35].
For the in vitro experiments, MEBV revealed a growth-inhibiting effect against Babesia and Theileria parasites, whereas AERC exhibited high growth-inhibiting effects with high IC 50 values against piroplasm parasites. Owing to the fact that Babesia, Theileria, and Plasmodium are classified into the same Apicomplexa phylum, the growth-inhibiting effect of MEBV and AERC against Babesia and Theileria parasites are consistent with those reported by Oryan. [16], and Rahimi-Madiseh et al. [18], who indicated that B. vulgaris extract was evaluated as a folk therapy for the treatment of fever and malaria. Additionally, Gathirwa et al. [29] showed that R. coriaria extract has antiplasmodial and antimalarial efficacy. In a separate report, the antimalarial action of R. coriaria was attributed to eriodictyol, 7-O-methylnaringenin, 7-O-methylluteolin, 30-O-dimethylquercetin, 7-O-methyl-apigenin, and di-O-methyl tetrahydroamentoflavone [30]. Further, diverse biological activities of MEBV have been attributed to the presence of berberine main active compounds [20]. Moreover, Fata et al. [25] investigated the effectiveness of alcoholic extracts of B. vulgaris against other parasites including cutaneous leishmaniasis. Interestingly, recent studies documented the antiprotozoal activities of various bioactive molecules identified in our GC-MS analysis. For instance, Le et al. [36] documented the antitrypanosomal activity of α-curcumene, the main chemical component in MEBV with IC 50 value of 13.38 µg/mL against Trypanosoma brucei brucei, whereas Andrade et al. [37] disclosed the antileishmanial efficacy of guaiol against Leishmania amazonensis promastigotes forms. Moreover, Colares et al. [38] and Le et al. [39] proved antileishmanial activity of eugenol and methyleugenol (the main chemical components in AERC) against promastigotes of L. amazonensis. In addition to that, β-guaiene and caryophyllene show antiprotozoal activity against P. falciparum, T. brucei, T. cruzi, L. amazonensis, and L. infantum with IC 50 values ranging from 2.2 to 56.6 µg/mL [40]. Sena-Lopes et al. [41] investigated the antiprotozoal effect of methyleugenol, β-caryophyllene and α-curcumene against the growth of T. vaginalis, with an IC 50 value of 100 µg/mL. The GC-MS analysis of Syzygium (S.) aromaticum documented the presence of eugenol, β-guaiene, caryophyllene, and α-ylangene which showed potent antipiroplasmic effect in vitro and in vivo [9,42]. Therefore, we hypothesized that α-curcumene, guaiol, α-guaiene, α-ylangene, caryophyllene, and methyleugenol are the main active compounds responsible for the antipiroplasmic activity of MEBV and AERC.
It is likely that more than one bioactive ingredient restricts piroplasm parasites multiplication, thus, elucidating the inhibitory effectiveness of MEBV and AERC would require further studies. As compared to previous studies, MEBV IC 50 values against Babesia parasites were less than those of S. aromaticum methanolic extract toward bovine Babesia (B. bovis and B. bigemina) [9]. While, AERC IC 50 values toward Babesia parasites were less than those of Uncaria tomentosa acetonic extract, Myrtus communis acetonic extract, Origanum vulgare ethanolic extract, and Cuminum cyminum methanolic extract toward bovine Babesia (B. bovis and B. bigemina) [6]. A viability study revealed that MEBV and AERC have the ability to restrict piroplasm parasites regrowth. For instance, B. caballi and B. divergens treated with MEBV did not revive at 4 × IC 50 concentration. While, AERC suppressed B. caballi, T. equi and B. divergens regrowth at 4 × IC 50 . However, there is a need for additional researches to isolate and identify the effective compounds from MEBV and AERC and evaluate the actual mode of action employed against the recovery of piroplasm parasites.
Additionally, MEBV and AERC treatment resulted in morphological changes in the treated parasites observed in micrographs taken at different incubation times. The captured micrographs revealed that Babesia and Theileria parasites were not able to eject and thus, died inside the iRBCs. Interestingly, recent studies revealed that the organic extract of B. vulgaris and R. coriaria extracts showed a significant antiparasitic effect toward P. falciparum and Leishmania with multiple morphological changes of the parasites [16,29,31]. Oryan. [16] showed that the IC 50 of B. vulgaris was 45.5 µg/mL with the highest inhibitory effect on promastigotes multiplication and morphological changes, demonstrating that, the extracts prepared from B. vulgaris showed more promising antileishmanial activity against both stages of the parasite. Gathirwa et al. [31] showed that the high activity of R. natalensis and chemosuppression 83.15% for R. coriaria against P. falciparum, explained the Rhus plant's potential.
The attempts to determine the cytotoxic effect of our extracts revealed that MEBV and AERC inhibited HFF, NIH/3T3, and MDBK cell viability at a slightly high selectivity index. Thus, suggesting that the tested extracts are able to act on Babesia and Theileria without affecting the host cells. These results are comparable with that of Oryan. [16], who reported the lower cytotoxicity of B. vulgaris against host cells (IC 50 = 390.5 µg/mL), with an excellent selectivity index equal to 24. Gathirwa et al. [31] showed that R. coriaria did not show any cytotoxic effect on mammalian cells with an EC 50 of 211.78 µg/mL and a selective index value of 2.78.
Nowadays, combination chemotherapies are used to treat serious ailments, including pulmonary tuberculosis, malignancy, immune deficiency syndrome, and some protozoal diseases to promote higher therapeutic efficacy [43]. The recent strategy to enhance the therapeutic effect in Chinese medicine which began a long time ago relies on the use of herb-herb and herb-compound combination [10]. MEBV and AERC combined with DMA exhibited an additive efficacy toward all species tested. The combined application of MEBV and AERC with ATV and CLF showed an additive and synergetic relationship toward T. equi, B. divergens, B. bigemina, B. caballi, and B. bovis. One possible explanation is that herbal extracts contain many bioactive ingredients that may interact differently resulting in synergistic action observed for combination treatment. The implication of this is that the plant extracts could be used as an adjuvant to the piroplasmosis therapy.
MEBV and AERC produced promising in vivo antibabesial activity. On the eighth-day post-infection, oral administration of 150 mg/kg of MEBV and AERC resulted in 66.7% and 70% inhibition compared to 92% inhibition showed by DMA at 25 mg/kg. MEBV exhibited chemotherapeutic efficacy higher than the 42.4% demonstrated by Camellia sinensis methanolic extract [9], while the inhibitory effect of AERC was higher than the 69.2% demonstrated by S. aromaticum methanolic extract [9]. Furthermore, neither the MEBV nor the AERC treatment showed any apparent toxic symptoms or promoted anemia in uninfected mice. The chemotherapeutical effectiveness induced by MEBV and AERC toward B. microti implicates that they may be alternate chemotherapy against B. microti infection in humans. Therefore, identifying the active compound is necessary for contriving a higher chemosuppression effect from these extracts for the future discovery of a novel potential drug against piroplasmosis.
The limitation of this study is the use of the whole extract to confirm the antiprotozoal activity, and it is recommended to evaluate the antipiroplasmic efficacy of the GC-MS identified compounds for the future discovery of a novel potential drug against piroplasmosis. And evaluate the actual mode of action employed against the recovery of piroplasm parasites.

