Untargeted metabolomics to evaluate antifungal mechanism: a study of Cophinforma mamane and Candida albicans interaction.

Microbial interactions between filamentous fungi and yeast are still not fully understood. To evaluate a potential antifungal activity of a filamentous fungus while highlighting metabolomic changes, co-cultures between an endophytic strain of Cophinforma mamane (CM) and Candida albicans (CA) were performed. The liquid cultures were incubated under static conditions and metabolite alterations during the course were investigated by ultra-performance liquid chromatography-tandem mass spectrophotometry (UPLC-MS/MS). Results were analyzed using MS-DIAL, MS-FINDER, METLIN, Xcalibur, SciFinder, and MetaboAnalyst metabolomics platforms. The metabolites associated with catabolic processes, including the metabolism of branched-chain amino acids, carnitine, and phospholipids were upregulated both in the mono and co-cultures, indicating fungal adaptability to environmental stress. Several metabolites, including C20 sphinganine 1-phosphate, myo-inositol, farnesol, gamma-undecalactone, folinic acid, palmitoleic acid, and MG (12:/0:0/0:0) were not produced by CA during co-culture with CM, demonstrating the antifungal mechanism of CM. Our results highlight the crucial roles of metabolomics studies to provide essential information regarding the antifungal mechanism of C. mamane against C. albicans, especially when the lost/undetected metabolites are involved in fungal survival and pathogenicity.

Anti-rheumatoid activity of a hexane-insoluble fraction from Plantago major in female Wistar rats induced by Complete Freund's Adjuvant.

Background and aim: Plantago major has long been used for medical purposes in Indonesia. However, reports on the anti-arthritic activities of P. major are limited. Experimental procedure: The anti-arthritic properties of an n-hexane-insoluble fraction of dichloromethane extracts of P. major (IPM) were evaluated using Complete Freund's Adjuvant (CFA)-induced arthritis induced in female Wistar rat by CFA. Diclofenac was used as a positive control. The volume of paw oedema, white blood cell count, lymphocytes, neutrophils, expression of TNF-α and Interleukin-6 and the histopathological features of the joint tissues were assessed to characterise IPM activity. Results: The IPM extract at doses of 280 and 420 mg/kg BW and diclofenac inhibited paw oedema by 15.70 %, 15.94 % and 19.71 % respectively. IPM also reduced the incidence of arthritis and arthritic index. Unlike untreated rats, animals treated with IPM showed a significant decrease in the number of neutrophils and decreased expression of TNF-α and Interleukin-6. Histopathological examination showed a reduction in the number of inflammatory cells and hyperplasia of the synovium after IPM treatment. Conclusion: This study showed that P. major displays anti-rheumatoid arthritis activity.

In vivo anti-inflammatory activities of Plantago major extract and fractions and analysis of their phytochemical components using a high-resolution mass spectrometry.

Background and purpose: Plantago major has been applied as a herbal remedy for centuries. However, studies on anti-inflammatory activities and their chemical ingredients are limited. The objective of this study was to investigate the anti-inflammatory properties of P. major in three animal models and its phytochemical contents. Experimental approach: Dichloromethane extract (DCM) of P. major was fractionated with n-hexane to yield the soluble (SHF) and insoluble (IHF) fractions. The anti-inflammatory activities of DCM, SHF, and IHF were evaluated using rat's paw edema induced by carrageenan, thioglycolate-induced leukocyte emigration in the mice, and rheumatoid arthritis (RA) induced by complete Freund's adjuvants in rats. The chemical constituents were analyzed using a high-resolution mass spectrometer (HRMS). Findings / Results: The DCM, SHF, and IHF inhibited paw edema in the rats and reduced the leukocyte migration in the mice. At dose 560 mg/kg, the percentage of inhibitory was 47.33%, 55.51%, and 46.61% for the DCM, IHF, and SHF, respectively. In the RA animal model, IHF at 280 and 560 mg/kg reduced osteoclast formation and COX-2 expression compared to diclofenac. Some compounds namely oleic acid, linoleic acid, palmitic acid, and oleamide identified in the DCM, IHF, and SHF may be responsible for these activities. Conclusion and implications: This study showed that P. major has several in-vivo anti-inflammatory activities.

Pharmacological validation of Solanum mammosum L. as an anti-infective agent: Role of solamargine.

