Biological activity of macrofungi in South Africa against respiratory and lung disease

Macrofungi represent an untapped source of natural bioactive compounds for various diseases, which have been targeted as potential therapeutic agents. The medicinal uses of macrofungi discovered to date include anticancer, antidiabetic, antioxidant, antimicrobial, and immunomodulatory properties. The knowledge regarding the medicinal uses of macrofungi in Africa is limited; however, it is believed that Africa may contain a large number of unidentified species of macrofungi. The objectives of this study were to: (i) screen the macrofungal extracts for antimicrobial activity against microorganisms responsible for respiratory diseases (e.g. Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Mycobacterium tuberculosis, HIV-1), (ii) determine the effect of macrofungal extracts on bacterial morphology, (iii) investigate the cytotoxicity of macrofungal extracts against human lung carcinoma cells, and to elucidate the mechanism/s of action of cytotoxicity/anticancer activity. In vitro bioassays for antimicrobial activity included: ρ-iodonitrotetrazolium chloride assays and microplate alamar blue assay (MABA), while the HIV-1 reverse transcriptase colorimetric ELISA and HIV-1 protease fluorometric assay kits were used for anti-HIV activity. Cytotoxicity of the macrofungal species against A549 lung cancer cell line was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and the IC50 values determined for the most cytotoxic macrofungal spp. The mechanism of cytotoxicity was investigated by cell cycle analysis and fluorescent staining to observe morphological and biochemical changes (i.e. acridine orange, caspase activation). Ethanol extracts of Amanita foetidissima, Gymnopilus junonius, Pisolithus tinctorius, Fomitopsis lilacinogilva, Stereum hirsutum and Pycnoporus sanguineus showed higher antimicrobial activity against the Gram-positive bacteria than aqueous extracts, with S. pneumoniae being the most susceptible. The ethanol extracts of Agaricus campestris, Chlorophyllum molybdites, Gymnopilus penetrans, Pseudophaeolus baudonii and Laetiporus sulphureus exhibited anti-TB (tuberculosis) activity with minimum inhibitory concentrations (MICs) ranging between 500-1,000 μg/mL. C. molybdites ethanol extract inhibited HIV-1 protease activity (IC50: 49.7 μg/mL). The macrofungal extracts did not inhibit HIV-1 reverse transcriptase activity. Ethanol extracts of F. lilacinogilva, G. junonius, P. sanguineus and the aqueous extract iv of P. baudonii were cytotoxic against A549 lung cancer cells at IC50 values of 69.2±3.6, 57±5, 7.4±1.1 and 53.6±1.1 μg/mL, respectively. Cell cycle arrest was observed in the G2 phase for both P. sanguineus and P. baudonii, and G2/M and early M phases for G. junonius and F. lilacinogilva, respectively. Apoptosis induced by macrofungal extracts was confirmed by fluorescent staining. Morphological and biochemical changes included chromatin condensation, membrane blebbing, loss of cytoskeletal structure, caspase activation and phosphatidylserine translocation. This study demonstrates the biological activities of selected macrofungal extracts and their potential mechanisms of action. Isolation and identification of active compounds require further analysis