The effects of trans-chalcone and chalcone hydrate on the growth of Babesia andTheileria

Background Chemotherapy is a principle tool for the control and prevention of piroplasmosis. The search for a new chemotherapy against Babesia and Theileria parasites has become increasingly urgent due to the toxic side effects of and developed resistance to the current drugs. Chalcones have attracted much attention due to their diverse biological activities. With the aim to discover new drugs and drug targets, in vitro and in vivo antibabesial activity of trans-chalcone (TC) and chalcone hydrate (CH) alone and combined with diminazene aceturate (DA), clofazimine (CF) and atovaquone (AQ) were investigated. Methodology/Principal findings The fluorescence-based assay was used for evaluating the inhibitory effect of TC and CH on five of Babesia and Theileria species, including B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi, the combination with DA, CF, and AQ on in vitro cultures, and on the multiplication of a B. microti–infected mouse model. The cytotoxicity of compounds was tested on Madin– Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3T3), and human foreskin fibroblast (HFF) cell lines. The half maximal inhibitory concentration (IC50) values of TC and CH against B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi were 69.6 ± 2.3, 33.3 ± 1.2, 64.8 ± 2.5, 18.9 ± 1.7, and 14.3 ± 1.6 µM and 138.4 ± 4.4, 60.9 ± 1.1, 82.3 ± 2.3, 27.9 ± 1.2, and 19.2 ± 1.5 µM, respectively. In toxicity assays, TC and CH affected the viability of MDBK, NIH/3T3, and HFF cell lines the with half maximum effective concentration (EC50) values of 293.9 ± 2.9, 434.4 ± 2.7, and 498 ± 3.1 µM and 252.7 ± 1.7, 406.3 ± 9.7, and 466 ± 5.7 µM, respectively. In the mouse experiment, TC reduced the peak parasitemia of B. microti by 71.8% when administered intraperitoneally at 25 mg/kg. Combination therapies of TC–diminazene aceturate and TC–clofazimine were more potent against B. microti infection in mice than their monotherapies. Conclusions/Significance In conclusion, both TC and CH inhibited the growth of Babesia and Theileria in vitro, and TC inhibited the growth of B. microti in vivo. Therefore, TC and CH could be candidates for the treatment of piroplasmosis after further studies. Author summary Protozoa of the genus Babesia are the second most common blood-borne parasites of mammals after the trypanosomes. Babesia and Theileria are the etiological agents of piroplasmosis, a tick-transmitted disease causing substantial losses of livestock and companion animals worldwide and has recently gained attention as one of the emerging zoonosis in humans. Diminazene aceturate and imidocarb dipropionate are still the first choices for the treatment of animals. However, these drugs cause many adverse effects. Furthermore, they are not approved for human medicine. Therefore, the development of alternative treatment remedies against babesiosis is urgently required. In the present study we evaluated the effects chalcone hydrate (CH) and trans-chalcone (TC), against the growth of four species of Babesia and T. equi. Furthermore, we studied the chemotherapeutic potential of TC on B. microti in mice. The effects of the combined treatment of TC with DA, CF and AQ revealed that TC was found to diminish the adverse effects of these drugs


2.4.
Combination treatment using CH or TC with DA, AQ, or CF in vitro 180 The combination studies were performed in accordance with the previously described to each well to a final concentration of 12.5-500 µM in triplicate, while for DA, AQ, and CF, 10 232 µL of twofold dilutions was added to each well to a final concentration of 100 µM in triplicate.

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The wells with only a culture medium were used as blanks, while the wells containing cells and a 234 medium with 0.4% DMSO were used as a positive control. The exposure of drugs was carried out for 24 h, followed by the addition of 10 µL of CCK-8. The plate was further incubated for 3 236 h, and the absorbance was measured at 450 nm using a microplate reader. The in vivo inhibitory effects of TC were evaluated against B. microti in mice as previously 240 described [23]. Briefly, 50 female BALB/c mice at 8 weeks of age were caged in 10 groups (five 241 mice/group). B. microti recovered from the frozen stock (stored at −80ºC) was thawed and 242 injected into two mice intraperitoneally. The parasitemia was monitored daily via microscopy.

