Metabolic investigation and activity of Cordyceps militaris and cordycepin in cancer cell lines

Inflammation is a normal reaction to infection or injury, but if the process is not resolved, it can lead to a variety of diseases, including cancer and arthritis. The available treatments for chronic inflammation are often insufficient or have unacceptable side effects, leading to a continued search for anti-inflammatory medicines with novel mechanisms of action. Natural sources are rich in anti-inflammatory compounds and are re-gaining interest. A common problem with natural medicines and natural compounds is their quality, consistency and purity. In this study, metabolomic analysis of Cordyceps militaris (L.) Fr., Cordyceps militaris extract was undertaken and thousands of compounds were identified by untargeted metabolomic analysis. Different batches from one producer were similar but had a somewhat variable cordycepin content and large differences in the cordycepin-potentiating compound pentostatin. There were much larger differences between batches from different suppliers. A comparison of the effects of cordycepin and Cordyceps extracts on inflammation in a macrophage cell line suggested that the extract contained additional activity. Untargeted metabolomics of commercially purified cordycepin (sold as 98% pure) revealed that six different batches had large numbers of significant contaminants, which were similar between suppliers. Remarkably, the batches of cordycepin differed in their cordycepin content as well as their potency. Some of the contaminants were tested in tissue culture for effects on inflammation, but many were commercially not available or even not fully identifiable. Pentostatin was also detected in cordycepin preparations. These data suggest that the reported biological activity of cordycepin may not be due to the presence of this compound alone

Metabolic investigation and activity of Cordyceps militaris and cordycepin in cancer cell lines

Inflammation is a normal reaction to infection or injury, but if the process is not resolved, it can lead to a variety of diseases, including cancer and arthritis. The available treatments for chronic inflammation are often insufficient or have unacceptable side effects, leading to a continued search for anti-inflammatory medicines with novel mechanisms of action. Natural sources are rich in anti-inflammatory compounds and are re-gaining interest. A common problem with natural medicines and natural compounds is their quality, consistency and purity. In this study, metabolomic analysis of Cordyceps militaris (L.) Fr., Cordyceps militaris extract was undertaken and thousands of compounds were identified by untargeted metabolomic analysis. Different batches from one producer were similar but had a somewhat variable cordycepin content and large differences in the cordycepin-potentiating compound pentostatin. There were much larger differences between batches from different suppliers. A comparison of the effects of cordycepin and Cordyceps extracts on inflammation in a macrophage cell line suggested that the extract contained additional activity. Untargeted metabolomics of commercially purified cordycepin (sold as 98% pure) revealed that six different batches had large numbers of significant contaminants, which were similar between suppliers. Remarkably, the batches of cordycepin differed in their cordycepin content as well as their potency. Some of the contaminants were tested in tissue culture for effects on inflammation, but many were commercially not available or even not fully identifiable. Pentostatin was also detected in cordycepin preparations. These data suggest that the reported biological activity of cordycepin may not be due to the presence of this compound alone

Protein Structure

Since the dawn of recorded history, and probably even before, men and women have been grasping at the mechanisms by which they themselves exist. Only relatively recently, did this grasp yield anything of substance, and only within the last several decades did the proteins play a pivotal role in this existence. In this expose on the topic of protein structure some of the current issues in this scientific field are discussed. The aim is that a non-expert can gain some appreciation for the intricacies involved, and in the current state of affairs. The expert meanwhile, we hope, can gain a deeper understanding of the topic

Mass Spectrometric Proteomics

As suggested by the title of this Special Issue, liquid chromatography-mass spectrometry plays a pivotal role in the field of proteomics. Indeed, the research and review articles  published in the Issue clearly evidence how the data produced by this sophisticated methodology may promote impressive advancements in this area. From among the topics discussed in the Issue, a few point to the development of  new procedures for the  optimization of the experimental conditions that should be applied  for the identification of proteins present in complex mixtures.  Other applications  described in these articles show  the huge potential of  these strategies in the protein profiling of organs and  range from  to the study of post-translational tissue modifications to the investigation of the molecular mechanisms behind human disorders and the identification of potential biomarkers of these diseases