ETHNOPHARMACOLOGICAL RELEVANCE: Fungal and bacterial infections remain a major problem worldwide, requiring the development of effective therapeutic strategies. Solanum mammosum L. (Solanaceae) ("teta de vaca") is used in traditional medicine in Peru to treat fungal infections and respiratory disorders via topical application. However, the mechanism of action remains unknown, particularly in light of its chemical composition. MATERIALS AND METHODS: The antifungal activity of TDV was determined against Trichophyton mentagrophytes and Candida albicans using bioautography-TLC-HRMS to rapidly identify the active compounds. Then, the minimum inhibitory concentration (MIC) of the fruit crude extract and the active compound was determined to precisely evaluate the antifungal activity. Additionally, the effects of the most active compound on the formation of Pseudomonas aeruginosa biofilms and pyocyanin production were evaluated. Finally, a LC-HRMS profile and a molecular network of TDV extract were created to characterize the metabolites in the fruits' ethanolic extract. RESULTS: Bioautography-TLC-HRMS followed by isolation and confirmation of the structure of the active compound by 1D and 2D NMR allowed the identification solamargine as the main compound responsible for the anti-Trichophyton mentagrophytes (MIC = 64 µg mL-1) and anti-Candida albicans (MIC = 64 µg mL-1) activities. In addition, solamargine led to a significant reduction of about 20% of the Pseudomonas aeruginosa biofilm formation. This effect was observed at a very low concentration (1.6 µg mL-1) and remained fairly consistent regardless of the concentration. In addition, solamargine reduced pyocyanin production by about 20% at concentrations of 12.5 and 50 µg mL-1. Furthermore, the LC-HRMS profiling of TDV allowed us to annotate seven known compounds that were analyzed through a molecular network. CONCLUSIONS: Solamargine has been shown to be the most active compound against T. mentoagrophytes and C. albicans in vitro. In addition, our data show that this compound affects significantly P. aeruginosa pyocyanin production and biofilm formation in our conditions. Altogether, these results might explain the traditional use of S. mammosum fruits to treat a variety of fungal infections and respiratory disorders.

Dynamics of Chemical Diversity during Co-Cultures: An Integrative Time-Scale Metabolomics Study of Fungal Endophytes Cophinforma mamane and Fusarium solani.

A rapid and efficient metabolomic study of Cophinforma mamane and Fusarium solani co-cultivation in time-series based analysis was developed to study metabolome variations during their fungal interactions. The fungal metabolomes were studied through the integration of four metabolomic tools: MS-DIAL, a chromatographic deconvolution of liquid-chromatography-mass spectrometry (LC/MS); MS-FINDER, a structure-elucidation program with a wide range metabolome database; GNPS, an effective method to organize MS/MS fragmentation spectra, and MetaboAnalyst, a comprehensive web application for metabolomic data analysis and interpretation. Co-cultures of C. mamane and F. solani induced different patterns of metabolite production over 10 days of incubation and induced production of five de novo compounds not occurring in monocultures. These results emphasize that co-culture in time-frame analysis is an interesting method to unravel hidden metabolome in the investigation of fungal chemodiversity.

How Histone Deacetylase Inhibitors Alter the Secondary Metabolites of Botryosphaeria mamane, an Endophytic Fungus Isolated from Bixa orellana.

Fungi are talented organisms able to produce several natural products with a wide range of structural and pharmacological activities. The conventional fungal cultivation used in laboratories is too poor to mimic the natural habitats of fungi, and this can partially explain why most of the genes responsible for the production of metabolites are transcriptionally silenced. The use of Histone Deacetylase inhibitors (HDACis) to perturb fungal secondary biosynthetic machinery has proven to be an effective approach for discovering new fungal natural products. The present study relates the effects of suberoylanilide hydroxamic acid (SAHA) and sodium valproate (VS) on the metabolome of Botryosphaeria mamane, an endophytic fungus isolated from Bixa orellana L. UHPLC/HR-MS analysis, integrated with four metabolomics tools: MS-DIAL, MS-FINDER, MetaboAnalyst and GNPS molecular networking, was established. This study highlighted that SAHA and VS changed metabolites in B. mamane, causing upregulation and downregulation of metabolites production. In addition, twelve compounds were detected in the extracts as metabolites structurally correlated to SAHA, indicating its important reactivity in the medium or its metabolism by the fungus. An addition of SAHA induced the production of eight metabolites while VS induced only two metabolites undetected in the control strain. This result illustrates the importance of adding HDACis to a fungal culture in order to induce metabolite production.

Thiodiketopiperazines with two spirocyclic centers extracted from Botryosphaeria mamane, an endophytic fungus isolated from Bixa orellana L.

Three thiodiketopiperazines, botryosulfuranols A-C (1-3) were isolated from the endophytic fungus Botryosphaeria mamane. The three compounds present sulfur atoms on α- and ß-positions of phenylalanine derived residues and unprecedented two spirocyclic centers at C-4 and C-2'. Their planar structures were determined by spectroscopic analysis and absolute configurations were achieved by X-ray diffraction analysis and ECD and NMR chemical shifts calculations. Botryosulfuranol A (1) was the most cytotoxic compound against four cancer cell lines (HT-29, HepG2, Caco-2, HeLa) and two healthy cell lines (IEC6, Vero) highlighting the importance of an electrophilic center for cell growth inhibition.