Activities of Mushroom and Herbal Tea Extracts Against Toxoplasma gondii and Their Mechanisms of Actions

Date of Award

Spring 2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Daniel A. Abugri

Second Advisor

Emmanuel Tadjuidje

Third Advisor

Muhammad Saleem

Abstract

Toxoplasma gondii is an obligate parasite that can infect diverse arrays of cold-blooded and warm-blooded vertebrate animals. Approximately 30–50% of the global population is supposed to be affected with T. gondii with the most susceptible group being people living with immunocompromised statuses such as individuals with HIV-AIDS or those accepting organ transplant, cancer therapy, blood transfusion or plasmapheresis in addition to pregnant women, fetuses, and infants. Toxoplasmosis, the infection caused by T. gondii , presents considerable challenges in diverse fields worldwide. Notably affected among those fields are medical, veterinary, food safety, public health, socio-economic and bioterrorism ones. No vaccine is available to control T. gondii and treatment regimens are either rife with toxicity (side effects) and ineffectiveness to control both forms: tachyzoites and bradyzoites of parasites or some do not apply to all groups of people infected. In this context, alternative nutraceuticals or drugs are urgently required to be developed for toxoplasmosis therapy. Centered on some verified shreds of evidence for antimicrobial activities shown by natural compounds, developing novel, safe, and effective drugs targeted against T. gondii by using mushroom or plant-derived bioactive compounds can be a favorable option for study. We conducted research to assess the effect of extract of higher fungi on T. gondii tachyzoites growth in vitro and possibly decipher its mechanism of action. Furthermore, we evaluated the extract's effect on human foreskin fibroblast viability. The methanol extract of Turkey tail (TT) mushroom was tested against T. gondii tachyzoites growth using an RH-RFP type I strain that expresses red fluorescent protein throughout culture in a dose-dependent manner using a fluorescent plate reader. Similarly, we tested the effect of the extract on host cell viability. In the research, we observed that TT extract inhibited tachyzoites growth with a 50% minimum inhibitory concentration (IC 50s ), IC 50 = 5.98 ± 1.22 µg/mL, and 50% cytotoxic concentration (CC 50s ), CC 50 = >100 µg/mL. It was discovered that TT extract induced strong mitochondria superoxide production (MitoSox) and disrupted mitochondria membrane potential (MMP) in T. gondii tachyzoites. Additionally, scanning electron microscopy (SEM) depicted that TT extract and pyrimethamine (PY) caused a morphological deformation of tachyzoites in vitro . In conclusion, TT methanol extract made up of phytosterols, bioactive sphingolipids, peptides, phenolic acids, and lactones could be a promising source of new compounds for the future development of anti- Toxoplasma gondii drugs. Extracts were non-cytotoxic, even at higher concentrations. In the second study, we report for the first time the anti- T. gondii activity of methanolic extract of Nature Herb for Chinese Soup Base (SB) tea in vitro . The mean 50% cytotoxic concentration (CC 50 ) value for hTERT cells was determined to be 65.43 μg/mL. Based on these results, we further investigated the effect of the methanolic SB extract against intracellular T. gondii growth, which gave a 50% inhibitory concentration (IC 50 ) value of 0.57 and 0.45 μg/mL for 48- and 72-hours exposure, respectively. The selectivity index (SI) value for SB extract was calculated to be 145. Also, Pyrimethamine (PY), Sulfadiazine (SZ) and PY/SZ combination (2:1) ratio were used as positive controls, which gave mean IC 50 values of 0.88 and 0.6010.24 μg/mL for PY, 0.39 and 0.24 μg/mL for SZ, and 0.077 and 0.036 μg/mL for PY/SZ at 48- and 72-hours interactions, respectively. Interestingly, we observed that the methanolic SB extract induced mitochondrial superoxide (MitoSOX) production, disrupted mitochondrial membrane potential (MMP), and impeded parasite invasion of host cells. Confirming our biochemical findings, we performed a scanning electron microscopy (SEM) data showed that methanolic SB extract caused intracellular T. gondii tachyzoite membrane disruption at 72 hours treatment. LC-MS/GC-MS chemical fingerprinting showed that the methanolic SB extract contained lipids/fatty acids, phenolics, terpenes, alkaloids, amino acids and carbohydrates. Together, our unique findings showed that SB extract inhibited intracellular parasite growth and contains potent compounds that could be investigated in future studies to assess their efficacy on in vivo parasite clearance and host safety.

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