4.4.3. In Vitro IC50 Determination

Each compound was dissolved in dimethyl sulfoxide and adjusted to 150, 75, 37.5, 18.75, and 9.375 μg/mL by adding a suspension of *E. histolytica* trophozoites at a logarithmic phase in PEHPS medium with 10% bovine serum. Vials were incubated for 72 h, and then chilled in cold water for 20 min, and the number of dead trophozoites per milliliter was evaluated using a hemocytometer. All assays were performed in triplicate. Metronidazole was used as a positive control, and as a negative control, an *E. histolytica* suspension in PEHPS medium with no extract added was used. The percentage of inhibition was estimated as the number of dead trophozoites compared to the negative controls. The 50% inhibitory concentration of each compound was calculated by using a Probit analysis, considering a 95% confidence level.

#### **5. Conclusions**

The isolated and pure flavonoids from *L. graveolens* showed significant growth inhibition against *E. histolytica* (52% to 97% at a concentration of 150 μg/mL). The IC50 values of these compounds ranged from 28.86 to 154.26 μg/mL, so they were not as effective as metronidazole (IC50 0.205 μg/mL), but these IC50 values can be used as a guideline for further research on this plant as a source of potential antiamoebic agents.

The main contribution of this research work lies in the fact that it has shown that the presence of the flavonoids described herein in *Lippia graveolens* has a direct relationship with the antiprotozoal activity of extracts of this plant against *Entamoeba histolytica*. These flavonoids could be used as biomarkers [78] for the quality control of phytotherapeutics developed based on this work.

The results of our research may also form the basis for directly incorporating the use of *Lippia graveolens* extracts into conventional and complementary medicine for the treatment of amebiasis, as well as other infectious diseases.

**Supplementary Materials:** The following are available online, Figures S1–S30: supplementary spectroscopic data.

**Author Contributions:** Conceptualization, R.Q.-L.; investigation, R.Q.-L., J.V.-V., M.J.V.-S., and V.M.R.-G.; writing—review and editing, R.Q.-L. and Á.D.T.-H.; R.Q.-L. conceived and designed the experiments; R.Q.-L. and M.J.V.-S. performed the extraction and isolation; V.M.R.-G. performed the spectral analysis and structure determination; J.V.-V. conceived, designed, and performed the antiprotozoal assay; R.Q.-L. wrote the paper. All authors read and approved the final manuscript.

**Funding:** We thank the Universidad Autónoma de Nuevo León (Mexico) for PAICYT grants CN-422-10, and CN-662-11.

**Acknowledgments:** For the spectroscopic measurements, we thank Noemi Waksman-Minsky of the College of Medicine from Universidad Autónoma de Nuevo León.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


**Sample Availability:** Not available.

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