Anti-Protozoal Activity of Hops Essential Oil and Myrcene Against Cryptosporidium Parvum in Cell Culture

Hops essential oil (HEO), a by-product of the brewing industry, has known antibacterial and antifungal properties, but its antiparasitic effects remain underexplored. This study evaluated the cytotoxicity of HEO and its predominant monoterpene, myrcene, in intestinal cells and assessed their ability to reduce <i>Cryptosporidium parvum</i> infection in vitro. The cytotoxicity (IC₅₀) of HEO and myrcene was determined in HCT-8 intestinal cells using flow cytometry and propidium iodide staining after 24 and 48 h of exposure. The anti-<i>Cryptosporidium</i> activity of HEO and myrcene was assessed by infecting confluent HCT-8 cells with <i>C. parvum</i> sporozoites (1 × 10⁴ sporozoites/mL) and treating them with bioactives below their IC₅₀ values. Two treatment modalities were tested: (1) immediate treatment during infection (invasion) and (2) treatment initiated 2 h after infection (growth). Parasite growth was quantified using an immunofluorescence assay with a fluorescence-conjugated anti-<i>Cryptosporidium</i> antibody. HEO exhibited low cytotoxicity (IC₅₀ = 382.7 µg/mL), while myrcene showed higher cytotoxicity (IC₅₀ = 240.6 µg/mL). HEO reduced <i>C. parvum</i> growth in a dose-dependent manner, with IC₅₀ values of 45.8 and 58.7 µg/mL under either modality, respectively. Myrcene alone demonstrated greater anti-<i>Cryptosporidium</i> activity, with IC₅₀ values lower under the invasion modality (17.7 µg/mL) than the growth modality (28.1 µg/mL) on average for both food-grade and analytical standards. HEO and myrcene exhibited significant in vitro anti-<i>Cryptosporidium</i> activity, highlighting their potential as novel therapeutic agents against cryptosporidiosis.