Caracterização estrutural de alguns polissacarídeos presentes no basidioma de Pleurotus pulmonarius e aplicações

Orientador : Prof. Dr. Marcello IacominiCo-orientadora : Drª Eliane R. CarboneroDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica. Defesa: Curitiba, 2008Inclui bibliografiaO presente trabalho teve por objetivo elucidar a estrutura química de alguns polissacarídeos presentes no basidioma de Pleurotus pulmonarius, bem como verificar os efeitos antinociceptivos e antiinflamatórios de uma manogalactana isolada e, analisar alguns parâmetros nutricionais. O corpo de frutificação do basidiomiceto seco, moído e deslipidificado foi submetido a extrações aquosas (a frio e a quente) e alcalina (KOH 1%). Os extratos aquosos foram primeiramente submetidos à precipitação com etanol, enquanto o extrato alcalino foi submetido a adição de ácido acético até pH 5,0. Os extratos polissacarídicos obtidos foram submetidos aos processos de purificação por congelamento e degelo, precipitação com solução de Fehling, ultrafiltração e diálise por membranas de diferentes porosidades, de maneira sequencial. A partir destes processos foram purificadas e caracterizadas diferentes estruturas. A fração MG obtida pela extração aquosa a frio apresentou-se pura e composta por manose, galactose e O-metil-galactose. Análises espectroscópicas e de metilação sugerem que essa molécula apresenta cadeia principal constituída de 3-O-Me-a-D-Galp e a-D-Galp ligadas (1 - 6), sendo que ambas as unidades podem estar substituídas em O-2 por terminais não redutores de b-D-Manp. Esse polissacarídeo apresentou um efeito analgésico, porém não demonstrou atividade antiinflamatória, quando avaliado em teste de contração muscular induzida por ácido acético em camundongos. A fração PHW obtida pela extração aquosa a quente, após tratamento com NaOH apresentou glucose como principal componente monossacarídico. Os espectros de RMN-¹³C e HMQC dessa fração apresentaram sinais característicos de uma b-glucana-(1 - 3), substituída em O-6 por b-Glcp, semelhante à lentinana. O extrato alcalino obtido foi tratado com solução de Fehling, originando uma fração sobrenadante (SF-GLC) e uma fração precipitada (PF-GLC) neste reagente. Ambas apresentaram espectros de RMN-¹³C semelhantes e característicos de b-glucanas. Entretanto, a análise de metilação mostrou que SF-GLC é composta por alto teor de ligações (1- 3) e (1 - 6), enquanto PF-GLC é composta principalmente por ligações (1 - 6). A análise dos parâmetros nutricionais desse cogumelo mostrou que este é rico em proteínas e aminoácidos essenciais apresentando baixos teores de lipídios. Além disso, é uma boa fonte de minerais apresentando cálcio, magnésio, cobre, zinco e manganês em sua composição.The present object work is an elucidation of chemical structures of some polysaccharides extracted from the fruiting bodies of Pleurotus pulmonarius, besides their biological applications and nutritional value. The dried and milled basidiomycete was submitted to aqueous extraction (cold and hot), and alkaline extraction (1% KOH). The aqueous extracts were firstly submitted to ethanol precipitation, and the alkaline extract was neutralized with acetic acid. The resulting polysaccharide extracts were submitted successively to a freezethawing process, precipitation with Fehling solution, ultrafiltration and dialysis with membranes of different Mr cut-offs. After these processes different polysaccharides were characterized. Fraction MG, obtained from the cold water extraction, was homogeneous and was composed of mannose, galactose and O-methyl-galactose. Spectroscopic and methylation analysis suggest that this molecule has a main chain composed of 3-O-Me-a-D-Galp e a-D-Galp (1 - 6)- linked units, with both substituted at O-2 by b-D-Manp non-reducing end units. This polysaccharide presented an analgesic effect, independently of an inflammatory response, when analyzed by a test of abdominal constriction induced by acetic acid in mice. Fraction PHW obtained via hot water extraction, after treatment with NaOH, had glucose as its principal monosaccharide component. The ¹³C-NMR and HMQC spectra of this fraction contained signals characteristic of a (1 - 3)-linked-b-glucan, substituted at O-6 by b-Glcp, similar to lentinan. The alkaline extract was treated with Fehling solution giving rise to a soluble (SF-GLC) and precipitated fraction (PF-GLC). Both fractions had similar ¹³C-NMR spectra and they were characteristic of b-glucans. Although methylation data showed that SF-GLC was composed of a high proportion of (1 - 3) and (1 - 6) linkages, and PF-GLC was composed mostly of (1 - 6) linkages. Analysis of the nutritional value of this mushroom showed that it is rich in protein and essential amino acids, besides containing lower levels of lipids. In addition, it is a good source of minerals such as calcium, magnesium, copper, zinc and manganese