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The mice were sacrificed and blood collected by cardiac puncture when the parasitemia was over     The positive symbol (+) indicates regrowth of the parasites, and the negative symbol (-) indicates 339 total clearance of the parasites on day 8 after withdrawing the drug pressure as seen in the 340 microscopy assay.

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Micrographs of TC-and CH-treated B. bovis (Fig 4) Table), as recommended in 362 the Chou-Talalay method [25], were combined at a constant ratio with DA, AQ, or CF. The showed an antagonistic effect (Table 3). 22

The chemotherapeutic effect of trans-chalcone against B. microti in mice
For further evaluation of TC efficacy in comparison with other drugs, the chemotherapeutic 398 effect of TC was examined in mice infected with B. microti (Fig 6). Furthermore, infection with B. microti reduces the RBC count (Fig 9A), hemoglobin 441 concentration (Fig 9B), and hematocrit percentage (Fig 9C) in mouse blood, as observed in the 442 DDW control group on days 8 and 12 p.i. Significant differences (p < 0. 05) in RBC count were 443 observed between the DDW control group and all drug-treated groups on days 8 and 12.

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The treatment of bovine and equine piroplasmosis is limited to diminazene aceturate (DA) 453 and imidocarb propionate, while clindamycin-quinine and atovaquone-azithromycin were assessed in vitro.

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In the current study, both CH and TC were effective against the Babesia and Theileria 463 parasites in vitro (Figs 2 and 3). It is noteworthy that CH and TC were most effective against T. metabolism of the parasites [12,15]. Since this enzyme is absent from mammalian cells, it could 476 be an important target for drugs against protozoan parasites. Based on the previous findings, it is 477 possible that chalcones also inhibit the mitochondrial respiratory chain enzymes in Babesia and

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Theileria parasites, which could be elucidated in future studies.

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The viability assay showed that TC and CH were more effective against Babesia parasites  Although the exact mode of action is yet to be elucidated, the parasites progressively lost their 492 shape and became smaller. This could be attributed to the ability of chalcones to interfere with 493 the metabolic pathway, as documented in P. falciparum and Leishmania parasites [21,28]. Theileria [29]. That study showed that the CF-DA combination has additive effects on the in The promising efficacy of TC in vitro prompted us to evaluate TC performance in vivo.

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TC administered intraperitoneally at a dose of 25 mg/kg resulted in a 71.8% inhibition of the 522 parasitemia on day 9 p.i. However, the inhibition rate was lower than those in the presence of 25 523 mg/kg DA, 20 mg/kg AQ, and 20 mg/kg CF, which were 92.5%, 90.8%, and 93.1%, respectively 524 (Fig 6). Certainly, the additive and synergistic effects in these combinations were indicated by 525 the high degree of association observed in vitro, which prompted studies in vivo. Therefore, the CF at a dose of 12.5 + 10 mg/kg resulted in a 94.4% inhibition in the parasitemia level at day 8 532 p.i. (Fig 7). The potentiation of TC that was achieved in in vivo combination therapy confirms parasite in vivo [27]. In addition to being efficacious, chalcones enhanced the production of nitric 538 oxide, which kills the intra-erythrocytic parasites and stimulates the host immune system [31].

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In order to confirm the ability of TC to eliminate B. microti, a PCR assay was performed 540 on samples collected on day 45 p.i. to be analyzed for the presence of DNA. Interestingly, this 541 study confirmed the absence of B. microti DNA in groups treated with a combination 542 chemotherapy of TC+DA or TC+CF (Fig 8) as compared to monotreatment. These results underscore the importance of combination chemotherapy in the effective control of 544 piroplasmosis. This finding further emphasizes the need for combination therapy to achieve the 545 most optimum efficacy and prevent the relapse of infection or development of a carrier state 546 [29]. Furthermore, TC did not show toxic side effects to mice (Fig 9A-C), consistent with a 547 previous study [14]. Taken together, the findings advocate that TC is a potential drug against 548 bovine and equine piroplasmosis.