Department of Infectious Disease and Global Health, Cummings School of Veterinary Medicine at Tufts University, North Grafton, MA, 01536, USA; Department of Parasitology, Bangladesh Agricultural University, Mymensingh, 2202, Bangladesh.
Department of Infectious Disease and Global Health, Cummings School of Veterinary Medicine at Tufts University, North Grafton, MA, 01536, USA; Department of Parasitology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt.
Exp Parasitol. 2020 May;212:107873. doi: 10.1016/j.exppara.2020.107873. Epub 2020 Mar 9.
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. IC 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 IC 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.
人参皂苷 Rh2 和葫芦素 B（CuB）分别为人参（Panax ginseng）和葫芦科植物的次生代谢产物。我们在隐孢子虫病的细胞培养模型中评估了这两种功能化合物的抗隐孢子虫活性。使用对宿主细胞无毒的每种化合物的最高浓度来评估其在感染/入侵和 HCT-8 细胞单层中生长期间对 C. parvum 的活性。单层被预先囊化的 C. parvum 卵囊感染。感染的单层在 37°C 下孵育 24 小时和 48 小时，同时存在不同浓度的每种测试化合物。通过在存在化合物的情况下孵育感染的单层 24 小时，然后在不存在化合物的情况下再孵育 24 小时来进行生长恢复测定。为了筛选入侵抑制活性，将新鲜囊化的 C. parvum 孢子前用不同浓度的化合物处理，然后将其添加到细胞单层中。作为阳性和阴性对照，分别使用已知的 C. parvum 抑制剂巴龙霉素和 DMSO。最初使用免疫荧光测定法评估感染水平，并通过实时 PCR 进行定量。两种化合物均以剂量依赖性方式强烈抑制 C. parvum 细胞内发育。在 24 小时发育期间，Rh2 的 IC 值为 25 μM，在 48 小时发育后为 5.52 μM，而对于 CuB，在相同的孵育期内，IC 值分别为 0.169μg/ml 和 0.118μg/ml。CuB 还有效地抑制了生长的恢复，而 Rh2 则没有观察到这种活性。CuB 在抑制脱囊和/或宿主细胞入侵方面更有效，表明该化合物还针对寄生虫的细胞外阶段。