Pressurized Liquid (PLE) Truffle Extracts Have Inhibitory Activity on Key Enzymes Related to Type 2 Diabetes (α-Glucosidase and α-Amylase)

An optimized PLE method was applied to several truffle species using three different solvent mixtures to obtain bioactive enriched fractions. The pressurized water extracts contained mainly (1 ? 3),(1 ? 6)-?-D-glucans, chitins, and heteropolymers with galactose and mannose in their structures. The ethanol extracts included fatty acids and fungal sterols and others such as brassicasterol and stigmasterol, depending on the species. They also showed a different fatty acid lipid profile depending on the solvent utilized and species considered. Ethanol:water extracts showed interesting lipids and many phenolic compounds; however, no synergic extraction of compounds was noticed. Some of the truffle extracts were able to inhibit enzymes related to type 2 diabetes; pressurized water extracts mainly inhibited the ?-amylase enzyme, while ethanolic extracts were more able to inhibit ?-glucosidase. Tuber brumale var. moschatum and T. aestivum var. uncinatum extracts showed an IC50 of 29.22 mg/mL towards ?-amylase and 7.93 mg/mL towards ?-glucosidase. Thus, use of the PLE method allows o bioactive enriched fractions to be obtained from truffles with antidiabetic properties.This research was funded by the fellowship Ibercaja-CAI Estancias de Investigación number CA 1/20. The research leading to these results has received funding from the European Union under “Horizon 2020—the Framework Programme for Research and Innovation (2014–2020)”. Grant Agreement of the Project: “Innovation in truffle cultivation, preservation, processing and wild truffle resources management”, INTACT, Project number 101007623.trufflesβ-D-glucansergosterolfatty acidsantidiabeticamylaseglucosidasePublishe

Antinociception of β-d-glucan from Pleurotus pulmonarius is possibly related to protein kinase C inhibition

Abstractβ-d-Glucan, a polysaccharide isolated from an edible mushroom Pleurotus pulmonarius (Fr.) Quel., presented antinociceptive activity in mice. This study evaluated the involvement of transient receptor potential (TRP) channels and protein kinase C (PKC) on antinociceptive effect of a (1→3),(1→6)-linked β-d-glucan (GL) in mice. Intraperitoneal administration of GL potently inhibited nociceptive responses induced by intraplantar injections of capsaicin, cinnamaldehyde, menthol, acidified saline and phorbol myristate acetate (PMA). Moreover, Western blot analysis revealed that GL treatment also prevented PMA-induced PKCɛ activation. Collectively, present results demonstrate that GL could constitute an attractive molecule of interest for the development of new analgesic drugs

Polissacarídeos produzidos por basidiomicetos e ascomiceto : caracterização estrutural e atividade imunomoduladora