Fungi and Fungal Metabolites for the Improvement of Human and Animal Nutrition and Health

The purpose of this book was not to provide a comprehensive overview of the vast arena of how fungi and fungal metabolites are able to improve human and animal nutrition and health; rather, we, as Guest Editors, wished to encourage authors working in this field to publish their most recent work in this rapidly growing journal in order for the large readership to appreciate the full potential of wonderful and beneficial fungi. Thus, this Special Issue welcomed scientific contributions on applications of fungi and fungal metabolites, such as bioactive fatty acids, pigments, polysaccharides, alkaloids, terpenoids, etc., with great potential in human and animal nutrition and health

Abstracts of Papers, 93rd Session, Iowa Academy of Science, April 24-25, 1981

Presentation abstracts from the annual meeting of the Iowa Academy of Sciencehttps://scholarworks.uni.edu/ias_docs/1049/thumbnail.jp

Selenized yeast in production of selenium enriched Pleurotus ostreatus mushroom with good flavour

The aim of this study was to investigate the potential influence of selenized yeast (Sel Plex, Alltech Inc., Lexington, USA) on chemical composition and flavour of solid state grown mushroom Pleurotus ostreatus. Amino acid composition that influences the flavour of selenium-enriched P.ostreatus P80 (137.84 ppm of selenium in d.w.) and non enriched cultivated strains with particular emphasis on selenomethionine was determined by HPLC method, after complete hydrolysis. Volatile flavour compounds of mushroom cultures were analyzed by GC-MS using Headspace sampler. In mushrooms with high selenium content, selenium in the form of L-selenomethionine was present. High selenium concentration in fruit body did not significantly change the amino acid composition of mushrooms. The major amino acids of fruit body sample were glutamic acid, alanine, aspartic acid and tryptophan. In the fruit body of P.ostreatus P80 control, 30 volatile compounds were detected, and in selenium enriched sample number of detected compounds was 25. Compounds that were detected in control sample, but not in eriched fruit body are 2-amino-N-ethylpropanamide, 2-methylpropyl pentan-2-yl sulfite, 2,3-pentanedione, heptan-2-one, pentan-1-ol, 2-methyldihydrofuran-3(2H)-one,1-hydroxyacetone, 3-hydroxy-2-butanon, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine and benzaldehyde. Compounds that were detected in enriched P.ostreatus P80, but not in control are acetone, ethylacetate, 2-propanol, acetonitrile, 2-methylbenzaldehyde and 2-hydroxyethylmethacrylate. Selenized yeast presents good source of selenium for selenium enriched mushroom production with good flavour, which is probably safety for consumption

Bioactive polysaccharides and small molecules from the native North American fungus Echinodontium tinctorium

