Phytochemical Screening and Antiprotozoal Effects of the Methanolic Berberis vulgaris and Acetonic Rhus coriaria Extracts

Abstract

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.

Keywords: Babesia; Berberis vulgaris; Rhus coriaria; Theileria; clinical studies; drug candidates; pharmacological activities.

Figure 1

Dose-response curves of MEBV against Babesia and Theileria parasites in vitro. The curves showing the correlation between the percentage of inhibition and the concentration of MEBV (µg/mL) on B. bovis, B. bigemina, B. divergens, B. caballi and T. equi. The values obtained from three separate trials were used to determine the IC₅₀’s using the non-linear regression (curve fit analysis) in GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA).

Figure 2

Dose-response curves of AERC against Babesia and Theileria parasites in vitro. The curves showing the correlation between the percentage of inhibition and the concentration of AERC (µg/mL) on B. bovis, B. bigemina, B. divergens, B. caballi and T. equi. The values obtained from three separate trials were used to determine the IC₅₀’s using the non-linear regression (curve fit analysis) in GraphPad Prism software.

Figure 3

Morphological changes and light micrographs captured for MEBV- and AERC-treated T. equi in an in vitro culture taken after 24 (a) and 72 h (b). The arrows show the abnormal dividing parasites at 24 h. While at 72 h, drug-treated cultures showed higher numbers of degenerated parasites than did the control cultures.

Figure 4

Growth inhibition effect of MEBV and AERC treatment against B. microti in vivo. The arrow indicates 5 consecutive days of treatment start from day 4 to 8 p.i. Asterisks indicate statistically significant (p < 0.05) differences of parasitemia between treated groups and the untreated group based on unpaired t-test analysis. Parasitemia was detected using Giemsa-stained thin blood smears by counting infected RBCs (iRBCs) among 2000 RBCs.

Figure 5

Hematology parameters changes in MEBV- and AERC-treated mice in vivo. Graphs showing the changes in RBCs count (A), HCT percentage (B), and HGB concentration (C) in mice treated with DMA and MEBV and AERC. Asterisks indicate statistical significance (p < 0.05) based on unpaired t-test analysis.

Figure 5

Hematology parameters changes in MEBV- and AERC-treated mice in vivo. Graphs showing the changes in RBCs count (A), HCT percentage (B), and HGB concentration (C) in mice treated with DMA and MEBV and AERC. Asterisks indicate statistical significance (p < 0.05) based on unpaired t-test analysis.