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. 2020 Aug 27;14(8):e0008489.
doi: 10.1371/journal.pntd.0008489. eCollection 2020 Aug.

In vitro and in vivo growth inhibitory activities of cryptolepine hydrate against several Babesia species and Theileria equi

Gaber El-Saber Batiha  1   2 , Amany Magdy Beshbishy  1 , Luay M Alkazmi  3 , Eman H Nadwa  4   5 , Eman K Rashwan  6   7 , Naoaki Yokoyama  1 , Ikuo Igarashi  1
Affiliations

In vitro and in vivo growth inhibitory activities of cryptolepine hydrate against several Babesia species and Theileria equi

Gaber El-Saber Batiha et al. PLoS Negl Trop Dis. .

Abstract

Piroplasmosis treatment has been based on the use of imidocarb dipropionate or diminazene aceturate (DA), however, their toxic effects. Therefore, the discovery of new drug molecules and targets is urgently needed. Cryptolepine (CRY) is a pharmacologically active plant alkaloid; it has significant potential as an antiprotozoal and antibacterial under different in vitro and in vivo conditions. The fluorescence assay was used for evaluating the inhibitory effect of CRY on four Babesia species and Theileria equi in vitro, and on the multiplication of B. microti in mice. The toxicity assay was evaluated 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 CRY on Babesia bovis, B. bigemina, B. divergens, B. caballi, and T. equi were 1740 ± 0.377, 1400 ± 0.6, 790 ± 0.32, 600 ± 0.53, and 730 ± 0.025 nM, respectively. The toxicity assay on MDBK, NIH/3T3, and HFF cell lines showed that CRY affected the viability of cells with a half-maximum effective concentration (EC50) of 86.67 ± 4.43, 95.29 ± 2.7, and higher than 100 μM, respectively. In mice experiments, CRY at a concentration of 5 mg/kg effectively inhibited the growth of B. microti, while CRY-atovaquone (AQ) and CRY-DA combinations showed higher chemotherapeutic effects than CRY alone. Our results showed that CRY has the potential to be an alternative remedy for treating piroplasmosis.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The dose-response curves of CRY against Babesia and Theileria parasites in vitro.
The curves show the growth inhibition of B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi treated with various concentrations of CRY. The result was determined by fluorescence assay after 96 h of incubation. The values obtained from three separate trials were used to determine the IC50s using nonlinear regression (curve fitting analysis) in GraphPad Prism software (GraphPad Software Inc., USA).
Fig 2
Fig 2. Growth of B. divergens with CRY-treated bovine RBCs (A) and growth of B. caballi with CRY-treated horse RBCs (B).
Fig 3
Fig 3. The growth inhibition of CRY on B. microti in vivo.
The inhibitory effects of DA-i.p., AQ-oral, and CRY-i.p. single treatments and combination treatments of CRY with DA and AQ as compared with the untreated group. The asterisks (*) indicate statistical significance (P < 0.05) between treated and untreated mice. The arrow indicates 5 consecutive days of treatment. Parasitemia was calculated by counting infected RBCs among 5000 RBCs using Giemsa-stained thin blood smears.
Fig 4
Fig 4. Hematology profiles of CRY-treated mice in vivo.
Graphs showing the changes in the HGB (A), HCT (B), and RBCs (C) of treated mice as compared with untreated mice. The values plotted are the mean ± standard deviation for two separate trials. Asterisks (*) indicate statistical significance (P < 0.05) based on the unpaired t-test analysis. The arrow indicates 5 consecutive days of treatment.
Fig 5
Fig 5. Molecular detection of parasite DNA in the blood of treated groups.
The image shows the molecular detection of parasites in the blood of treated groups on day 49. M indicates the marker, NC indicates the untreated-uninfected group used as a negative control, and PC indicates the untreated-infected group used as the positive control. The arrow shows the expected band length of 154 bp for positive cases of B. microti.
See this image and copyright information in PMC

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References

    1. Batiha GE-S, Beshbishy AM, Guswanto A, Nugraha AB, Munkhjargal T, Abdel-Daim MM, et al. Phytochemical characterization and chemotherapeutic potential of Cinnamomum verum extracts on the multiplication of protozoan parasites in vitro and in vivo. Molecules. 2020a; 25(4): 996 10.3390/molecules25040996 - DOI - PMC - PubMed
    1. Beshbishy AM, Batiha GE-S, Alkazmi L, Nadwa E, Rashwan E, Abdeen A, et al. Therapeutic effects of atranorin towards the proliferation of Babesia and Theileria parasites. Pathogen. 2020; 9(2): E127 10.3390/pathogens - DOI - PMC - PubMed
    1. Rajapakshage BKW, Yamasaki M, Hwang SJ, Sasaki N, Murakami M, Tamura Y, et al. Involvement of mitochondrial genes of Babesia gibsoni in resistance to diminazene aceturate. J Vet Med Sci. 2012; 74: 1139–1148. 10.1292/jvms.12-0056 - DOI - PubMed
    1. Hines SA, Ramsay JD, Kappmeyer LS, Lau AO, Ojo KK, Van Voorhis WC, et al. Theileria equi isolates vary in susceptibility to imidocarb dipropionate but demonstrate uniform in vitro susceptibility to a bumped kinase inhibitor. Parasite Vectors. 2015; 8: 33 10.1186/s13071-014-0611-6 - DOI - PMC - PubMed
    1. Zintl A, Mulcahy G, Skerrett HE, Taylor SM, Gray JS. Babesia divergens, a bovine blood parasite of veterinary and zoonotic importance. Clin Microbiol Rev. 2003; 16:622–663. 10.1128/cmr.16.4.622-636.2003 - DOI - PMC - PubMed

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Grants and funding

This study was supported by the Ministry of Higher Education, Egypt, and the Japan Society for the Promotion of Science (JSPS) KAKEN, Grant Number 18 H02337 to Prof. Ikuo Igarashi. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.