Mushrooms, the fruiting bodies of fungi, are known to be powerful sources of nutraceuticals and pharmaceuticals but there are limited studies focusing on exploring the medicinal value of mushrooms native to North America. Here, I describe the isolation of two novel bioactive polysaccharides from the aqueous extracts of the fungus Echinodontium tinctorium: an immunostimulatory complex polysaccharide (EtISPFa) of 1354 kDa, and a growth-inhibitory β-glucan of 275 kDa. In addition, six small molecules including a phenol derivative, a new diphenylmethane derivative and three lanostane-type triterpenes were isolated from the organic extracts of E. tinctorium. The molar mass of these isolated small molecules (labelled 1-6) was determined to be 124, 260, 506, 498, 496, and 440 g/mol respectively. Phase separation, Sephadex LH-20 size exclusion, Sephadex DEAE ion exchange chromatography, Sephacryl S-500 HR size exclusion, silica column chromatography, and HPLC were used for bioactivity-guided purification. Chemical structures and linkages of EtISPFa and EtGIPL1a polysaccharides were determined by gas chromatography mass spectrometry (GCMS) and nuclear magnetic resonance (NMR). Final structures of small molecules were determined by Fourier transform infrared spectroscopy (FTIR), electrospray ionization mass spectrometry (ESI-MS), NMR, and X-ray crystallography. Immuno-stimulatory activity of EtISPFa was assessed by immunoassay in Raw 264.7 murine macrophage cells and growth-inhibitory activity of EtGIPL1a and small molecules were assessed by MTT growth-inhibitory assay in cancer cell lines. The mechanism of growth inhibition was assessed via apoptosis and cell cycle assays. EtISPFa stimulated the immune response by inducing TNF-α and other inflammatory cytokines in murine macrophage cells. In contrast, EtGIPL1a showed promising anti-proliferative activity against U251 glioblastoma cells and on ten other cancer cell lines. EtGIPL1a induced apoptosis in U251 cells with an increased cleaved caspase-3 apoptotic marker and significant DNA fragmentation in cell cycle analysis. Amongst the small molecules, compounds (2), (4) and (5) caused growth-inhibition in U251 cells; compound (4) also showed promising effects on multiple other cancer cell lines; all its bioactivities are reported here for the first time. The crystal structures of compounds (2), (4) and (5) have also been reported for the first time. Molecular targets of (1), (2), (4) and (5) by MolTarPred were predicted and warrants further experimental investigation

Advances in mycotechnology : bioprospection of native macrofungi strains, molecular identification and cryopreservation of mycelia, production of antioxidant enzymes, bioprocessing of organic residues and bioprocesses automation and control using free hardware and software