Conclusions
MEBV and AERC demonstrated the in vitro and in vivo chemotherapeutic potentials against the multiplication of several piroplasm parasites both in vitro and in vivo, implying that both extracts have potential value in treating clinical diseases in animals and humans either alone or in combination with other drugs. The GC-MS analysis documented the presence of many phytochemical compounds that may be responsible for the babesicidal activities of MEBV and AERC.

Ethical Statement
The in vivo studies were conducted based on the local guidelines for animal experimentation, as approved by the Obihiro University of Agriculture and Veterinary Medicine, Japan (accession numbers 28-111-2, 28-110, and 1417-2). This ethical approval was developed through the basic guidelines for the proper conduct of animal experimentation and related activities in Academic Research Institutions, Ministry of Education, Culture, Sports and Technology (MEXT), Japan.

Chemical Reagents
Stock solutions (100 mg (crude extract)/1 mL (DMSO) and 10 mM) in dimethyl sulfoxide (DMSO) of crude extract and DMA (Ciba-Geigy Japan limited, Tokyo, Japan), ATV and CLF (Sigma-Aldrich, Tokyo, Japan), respectively were stored at −30 • C and used for antibabesiosis evaluation. Reference drugs including DMA, CLF, and ATV were used either singly and/or in combination with the two extracts for both the in vivo and in vitro experiments. For the fluorescence assay, SYBR Green I (SGI) stain (10,000×, Lonza, Alpharetta, GA, USA) was mixed with the lysis buffer containing saponin (0.016% w/v), EDTA (10 mM), Triton X-100 (1.6% v/v), and Tris (130 mM at pH 7.5) which was filtered using a polyethersulfone (0.22 µm) and kept at 4 • C.

