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Sanah Malomile Nkadimeng,1 Christiaan ML Steinmann,2 Jacobus N Eloff1 1Phytomedicine Programme, Paraclinical Sciences Department, University of Pretoria, Onderstepoort, Pretoria, Gauteng, 0110, South Africa; 2Physiology Department, Sefako Makgatho Health Sciences University, Ga-Rankuwa, Gauteng, 0208, South AfricaCorrespondence: Sanah Malomile NkadimengPhytomedicine Programme, Paraclinical Sciences Department, University of Pretoria, P/Bag X04, Onderstepoort, Pretoria, Gauteng, 0110, South AfricaTel +27 12 529 8187Email sanah.nkadimeng@up.ac.zaPurpose: During a pathological inflammation, macrophages are activated to produce accumulation of inflammatory mediators such as induced-cyclooxygenase-2 (COX-2), 15-lipoxygenase (15-LOX) and pro-inflammatory cytokines. Pathological inflammation is a significant problem in many chronic diseases. As a result, more research into natural remedies with anti-inflammatory potential is crucial. Since ancient times, psilocybin-containing mushrooms, also known as magic mushrooms, were used for mind healing and also to advance the quality of life. However, not much is known about their anti-inflammatory potential. This study aimed at investigating the anti-inflammatory effects of four psilocybin-containing mushrooms (Panaeolus cyanescens, Psilocybe natalensis, Psilocybe cubensis and Psilocybe cubensis leucistic A+ strain) from genus Panaeolus and Psilocybe for the first time in vitro on 15-LOX activity and also on lipopolysaccharide (LPS)-induced inflammation in human U937 macrophage cells.Methods: Mushrooms were grown and extracted with boiling hot water. Effects of the four water extracts on 15-LOX activity were determined. Confluent human U937 cells were differentiated with phorbol 12-myristate 13-acetate and treated with the hot-water extracts (25 and 50 μg/mL) 2 hours before being stimulated with 1 μg/mL LPS over 24 hours. Quercetin was used as a positive control. Control cells were differentiated but not LPS-induced nor treated. Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10 concentrations were measured. Levels of COX-2 and mitochondrial activity were also determined.Results: The four water extracts had poor 15-LOX inhibition activity with IC50 > 250 μg/mL. Extracts were safe at the concentration studied and inhibited the LPS-induced production of pro-inflammatory mediators, TNF-α and IL-1β significantly and lowered IL-6 and COX-2 concentrations in treated human U937 macrophage cells. Water extracts also increased percentage viability of treated cells and levels of anti-inflammatory IL-10 non-significantly.Conclusion: The study suggested that the hot-water extracts of the four psilocybin-containing magic mushrooms have potential anti-inflammatory effects executed by down-regulating pro-inflammatory mediators.Keywords: Panaeolus cyanescens, Psilocybe natalensis, Psilocybe cubensis, Psilocybe cubensis A+ strain, pro-inflammatory cytokine, COX-2, 15-LOX