Gas chromatography-mass spectrometry analysis, phytochemical screening and antiprotozoal effects of the methanolic Viola tricolor and acetonic Laurus nobilis extracts

Abstract

Background: The antiprotozoal and antioxidant activities of Viola tricolor and Laurus nobilis have been reported recently. Thus, the existing study pursued to assess the growth inhibition effect of methanolic extract of V. tricolor (MEVT) and acetonic extract of L. nobilis (AELN) against five Babesia parasites and Theileria equi in vitro and in vivo.

Results: MEVT and AELN suppressed Babesia bovis, B. bigemina, B. divergens, B. caballi, and T. equi growth at half-maximal inhibitory concentration (IC₅₀) values of 75.7 ± 2.6, 43.3 ± 1.8, 67.6 ± 2.8, 48 ± 3.8, 54 ± 2.1 μg/mL, and 86.6 ± 8.2, 33.3 ± 5.1, 62.2 ± 3.3, 34.5 ± 7.5 and 82.2 ± 9.3 μg/mL, respectively. Qualitative phytochemical estimation revealed that both extracts containing multiple bioactive constituents and significant amounts of flavonoids and phenols. The toxicity assay revealed that MEVT and AELN affected the mouse embryonic fibroblast (NIH/3 T3) and Madin-Darby bovine kidney (MDBK) cell viability with half-maximum effective concentrations (EC₅₀) of 930 ± 29.9, 1260 ± 18.9 μg/mL, and 573.7 ± 12.4, 831 ± 19.9 μg/mL, respectively, while human foreskin fibroblasts (HFF) cell viability was not influenced even at 1500 μg/mL. The in vivo experiment revealed that the oral administration of MEVT and AELN prohibited B. microti multiplication in mice by 35.1 and 56.1%, respectively.

Conclusions: These analyses indicate the prospects of MEVT and AELN as good candidates for isolating new anti-protozoal compounds which could assist in the development of new drug molecules with new drug targets.

Keywords: Drug discovery; Medicinal biochemistry; Natural products; Parasitic diseases.

Fig. 1

The relationship between the relative fluorescence units (RFUs) and the log concentrations of methanolic V. tricolor (MEVT) (μg/mL) on T. equi, B. divergens, B. bigemina, B. caballi, and B. bovis. The non-linear regression (curve fit analysis) in GraphPad Prism software (GraphPad Software Inc. USA) used for IC₅₀’s calculation. The percentage of parasite growth inhibitory efficacy is calculated as the percentage of parasites inhibited divided by that of the positive control wells and the result was subtracted from the negative control wells

Fig. 2

The relationship between the relative fluorescence units (RFUs) and the log concentrations of acetonic L. nobilis (AELN) (μg/mL) on T. equi, B. divergens, B. bigemina, B. caballi, and B. bovis. The non-linear regression (curve fit analysis) in GraphPad Prism software (GraphPad Software Inc. USA) used for IC₅₀’s calculation. The percentage of parasite growth inhibitory efficacy is calculated as the percentage of parasites inhibited divided by that of the positive control wells and the result was subtracted from the negative control wells

Fig. 3

The growth of B. bovis and T. equi on RBCs pretreated with methanolic V. tricolor and acetonic L. nobilis in vitro. The curves showing the growth of B. bovis (a) and T. equi (b) on RBCs pretreated with 400 μg/mL of MEVT and AELN. The result was determined by counting using a microscope. The values obtained from three separate trials were used to determine the significance

Fig. 4

Gas chromatography-mass spectrometry analysis in the methanolic extract of Viola tricolor (a) and acetonic extract of Laurus nobilis (b)

Fig. 5

The growth inhibition of methanolic V. tricolor and acetonic L. nobilis on B. microti in vivo. Inhibitory effect of MEVT and AELN on the growth of B. microti in mice, based on observations taken from five mice per experimental group. The arrow indicates 5 consecutive days of treatment. Asterisks indicate statistically significant (p < 0.05) differences of parasitemia between treated groups and the untreated control group based on one-way ANOVA Tukey’s test using GraphPad Prism version 5.0 for Windows (GraphPad Software Inc., San Diego, CA, USA). Parasitemia was calculated by counting iRBCs among 2000 RBCs using Giemsa-stained thin blood smears. The data were the mean and standard deviation from two separate experiments

Fig. 6

The hematology parameters changes in methanolic V. tricolor (MEVT) and acetonic L. nobilis (AELN)-treated mice in vivo. Graphs showing the changes in hematocrit count (a), hemoglobin percentage (b), and red blood cell count (c) in mice treated with DMA and MEVT and AELN. The arrow indicates 5 consecutive days of treatment. Asterisks indicate statistical significance (p < 0.05) based on one-way ANOVA Tukey’s test using GraphPad Prism version 5.0 for Windows (GraphPad Software Inc., San Diego, CA, USA). The data were the mean and standard deviation from two separate experiments (five mice per group)