Herbal Plants
Berberis vulgaris and R. coriaria were collected from Delta, Egypt and identified by the members of the Pharmacology and Chemotherapeutics Department, Faculty of Veterinary Medicine, Damanhour University, Egypt. B. vulgaris and R. coriaria voucher specimen numbers are A0177105 (DPV) and A 0177106 (DPV), respectively. An electric dryer (Sanyo Electric Co., Ltd., Osaka, Japan) was used to dry the plants at a temperature of 30 • C, then ground using a 60-80 mm mesh to a fine powder.
Subsequently, fine plant powder (100 g) was dissolved in methanol (99.8%) (Wako Pure chemical Industrial, Ltd, Osaka, Japan) or acetone (99.5%) (Nacalai Tesque, Kyoto, Japan) (50 mL) and incubated for 72 h at a temperature of 30 • C. The preparation of slurry extract was performed following the method as previously described [9,18,44] and the extracted stock (100 mg/1 mL DMSO) was stored at −30 • C and used for antibabesiosis evaluation. The obtained extracts of the MEBV and AERC were 7.31 and 8.06 g, respectively, and the yield percentage was calculated using the following Equation [45]: Percentage yield of extract = Weight of extracted material Weight of original plant material used × 100

Phytochemical Examination of Plant Extracts
MEBV and AERC were examined for the existence of terpenoids, saponins, tannins, and alkaloids using several qualitative tests as previously reported elsewhere [46].

Determination of Total Phenolic Content
The concentration of total phenolic content (polyphenols) present in MEBV and AERC was detected using Folin-Ciocalteu (FC) reagent method as described elsewhere [47]. A volume of 0.5 mL of both extracts (1 mg/mL) was added to 1.5 mL of 10% FC reagent (diluted 1:10 with de-ionized water) and mixed for 5 min. After that, an aliquot (3 mL) of 7.5% Na 2 CO 3 solution was added and further incubated at 30 • C for 2 h. Finally, the absorbance was calculated at 760 nm and the content of total phenolic compounds was detected from a gallic acid standard curve and expressed as mg/g gallic acid equivalent (GAE) of the dry weight of the extract (mg GAE/g DW).

Determination of Total Flavonoid Content
Aluminium chloride (AlCl 3 ) colorimetric method was used for the examination of total flavonoid content in MEBV and AERC as previously determined [47]. Briefly, an aliquot (1 mL) of both extracts was added to 3 mL of solvent extracts, 3.8 mL of distilled water, 200 µL of 1 M potassium acetate and 200 µL of 10% AlCl 3 and incubated for 30 min. The absorbance was measured at 420 nm and the flavonoid content was detected from a catechin standard curve and expressed as mg/g catechin equivalents of the dry weight of individual extract (mg CAE/g DW).

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
The chemical composition of MEBV and AERC was performed using Trace GC-ISQ mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG-5MS (30 m × 0.25 mm × 0.25 µm film thickness) as previously described [48]. The column oven temperature was initially held at 50 • C and then increased by 5 • C /min to 250 • C withhold 1 min then increased to 300 at rate of 30 • C /min. The injector temperatures were kept at 260 • C. Helium was used as a carrier gas at a constant flow rate of 1 mL/min. The solvent delay was 4 min and diluted samples of 1 µL were injected automatically using an AS3000 Autosampler coupled with GC in the split mode. EI mass spectra were collected at 70 eV ionization voltages over the range of m/z 50-650 in full scan mode. The ion source and transfer line were set at 250 • C and 270 • C, respectively. The components were identified by comparison of their retention times and mass spectra with those of WILEY 09 and NIST 11 mass spectral databases.

Parasites and Mice
For conducting in vitro experiments, B. bigemina Argentine strain [49], B. divergens Germany strain, Texas strain B. bovis were cultured in cattleRBCs, whereas United States Department of Agriculture (USDA) strains B. caballi and T. equi were maintained in horse RBCs; for conducting in vivo study, eight-week-old female BALB/c mice weighing 25 grams (g) were obtained from CLEA Japan and infected with rodent-borne parasitic nonzoonotic agent, B. microti Munich strain (AB071177) [49][50][51].
All parasites were incubated and maintained at 37 • C in a humidified chamber under 90% N 2 , 5% O 2 and 5% CO 2 atmosphere using a microaerophilic stationary-phase culture [5].

