Mycochemical characterization and bioactivity screening of microporus affinis Blume and T Nees Kunt and M xanthopus Fr Kunt

Abstract

Bracket fungi are valuable in research for their potential to develop newpharmaceuticals. In this study, the Polyporaceae family of Basidiomycetes wasexplored for its bioactivities. Microporus, a less studied genus in Polyporaceae, hasyet to be examined in detail for medicinal impacts. This research focuses on M.xanthopus (Fr.) Kunt. and M. affinis (Blume & T. Nees) Kunt., distinguished by thelateral stipe in M. affinis and the central stipe in M. xanthopus.Two species of Microporus were screened for chemical constituents.Chemical characterization was done in four sections: preliminary qualitativemycochemical (fungal chemical) screening, quantitative estimation (measuring theamount of specific chemicals), determination of volatile chemical constituentsthrough GC/MS analysis (Gas Chromatography/Mass Spectrometry), andidentification of non-volatile chemicals through HR-LC/MS analysis (HighResolution Liquid Chromatography/Mass Spectrometry). Terpenoids, steroids, andalkaloids were observed in both species in preliminary studies. Glycosides andsaponins were reported only in M. xanthopus. Alkaloids, terpenoids, carbohydrates,and proteins were determined quantitatively. GC/MS analysis of M. xanthopusrevealed 45 compounds, while M. affinis had 46. In both species, fatty acids were theleading class, and linoleic acid had the highest peak area. HR-LC/MS in M. xanthopusreported 31 compounds, while M. affinis had about 9 mycoconstituents. Differenttypes of fungal sterols (a group of steroids) with a wide spectrum of bioactivities havealso been reported.Because of its diverse chemical constituents, Microporus was studied forbioactivities such as antioxidant (combating cell damage from oxidants), cytotoxic(toxic to cells), antiproliferative (preventing cell growth), and neuroprotective(protecting nerve cells) effects. Special emphasis was given to extendedneuroprotective studies of M. xanthopus, confirmed by techniques like ROS (ReactiveOxygen Species) measurement using DCFDA (a fluorescent dye), Annexin V-FITCapoptosis (detection of programmed cell death using a specific marker) using flowcytometry (cell analysis technique), autophagy (cellular self-digestion), indirectELISA (a lab method to detect proteins), and gene expression studies using qRT-PCR(quantitative Reverse Transcription Polymerase Chain Reaction).The fungal extracts were subjected to free radical scavenging assays, likeDPPH, hydroxyl radicals, nitric oxide, and reducing power. In this scenario, M. affiniswas found to be more potent than M. xanthopus even though both species are havingantioxidant potential. Oxidative damage has a key role in the aging process and islinked to diseases like Cancer, Alzheimer's, and Parkinson's. Free radical scavengerscan help to mitigate these effects. So, the study was carried out on cytotoxic,antiproliferative, and neuroprotective areas. Cytotoxic efficiency was tested in Alliumcepa, together with the degree of mitotic index and percentage of chromosomalaberrations. Fungal extract was tested in vitro for antiproliferation on the DLD1 cellline (colorectal cancer) using the MTT assay. Comparatively, the lower LC50 value ofM. affinis indicates its higher cytotoxic potential towards the DLD1 cell line than thatof M. xanthopus. It was compared with the normal cell line L929. The degree ofapoptosis was confirmed by the AO/EtBr double staining method. The presence ofterpenoids in Microporus supports neuroprotective activities. Hence, the fungalextracts were subjected to the in vitro study of neuroprotective efficacy on IMR-32(human neuroblastoma cell line). Cell viability was assessed by using the MTT assay.Neurodegeneration was induced by using β-amyloid. Apoptosis determinationusing the AO/EtBr method clearly revealed the neuroprotective efficacy of M.xanthopus. A dose dependent increase in cell viability was noticed in M. xanthopusonly. So, further validations on neuroprotection were carried out in M. xanthopusonly. From the study conducted, it was clear that the increased ROS level could becontrolled by using fungal extract on amyloid treated cells. It indirectly supports therecovery of cells with neurodegeneration. In indirect ELISA, the primary antibodies,ChAt, Glutamate Receptor 2, Tau, and alpha synuclein were used. HRP conjugate wasused as the secondary antibody. In a study of Annexin V- FITC, it was clear that thesample treatment was able to reduce the late apoptotic cells, which indicates itspotential role in neuroprotection. The effect of the parkin gene was also comparedwith the housekeeping gene actin. The present study proved that the increased cellviability, decreased apoptosis, and lower ROS levels are good for neuroprotection.Even though it is positively supporting neuroprotection, in vivo studies are alsorequired to substantiate the neuroprotective efficacy of this fungal material.