Orientador : Prof. Dr. Marcello IacominiTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica. Defesa: Curitiba, 27/03/2012Bibliografia: fls. 115-125Resumo: A presente pesquisa visou isolar e caracterizar polissacarídeos presentes em cogumelos comestíveis, bem como determinar novos métodos de obtenção dessas moléculas através de cultivo submerso desses fungos. O objetivo de se estudar esses polissacarídeos, em especial as p-glucanas, é devido à capacidade dos mesmos em atuar como imunomoduladores no organismo humano, propiciando a melhora para algumas doenças como tumores, artrite reumatóide, alergias, entre outras. Foram testados alguns polissacarídeos isolados em um modelo de estudo para a resposta imune, utilizando a linhagem monocítica de células humanas THP-1. Inicialmente foram determinadas algumas condições de cultivo do basidiomiceto Pleurotus pulmonarius, sendo possível obter polissacarídeos semelhantes aos isolados do basidioma, com rendimento superior. Também foram utilizados os cogumelos Agaricus bisporus, A. brasiliensis e Cordyceps militaris, com os quais se procederam a extrações aquosas e alcalinas, seguidas de processos de purificação para obtenção de seus polissacarídeos. Os dois primeiros também são basidiomicetos e apresentaram polissacarídeos semelhantes, considerando que pertencem ao mesmo gênero. Foi observada a presença de uma p-glucana-(1->6), de uma a-glucana- (1->4)-(1->6) e de uma manogalactana em ambos os cogumelos. Entretanto, as proporções de cada um variaram entre eles, sendo que A. bisporus apresentou maior teor do heteropolissacarídeo, enquanto A. brasiliensis apresentou maior teor de p-glucana. Extratos brutos de ambos os basidiomicetos foram testados em macrófagos, sendo seus efeitos semelhantes, independentemente da composição polissacarídica apresentar essa variação na proporção. Pôde-se observar moderado aumento na expressão de IL-ip, TNF-a e COX-2 e inibição dos efeitos inflamatórios de LPS quando as células foram tratadas com esses extratos. As p-glucanas-(1->6) de Agaricus spp. apresentaram elevado aumento da expressão dos genes pró-inflamatórios testados e moderada inibição dos efeitos de LPS. Também foram isolados uma galactomanana com alto teor de ramificação de C. militaris e uma p-glucana-(1->3) linear. Ambos os polissacarídeos são comumente encontrados em ascomicetos, grupo ao qual este cogumelo pertence. A p-glucana-(1->3) foi analisada quanto a sua capacidade de estimular o sistema imune, mostrando ser incapaz de estimular a expressão de IL-ip, TNF-a, COX-2 e NF-KB. Entretanto, essa mesma glucana apresentou grande inibição dos efeitos desencadeados por LPS, mostrando uma capacidade antiinflamatória. Sugere-se que as p-glucanas-(1->6), por serem solúveis, estejam ativando receptores do tipo Toll like (TLRs), os quais ativam a expressão de NF-KB, que por sua vez, estimula a produção de outras moléculas pró-inflamatórias. A p-glucana-(1->3) foi, entretanto, incapaz de ativar esses receptores. A insolubilidade em água dessa molécula sugere que a ligação ocorra com o receptor dectina-1, entretanto ainda precisa ser esclarecida qual via de ativação é desencadeada por essa ligação, visto que os efeitos gerados são antiinflamatórios. Moléculas solúveis também podem ligar-se a dectina-1, porém somente moléculas insolúveis são capazes de ativar esse receptor. Outros estudos são necessários para se determinar como essas p-glucanas estão inibindo a ação inflamatória do LPS, sendo uma possível explicação, a competição pela ligação com o mesmo receptor de membrana. Os resultados observados nessa pesquisa mostraram a importância da determinação da estrutura química dos polissacarídeos para uma eficiente avaliação das suas propriedades terapêuticas, visando esclarecer mecanismos de ação, vias de ativação, bem como os efeitos proporcionados pelo tratamento com essas moléculas.Abstract: The present study aimed to isolate and characterize the polysaccharides from edible mushrooms, as well as to determine new approaches to obtain these molecules by using submerged culture of fungi. The objective of studying these polysaccharides, specially pglucans, is related to their immunomodulatory activities in the human body, providing an improvement in some diseases such as tumors, arthritis, allergies, and others. Some of the isolated polysaccharides were tested by using an immune response model of human acute monocytic leukemia cell line, THP-1. Initially, some conditions for the submerged culture of Pleurotus pulmonarius were determined, which provided similar polysaccharides comparing to the fruit body ones, although the yield was higher. Some other mushrooms, as Agaricus biporus, A. brasiliensis, and Cordyceps militaris, were also studied. They were submitted, separately, to aqueous and alkaline extractions, followed by purification procedures to obtain the isolated polysaccharides. The Agaricus spp. belong to basidiomycetes group and both presented similar polysaccharides, which is in accordance as they also belong to the same genera. It was observed the presence of a p-glucan-(1->6), an a-glucan-(1->4)-(1->6), and a mannogalactan in both mushrooms. Although the proportion of each polymer in each mushroom was different: the A. bisporus contained higher amounts of mannogalactan and A. brasiliensis presented higher level of p-glucan. Crude extracts of these mushrooms were tested in macrophages, and besides their polysaccharide proportions were different, their effects were similar. It was observed a moderate increase in the expression of IL-ip, TNF-a, and COX-2 while the cells were treated with the extracts, and they also inhibited the LPS inflammatory effect in these cells. The p-glucans-(1->6) of Agaricus spp. presented high increase of the expression of the pro-inflammatory genes and moderate inhibition of the LPS effects. A highly branched galactomannan and a linear p-glucan (1->3)-linked were isolated from C. militaris. Both polysaccharides are commonly found in ascomycetes, in which C. militaris is also included. The p-glucan-(1->3) was analyzed to verify its capacity of immune stimulation and it failed in stimulating the expression of IL-ip, TNF-a, COX-2, and NF-KB. Nevertheless, this same glucan presented strong inhibition of the LPS inflammatory effects, showing an anti-inflammatory activity. It is possible that the p-glucans-(1->6), which are very soluble, can activate Toll like receptors (TLRs), that activate the NF-KB expression, and subsequently augment other pro-inflammatory molecules. Although, the p-glucan-(1->3) was unable of activating these receptors. The insolubility in water of this molecule suggests that it can bind dectin-1 receptor, although it still remains unclear which is the activation role initiated by this complex, considering that the observed effects were anti-inflammatory. Soluble molecules can also bind dectin-1 receptor, however only insoluble molecules can activate it. More studies are necessary to determine how the p-glucans are inhibiting the inflammatory effects of LPS, considering as a possible explanation, the competition for the same receptor linkage site. The observed results of this research showed the importance of determining the chemical structure of polysaccharides to evaluate efficiently their therapeutic properties, aiming to clarify the action mechanisms, the activation roles, as well as the effects promoted by the treatment with these molecules