Culture Conditions
GIT medium replenished with 40% horse serum was used as a growth medium for B. caballi parasite culture, while T. equi was grown in medium 199 (M199) supplemented with hypoxanthine (MP Biomedicals, Santa Ana, CA, USA; final concentration 13.6 µg/mL). Babesia divergens was preserved in Roswell Park Memorial Institute 1640 (RPMI 1640; Sigma-Aldrich, Tokyo, Japan) medium, replenished with 40% cattle serum and culture medium M199 was used as a growth medium for B. bovis and B. bigemina replenished with 40% cattle serum. To ensure free-bacterial contamination, all medium was supplemented with amphotericin B (0.15 µg/mL) (Sigma-Aldrich, St. Louis, MO, USA), streptomycin (60 U/mL) and penicillin G (60 U/mL).

The Inhibition Assay of MEBV and AERC In Vitro
The Babesia fluorescent assay was carried out on the in vitro culture as previously reported elsewhere [5,52,53]. RBCs' stock supply at 1% parasitemia was prepared by diluting parasite-iRBCs with uninfected RBCs. Briefly, in three separate trials, using two-fold dilution, different concentrations of MEBV, AERC, ATV, CLF, and DMA were prepared in the culture medium and added in 96-well plates in triplicate with 1% parasitemia and 2.5% HCT for B. bigemina and B. bovis and 5% HCT for T. equi, B. caballi and B. divergens. The positive control had iRBCs with final concentration of 0.3% of DMSO, whereas uninfected RBCs and the culture medium acted as the negative control. Afterward, parasite cultures were cultivated for 4 consecutive days without changing medium at 37 • C humidified multi-gas incubator in 90% N 2 , 5% O 2 , and 5% CO 2 atmosphere. On day four of culture, an aliquot of lysis buffer (100 µL) was mixed with 0.2 µL/mL SG1 and gently added per well; subsequently, it was covered with aluminum foil to prevent exposure to light. After a 6-h incubation at 37 • C, fluorescence readings were acquired on a spectrofluorimeter (Fluoroskan Ascent, Thermo Fisher Scientific, Oceanside, CA, USA) with an excitation wavelength of 485 nm and an emission wavelength of 518 nm.

Parasite Viability Test In Vitro and Morphological Changes
The viability studies were monitored via microscopy as described previously [4,9,52]. Briefly, an aliquot (20 µL) of infected RBCs (1% parasitemia) was cultivated in 200 µL of media containing various concentrations of MEBV and AERC for 4 successive days, changing media daily. The concentrations used in this experiment were 0.25×, 0.5×, 1×, 2×, and 4× the IC 50 . On the fifth day, a mixture of iRBCs (6 µL) from each well and fresh equine or bovine RBCs (14 µL) was transferred to another plate, cultured in a medium free from drug and then left for an additional six days. The total parasite clearance was recorded as negative, while the relapse of parasites was recorded as positive.

Evaluation of The Impacts of MEBV and AERC on RBCs of Host
The hemolytic efficacy of MEBV and AERC on cattle and horse RBCs was evaluated in vitro as previously reported elsewhere [51]. Initially, MEBV and AERC at a concentration of 300 µg/mL were cultivated at 37 • C with purified bovine and equine RBCs for 3 h. Afterward, the pretreated-RBCs were added to T. equiand B. bovis-iRBCs after washing thrice with phosphate-buffered saline (PBS) to achieve 1% parasitemia. Thereafter, using a 24-well plate, an aliquot of iRBCs (100 µL) was mixed with culture media (900 µL); the control RBCs were left untreated. To monitor the parasitemia and any side effects due to the pretreatment, Giemsa-stained smears were prepared every 24 h for four days.

