Identification and in vitro evaluation of lipids from sclerotia of lignosus rhinocerotis for antioxidant and anti-neuroinflammatory activities

Lignosus rhinocerotis (Cooke) Ryvarden (Tiger milk mushroom) is traditionally used to treat inflammation triggered symptoms and illnesses such as cough, fever and asthma. The present study evaluated the in vitro antioxidant, cytotoxic and anti-neuroinflammatory activities of the extract and fractions of sclerotia powder of L. rhinocerotis on brain microglial (BV2) cells. The ethyl acetate fraction had a total phenolic content of 0.30 ± 0.11 mg GAE/g. This fraction had ferric reducing capacity of 61.8 ± 1.8 mg FSE/g, ABTS+ scavenging activity of 36.8 ± 1.8 mg TE/g and DPPH free radical scavenging activity of 21.8% ± 0.7. At doses ranging from 0.1 μg/mL - 100 μg/mL, the extract and fractions were not cytotoxic to BV2 cells. At 100 μg/mL, the crude hydroethanolic extract and the ethyl acetate fraction elicited the highest nitric oxide reduction activities of 68.7% and 58.2%, respectively. Linoleic and oleic acids were the major lipid constituents in the ethyl acetate fraction based on FID and GC-MS analysis. Linoleic acid reduced nitric oxide production and down regulated the expression of neuroinflammatory iNOS and COX2 genes in BV2 cells

Anti-neuroinflammatory activities of lipid components from sclerotia of Lignosus rhinocerotis and stroma of Cordyceps militaris in microglia BV2 cells / Neeranjini S. Nallathamby

Inflammation in the brain, which is characterised by activation of microglia cells, plays an important role in the progression of many central nervous system (CNS) diseases. Many medicinal and edible mushrooms have been studied intensively for the treatment of neuroinflammatory conditions. In this study, an assay guided approach was adopted to identify the active ingredients of L. rhinocerotis and C. militaris responsible for its anti-neuroinflammatory activity. Preliminary in vitro antioxidant investigations of the extracts revealed that the ethanol extract of L. rhinocerotis had higher antioxidant capabilities compared to the aqueous extracts in all the three assays tested viz., DPPH, TEAC and FRAP; however, the aqueous extract of C. militaris exhibited higher antioxidant activities compared to the ethanol extract. The MTS assay revealed that both the (ethanolic and aqueous) extracts of the mushrooms did not have cytotoxic effects on the BV2 cells up to 100 μg/mL concentrations. The ethanol extracts of both mushrooms decreased NO production of LPS stimulated BV2 cells by more than 60% compared to their aqueous extracts at 100 μg/mL. Further fractionation of the ethanol extract to the hexane and ethyl acetate fractions showed that the ethyl acetate fractions of both mushrooms had a significant effect on NO production inhibition (>50%) compared to their hexane fractions at 10 μg/mL. Two bioactive subfractions, CE2 and CE3 from C. militaris and TE3 from L. rhinocerotis from the ethyl acetate fraction were obtained through vacuum liquid chromatography. These subfractions also showed more than 45% reduction in NO production at 100 μg/mL. Twenty two lipid components were identified by GCMS analysis whereby eight (8) components from TE3, eleven (11) components from CE2 and three (3) components from CE3. All these components had synergistic effect to reduce neuroinflammation. The qPCR results showed that all subfractions down regulated the proinflammatory genes (iNOS, COX2 and IL-1β) and only TE3 and CE2 up-regulated the HO-1 and NQO-1 anti-inflammatory genes. Subfractions TE3 and CE2 inhibited neuroinflammation via NRF2 and NFκB pathways while the CE3 subfraction inhibited neuroinflammation via NFκB pathway in LPS triggered BV2 cells

Synthesized 2-Trifluoromethylquinazolines and Quinazolinones Protect BV2 and N2a Cells against LPS- and H2O2-induced Cytotoxicity

BACKGROUND: Microglia are associated with neuroinflammation, which plays a key role in the pathogenesis of neurodegenerative diseases. It has been reported that some quinazolines and quinazolinones possess anti-inflammatory properties. However, the pharmacological properties of certain quinazoline derivatives are still unknown. OBJECTIVE: The antioxidant, cytotoxic, and protective effects of a series of synthesized 2-trifluoromethylquinazolines (2, 4, and 5) and quinazolinones (6-8) in lipopolysaccharide (LPS)-murine microglia (BV2) and hydrogen peroxide (H2O2)-mouse neuroblastoma-2a (N2a) cells were investigated. METHOD: The antioxidant activity of synthesized compounds was evaluated with ABTS and DPPH assays. The cytotoxic activities were determined by MTS assay in BV2 and N2a cells. The production of nitric oxide (NO) in LPS-induced BV2 microglia cells was quantified. RESULTS: The highest ABTS and DPPH scavenging activities were observed for compound 8 with 87.7% of ABTS scavenge percentage and 54.2% DPPH inhibition. All compounds were non-cytotoxic in BV2 and N2a cells at 5 and 50 μg/mL. The compounds which showed the highest protective effects in LPS-induced BV2 and H2O2-induced N2a cells were 5 and 7. All tested compounds, except 4, also reduced NO production at concentrations of 50 μg/mL. The quinazolinone series 6-8 exhibited the highest percentage of NO reduction, ranging from 38 to 60%. Compounds 5 and 8 possess balanced antioxidant and protective properties against LPS- and H2O2-induced cell death, thus showing great potential to be developed into anti-inflammatory and neuroprotective agents. CONCLUSION: Compounds 5 and 7 were able to protect the BV2 and N2a cells against LPS and H2O2 toxicity, respectively, at a low concentration (5 μg/mL). Compounds 6-8 showed potent reduction of NO production in BV2 cells

A Status Review of the Bioactive Activities of Tiger Milk Mushroom Lignosus rhinocerotis (Cooke) Ryvarden

Edible and medicinal mushrooms are regularly used in natural medicines and home remedies since antiquity for ailments like fever, inflammation, and respiratory disorders. Lignosus rhinocerotis (Cooke) Ryvarden is a polypore found in Malaysia and other regions in South East Asia. It can be located on a spot where a tigress drips milk while feeding, hence the name “tiger's milk mushroom.” The sclerotium of L. rhinocerotis is highly sought after by the native communities in Malaysia to stave off hunger, relieve cough and asthma, and provide stamina. The genomic features of L. rhinocerotis have been described. The pharmacological and toxicity effects, if any, of L. rhinocerotis sclerotium have been scientifically verified in recent years. In this review, the validated investigations including the cognitive function, neuroprotection, immune modulation, anti-asthmatic, anti-coagulation, anti-inflammatory, anti-microbial/ anti-viral, anti-obesity, anti-cancer/ anti-tumor, and antioxidant properties are highlighted. These findings suggest that L. rhinocerotis can be considered as an alternative and natural medicine in the management of non-communicable diseases. However, there is a paucity of validation studies including human clinical trials of the mycochemicals of L. rhinocerotis