Production of a β-d-glucan-rich extract from Shiitake mushrooms (Lentinula edodes) by an extraction/microfiltration/reverse osmosis (nanofiltration) process

A pilot-scale process combining extraction of Shiitake mushroom (Lentinula edodes) powder in water (98 °C, 1 h), cross-flow microfiltration and reverse osmosis (nanofiltration) was performed to obtain β-glucan-rich extracts. Suspensions (45–80 L) obtained after 3 extractions were clarified by microfiltration reducing their turbidity to <1 NTU. Membrane flux was completely recovered after filtration. One of the clarified extracts was concentrated (to 6–7 L) by reverse osmosis (Nanomax95) and the other two by nanofiltration (Nanomax50 and ALNF99-2517). Different physicochemical parameters (permeate flux, total soluble substances, total suspended particles and electrical conductivity) were monitored during filtration and the bioactive compounds present in the obtained fractions (β-glucans, total carbohydrates, chitins, eritadenine, lenthionine, ergosterol) were analyzed. The more adequate membrane for Shiitake extract concentration was Nanomax50 because it showed higher filtration flux and higher values of bioactive compounds in the obtained extract than the extracts obtained with the other two membranes.This research was supported by national R + D program from the Spanish Ministry of Science and Innovation (project AGL2014-56211-R) and the regional program from the Community of Madrid, Spain (S2013/ABI-2728).Peer reviewe

Strengths and weaknesses of the aniline-blue method used to test mushroom (1→3)-β-D-glucans obtained by microwave-assisted extractions

The parameters to extract polysaccharide-enriched fractions (PEF) from mushrooms using MAE (microwave-assisted extraction) were adjusted following a full factorial 3 2 experimental design. The highest yield and total carbohydrate values, using Lentinula edodes as model mushroom, were obtained at 180 °C and 30 min. Several mushroom species were submitted to MAE and their PEF yields ranged between 12.1–44.2%. (1→3)-β-Glucans determination using a conventional fluorimetric method changed depending on the standard utilized. NMR analyses of PEF indicated that the presence of other polysaccharides in the extracts or their specific folding, might impair the proper determination of (1→3) linkages by the fluorophore. Mushrooms from Cantharellales order contained (1→3)-β-glucans but they were not detected with the fluorimetric method. Therefore, although the method (after adjustments) was sensitive enough to detect their presence in many mushroom extracts, it cannot be used for all species and it is also not recommended for quantitative determinations

Testing the effect of combining innovative extraction technologies on the biological activities of obtained β-glucan-enriched fractions from Lentinula edodes