In Vitro Efficacy of The Drug Combination Treatment
The combined efficacy of MEBV and AERC with DMA or CLF or ATV was examined using the fluorescence inhibition assay as reported previously elsewhere [4,9,52,53]. Briefly, two-drug combinations of MEBV and AERC with DMA or CLF or ATV at five selected concentrations 0.25×, 0.5×, 1×, 2×, and 4× the IC 50 (Table S1) were added in 96 well-plates in duplicate. The drug cultivation and the fluorescence values were detected after the addition of 2×SGI mixed with lysis buffer to each well of the 96-well plate as described above. CompuSyn software was used for combination index (CI) values calculation and the synergetic degree was established as the average weighted CI values by using the following formulae; ((1 × IC 50 ) + (2 × IC 75 ) + (3 × IC 90 ) + (4 × IC 95 ))/10) and the resulted values were demonstrated using the recommended CI scale; lower than 0.90 was considered synergetic, between 0.90-1.10 was considered additive, while higher than 1.10 was considered antagonistic developed previously [53,54].

Cytotoxicity Assay of MEBV and AERC on Normal Cells
The cell viability test was conducted in a 96-well plate as described elsewhere [4,9,51,52]. Briefly, an aliquot of (100 µL) cells was implanted at a concentration of 5 × 10 4 cells/mL in DMEM or MEM with fetal bovine serum and incubated overnight under atmosphere 5% CO 2 at 37 • C for attachment. Using two-fold dilutions, aliquots (10 µL) were added in triplicate to each well to attain final concentrations of 15.8 to 1500 µg/mL and incubated for an additional 24 h. The positive control wells containing cells mixed with the medium in 0.4% DMSO, whereas the negative control wells containing culture medium only. After a 24-h incubation, Cell Counting Kits-8 (CCK-8) (10 µL) was added per well followed by the incubation of the plate for another 3 hours and a microplate reader was used to measure the absorbance at 450 nm.

In Vivo Chemotherapeutic Effects of MEBV and AERC
MEBV and AERC were examined for their in vivo chemotherapeutic efficacy using B. microti-infected BALB/c mice according to a procedure described elsewhere [49,51]. Briefly, twenty-five mice were placed in an environment free from pathogens with 22 • C temperature and adjusted humidity and under 12 h light and 12 h darkness and randomly distributed into five groups. All mice in groups 2 through 5 obtained 500 µL of 1 × 10 7 B. microti-iRBC by intraperitoneal (i.p.) injection. Group 1 served as a negative control and was neither infected nor treated. At 1% parasitemia, drug treatment of the mice by i.p. continuing for five days. Group 2 act as a positive control group and received 95% double-distilled water (DDW) and 5% DMSO. Groups 3 and 4 administered 150 mg/kg body weight (BW) of MEBV and AERC by the oral route, respectively. While group 5 received 25 mg/kg BW of DMA to act as a reference to drug control. The drug administration lasted for five days starting from the fourth day to the eighth day post-infection (p.i.), and parasitemia was monitored by preparing Giemsa-stained blood smears every two days in about 5000 RBCs by microscopy until day 32 p.i. Furthermore, the hematological parameters, including HGB, RBCs, and HCT percentage were examined every four days using an automatic hematology analyzer (Celltac α MEK-6450, Nihon Kohden, Tokyo, Japan) to detect the effect of MEBV and AERC on the retrogression of anemia related to Babesia infection. At the end of the in vivo experiment, an anesthetic system using an inhaler containing isoflurane was used to euthanize all mice by placing them in the induction chamber, adjusting the oxygen flowmeter to 0.8 to 1.5 L/min and vaporizer to 3% to 5%. When mice were completely anesthetized, all of them were killed by cervical dislocation according to the ethical approval confirmed by the Basic Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions, the Ministry of Education, Culture, Sports and Technology (MEXT), Japan.

Statistical Analysis
The nonlinear regression curve fit on a GraphPad Prism (GraphPad Software Inc., USA) was used to determine the IC 50 values of MEBV, AERC, DMA, CLF, and ATV from in vitro growth of the parasites. The significant variations (p < 0.05) among group mean values on parasitemia and Student's t-test, available in the GraphPad Prism software was used to analyze hematology profiles in mice infected with B. microti.

Supplementary Materials:
The following are available online. Table S1. The concentrations of the in vitro combination treatment of methanolic B. vulgaris and acetonic R. coriaria extracts with ATV, DMA, and CLF toward piroplasm parasites. Table S2. The IC 50 and selective indexes value of DMA, CLF, and ATV. Figure S1. Gas chromatography-mass spectrometry analysis in the methanolic extract of Berberis vulgaris. Figure S2. Gas chromatography-mass spectrometry analysis in the acetonic extract of Rhus coriaria.