Orientador: Prof. Dr. Carlos Ricardo SoccolTese (doutorado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Defesa : Curitiba, 29/05/2014Inclui referências: p.211-252Resumo: A presente tese de doutorado abrange múltiplas contribuições para o desenvolvimento da micotecnologia. Aproximadamente 70 tentativas de isolamento foram realizadas e pelo menos 15 cepas estáveis foram obtidas. Quatro destas culturas foram identificadas com base no sequenciamento de regiões ITS. Foram desenvolvidos novos métodos para criopreservação de micélios, utilizando vermiculite como suporte. Pelo menos 12 cepas foram reativadas após 12 meses de armazenamento, usando estas técnicas. Duas destas cepas foram avaliadas e mantiveram inalteradas a taxa de crescimento radial (0.58±0.38 cm/ dia) e o rendimento de exopolissacarídeos (250±110 mg/ L). Alguns fatores foram avaliados no cultivo de espécies do gênero Pleurotus, utilizando resíduos de pupunha como substrato. A fração externa do resíduo se mostrou excessivamente ácida (pH 3,21±0,15) para o crescimento micelial. P. ostreatus (1,38±0,16 cm/dia) e P. djamor (1,38±0,1 cm/dia) se adaptaram bem à fração interna do substrato. Taxas de inoculação altas (40%) e maior número de furos (4) produziram aumentos significativos (50%) na concentração protéica dos carpóforos. O processamento de resíduos de pupunha por cultivo micelial melhorou a qualidade deste material como fertilizante para o cultivo de alfaces (até 40% de aumento na produtividade). A cinética de produção das enzimas SOD (superóxido dismutase) e CAT (catalase) foi determinada para o cultivo submerso de P. ostreatus (PO). Seis substâncias foram testadas como indutores destas duas enzimas. Sulfonato de lignina 10 g/L resultou em um aumento na atividade de SOD de 700%. A atividade de CAT aumentou em 46% com a adição de H2O2 90mM. Duas cepas de P. ostreatus (PO e PL24) e três de outras espécies: P. djamor (PD), P. eryngii (PE) e P. pulmonarius (PP) foram comparadas quanto à produção de SOD e CAT em cultivo submerso. PD e PO apresentaram mais que o dobro da atividade de SOD das outras cepas testadas. As atividades de CAT de PE, PL24 e PP foram aproximadamente 59% mais altas que as de PO. PD apresentou atividade de CAT praticamente nula. As mesmas três isoformas de SOD foram encontradas em todas as cepas avaliadas. Técnicas para a recuperação do sulfonato de lignina foram desenvolvidas para purificação parcial de SOD para as análises zimográficas. Este objetivo foi alcançado, mantendo aproximadamente 33% da atividade enzimática inicial. Finalmente, foram desenvolvidos aparatos para cultivo micelial submerso. Hardware e software livres foram utilizados para construir protótipos de biorreatores com sistemas de automação e controle. Um controlador de fotoperíodo, um dispositivo termostático e um sistema de inferência da concentração de biomassa on-line, baseado na absorção de laser, foram projetados, programados e construídos. Espera-se que os avanços propostos contribuam para o desenvolvimento de novos processos, permitindo a obtenção de produtos que tragam benefícios à saúde e à qualidade de vida da população. Palavras-chave: cogumelos. antioxidantes. SOD. CAT. automação.Abstract: The present doctorate thesis covers multiple contributions for the development of mycotechnology. Approximately 70 isolation trials were performed and at least 15 stable strains were obtained. Four of these cultures were identified based in ITS regions sequencing. New methods for mycelia cryopreservation were developed, with the use of vermiculite as a carrier material. At least 12 strains were reactivated after twelve months of storage, using these techniques. Two of these strains were evaluated and kept metabolic characteristics unaltered, such as radial growth rate (0.58±0.38 cm/ day) and exopolysaccharide yield (250±110 mg/ L). Several factors were evaluated in the cultivation of some species of the Pleurotus genus, using pejibaye palm sheath residues as substrate. The external fraction of the residue proved excessively acid (pH 3,21±0,15) for mycelial growth. P. ostreatus (1,38±0,16 cm/day) and P. djamor (1,38±0,1 cm/day) adapted well to the internal fraction of the substrate. High levels of both inoculum rate (40%) and number of holes (4) produced significative increases in the protein content (50% increase) of the carpophores. The processing of pejibaye residues by mushrooms cultivation, improved its quality as fertilizer for lettuce cultivation (up to 40% increase in lettuce yield). The production kinetics of the antioxidant enzymes SOD (superoxide dismutase) and CAT (catalase) by the submerged mycelial cultivation of P. ostreatus (PO strain) were determined. Six substances were evaluated as inducers of these two enzymes. Lignin sulfonate was the best SOD inducer and hydrogen peroxide was the best CAT inducer. Lignin sulfonate 10 g/L increased SOD activity in approximately 700%. H2O2 90 mM increased CAT activity in up to 46%. Two P. ostreatus strains (PO and PL24) and three other species - P. djamor (PD), P. eryngii (PE) and P. pulmonarius (PP) - were compared in respect to SOD and CAT production by submerged cultivation. PD and PO presented more than double the SOD activity than the other assayed strains. PE, PL24 and PP CAT activities were about 59% higher than that of PO. PD showed virtually no measurable CAT activity. The same three isoforms were detected for these five strains. Lignin sulfonate recovering techniques were developed in order to partially purificate SOD sufficiently for zymographic methods. This objective was accomplished, maintaining nearly 33% of the enzymatic activity of the product. Finally, apparatuses for macromycetes mycelia submerged cultivation were developed. Free hardware and software were used for building bioreactors prototypes with automation and control systems. A photoperiod controller, a thermostatic device and an on-line biomass concentration estimation system, based in laser absorption were designed, programmed and built. It is expected that the proposed advances can contribute for new processes development, allowing the obtention of products that are intended to bring health benefits and improvements in populations' life quality. Keywords: mushrooms. antioxidants. SOD. CAT. automation