Innovative technologies as ultrasound-assisted extraction (UAE) (550 W, 60% amplitude, 50 °C) or subcritical water extraction (SWE) (200 °C, 11.7 MPa) were more effective than hot water extractions to obtain β-glucan-enriched fractions from shiitake mushrooms. UAE required longer extraction time (60 min) than SWE (15 min). Combination of UAE + SWE or pre-treatment of the raw material with supercritical CO2 (SFE) (40 °C, 35 MPa, 3 h) before both extractions yielded extracts containing larger β-glucan concentrations. Fluorimetric/colorimetric determinations indicated that obtained fractions contained (1 → 3)- and (1 → 3),(1 → 6)-β-glucans. NMR confirmed their presence as well as (1 → 3)-α-glucans and heteropolymers including mannose and galactose. SWE (15 min), SFE + SWE or UAE + SWE extracts showed larger glucose levels and lower mannose and galactose residues than the other extractions suggesting certain extraction specificity towards β-glucans. They also included more chitin-derivatives than UAE. The extracts obtained after combination of technologies partially retained their immunomodulatory properties but they showed high hypocholesterolemic activities according to in vitro studies.This research was supported by national R+ D program from the Spanish Ministry of Science and Innovation (project AGL2014-56211-R) and the regional program from the Community of Madrid, Spain (S2013/ABI-2728).Peer reviewe

Isolation and comparison of α- and β-D-glucans from shiitake mushrooms (Lentinula edodes) with different biological activities

A polysaccharide-enriched extract obtained from Lentinula edodes was submitted to several purification steps to separate three different D-glucans with β-(1→6), β-(1→3),(1→6) and α-(1→3) linkages, being characterized through GC–MS, FT-IR, NMR, SEC and colorimetric/fluorimetric determinations. Moreover, in vitro hypocholesterolemic, antitumoral, anti-inflammatory and antioxidant activities were also tested. Isolated glucans exerted HMGCR inhibitory activity, but only β-(1→6) and β-(1→3),(1→6) fractions showed DPPH scavenging capacity. Glucans were also able to lower IL-1β and IL-6 secretion by LPS-activated THP-1/M cells and showed cytotoxic effect on a breast cancer cell line that was not observed on normal breast cells. These in vitro results pointed important directions for further in vivo studies, showing different effects of each chemical structure of the isolated glucans from shiitake mushrooms.This research was supported by national R + D program from the Spanish Ministry of Science and Innovation (Project AGL2014-56211-R) and the regional program from the Community of Madrid, Spain (S2013/ABI-2728). Part of this study was also financed by the Brazilian funding agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) – Finance Code 001 and the Fundação Araucária.Peer reviewe

Evaluation of microwave-assisted and pressurized liquid extractions to obtain β-D-glucans from mushrooms

Microwave-assisted extraction (MAE) and pressurized liquid extraction (PLE) were compared as advanced technologies to obtain polysaccharides (particularly biologically active β-glucans) from Pleurotus ostreatus and Ganoderma lucidum fruiting bodies. Extraction effectiveness was compared by a full-factorial experimental design (response surface methodology, RSM), using water as extraction solvent. Total carbohydrate content of the obtained extracts and polysaccharide yields were the variable responses investigated, while temperature and extraction time were the experimental factors. Temperature showed stronger influence in the polysaccharide extraction than time. The latter factor slightly affected MAE but not PLE extractions. Optimal conditions within the studied range were determined for each extraction method and species based on the desirability functions. Regarding the polysaccharide composition, the main differences between the species were more quantitative rather than qualitative, since NMR analyses indicated that all extracts contained mainly β- and α-glucans and heteropolysaccharides. Both extraction systems were effective for polysaccharide extraction from mushrooms.The authors would like to thank Juncao Brazil (Taboão da Serra-SP, Brazil), for the donation of Ganoderma lucidum, to CTICH (Centro Tecnológico de Investigación del Champiñón de La Rioja, Autol, Spain) for the Pleurotus ostreatus strain, and to Anton-paar GmbH for access to Monowave EXTRA instrument. F.R.S. thanks to the Brazilian funding agencies CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and the Fundação Araucária. B.G.L. thanks Spanish MINECO (Ministerio de Economía y Competitividad) for her Juan de la Cierva postdoctoral research contract (ref. JCI-2012-12972). The research was also supported by national R + D program from the Spanish Ministry of Science and Innovation (project AGL2014-56211-R) and the regional program from the Community of Madrid, Spain (S2013/ABI-2728).Peer Reviewe