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

## TL;DR

This study shows that hops essential oil and myrcene can reduce Cryptosporidium parvum infection in intestinal cells, suggesting they may be useful as new treatments for cryptosporidiosis.

## Contribution

The novel contribution is the discovery of hops essential oil and myrcene's anti-Cryptosporidium activity in cell culture.

## Key findings

- Hops essential oil reduced Cryptosporidium parvum growth with IC50 values of 45.8 and 58.7 µg/mL.
- Myrcene showed stronger anti-Cryptosporidium activity with IC50 values of 17.7 and 28.1 µg/mL.
- Both compounds exhibited low cytotoxicity in intestinal cells.

## Abstract

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 Cryptosporidium parvum 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-Cryptosporidium activity of HEO and myrcene was assessed by infecting confluent HCT-8 cells with C. parvum 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-Cryptosporidium antibody. HEO exhibited low cytotoxicity (IC50 = 382.7 µg/mL), while myrcene showed higher cytotoxicity (IC50 = 240.6 µg/mL). HEO reduced C. parvum 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-Cryptosporidium 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-Cryptosporidium activity, highlighting their potential as novel therapeutic agents against cryptosporidiosis.