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Effect of Ginsenoside-Rh2 and Curcurbitacin-B on Cryptosporidium parvum in vitro

Shahiduzzaman, Md, Ras, Refaat, Widmer, Giovanni
Experimental parasitology 2020 v.212 pp. 107873
Cryptosporidium parvum, Cucurbitaceae, Panax ginseng, active ingredients, cell culture, cryptosporidiosis, dimethyl sulfoxide, dose response, excystation, fluorescent antibody technique, host cell invasion, inhibitory concentration 50, oocysts, parasites, quantitative polymerase chain reaction, secondary metabolites, sporozoites, toxicity
Ginsenoside-Rh2 and cucurbitacin-B (CuB) are secondary metabolites of Ginseng (Panax ginseng) and Cucurbitaceae plants respectively. We assessed the anticryptosporidial activity of these two functional compounds in a cell culture model of cryptosporidiosis. The highest concentration of each compound that was not toxic to the host cells was used to assess the activity against C. parvum during infection/invasion and growth in HCT-8 cell monolayers. Monolayers were infected with pre-excysted C. parvum oocysts. Infected monolayers were incubated at 37 °C for 24 h and 48 h in the presence of different concentrations of each test compound. A growth resumption assay was performed by incubating infected monolayers in the presence of compounds for 24 h followed by a second 24-h incubation in the absence of compound. To screen for invasion inhibiting activity, freshly excysted C. parvum sporozoites were pre-treated with different concentrations of compounds prior to adding them to the cell monolayers. Paromomycin, a known inhibitor of C. parvum, and DMSO were used as positive and negative control, respectively. The level of infection was initially assessed using an immunofluorescent assay and quantified by real-time PCR. Both compounds were found to strongly inhibit C. parvum intracellular development in a dose-dependent manner. IC₅₀ values of 25 μM for a 24 h development period and 5.52 μM after 48 h development were measured for Rh2, whereas for CuB an IC₅₀ value of 0.169 μg/ml and 0.118 μg/ml were obtained for the same incubation periods. CuB also effectively inhibited resumption of growth, an activity that was not observed with Rh2. CuB was more effective at inhibiting excystation and/or host cell invasion, indicating that this compound also targets extracellular stages of the parasite.