Biološki potencijal i analiza metabolita jestivih i lekovitih gljiva iz rodova Agrocybe, Laetiporus, Pleurotus i Polyporus sa teritorije Srbije

Abstract

Makromicete su gljive iz podrazdela Ascomycotina i Basidiomycotina koje karakterišu krupna plodonosna tela, dovoljno velika da se vide golim okom i uberu rukom. U ovoj doktorskoj disertaciji makromicete iz rodova: Agrocybe, Laetiporus, Pleurotus i Polyporus, sakupljene na teritoriji Srbije, detaljno su analizirane. Ispitivan je sadržaj makronutrijenata i odabranih metabolita u makromicetama i utvrđen je njihov biološki potencijal. Identifikacija i određivanje sadržaja nutrijenata (ukupnih ugljenih hidrata, proteina, masti i pepela), šećera, tokoferola, masnih, fenolnih i organskih kiselina izvedeno je putem HPLC-a sa različitim detektorima u zavisnosti od jedinjenja od interesa. Radi detaljne procene biološkog potencijala ekstrakata makromiceta, izvedeni su testovi in vitro antioksidativne, antimikrobne, anti-quorum sensing i citotoksične aktivnosti, a iz bazidiokarpa Laetiporus sulphureus 1 izolovan je i okarakterisan lektin i procenjen njegov antibakterijski potencijal. Rezultati analize makronutrijenata pokazali su da testirane makromicete imaju povoljan profil nutrijenata zbog čega se mogu preporučiti u ishrani. Karakteriše ih znatna količina ugljenih hidrata i proteina kao i nizak sadržaj masti. Analizom šećera identifikovane su: ramnoza, fruktoza, manitol, glukoza i trehaloza koja je bila i najzastupljeniji šećer sa 16,50 g/100 g suve mase kod Pleurotus ostreatus K i 32,60 g/100 g suve mase kod P. ostreatus S. U uzorcima testiranih vrsta makromiceta određen je sadržaj četiri izoforme tokoferola (α-, β-, γ- i δ- tokoferol), dok je analizom masnih kiselina potvrđeno prisustvo 27 masnih kiselina, među kojima je nutritivno poželjna linolna kiselina najviše zastupljena u uzorku Agrocybe aegerita 1 (78,40%). Među prostim organskim kiselinama utvrđeno je prisustvo: oksalne, kininske, jabučne, limunske i fumarne kiseline, dok su analizom organskih kiselina sa fenolnom grupom identifikovane protokatehinska, p-hidroksibenzoeva, p-kumarna i cinaminska kiselina. Izoloacijom i karakterizacijom lektina iz bazidiokarpa L. sulphureus 1 pokazano je da je lektin multimerne građe, pI vrednosti ~ 4,6. Rezultati antioksidativne aktivnosti (redukciona sposobnost, DPPH· aktivnost, β-karoten/linolna kiselina i TBARS test) pokazali su da su testirane makromicete moćni in vitro antioksidativni agensi više ili manje efikasni u neutralizaciji slobodnih radikala, inhibiciji lipidne peroksidacije i redukcionoj sposobnosti (EC50/ EC0.5 vrednosti su u opsegu 0,25 - 23,78 mg/mL). Etanolni i metanolni ekstrakti bazidiokarpa testiranih makromiceta pokazali su visok antimikrobni potencijal (u odnosu na vodeni ekstrakt) na testirane mikroorganizme, a utvrđeno je da pri testiranim koncentracijama lektin ne deluje na bakterije, što ide u prilog tvrdnji da supstance prirodnog porekla biološki efekat ostvaruju zahvaljujući aditivnim i sinergističkim interakcijama većeg broja prisutnih jedinjenja. Metanolni ekstrakti testiranih makromiceta su na subinhibitornom nivou efikasno ometali međućelijsku komunikaciju oportunističkog patogena Pseudomonas aeruginosa PAO1, što je zaključeno nakon smanjenja dijametra njegove kolonije, struktura za kretanje i sinteze pigmenta piocijanina koji je ujedno i faktor virulentnosti. Najbolji antibiofilm efekat postigao je uzorak metanolnog ekstrakta Polyporus squamosus 1 (88,30%). Umereni citotoksični potencijal na tumorske ćelijske linije humanog porekla HeLa, NCI-H460 i MCF-7 pokazao je samo polisaharidni ekstrakt uzorka L. sulphureus 1. Rezultati preliminarne analize hepatotoksičnosti predočili su da ekstrakti nisu toksični za primarne svinjske hepatocite čime je otvoren put za sigurnu primenu preparata na bazi testiranih makromiceta kod ljudi. Rezultati sprovedenih istraživanja su pokazali da testirane makromicete imaju potencijal funkcionalne hrane zahvaljujući produkciji biološki aktivnih jedinjenja koja mogu pozitivno delovati na zdravlje, a zahvaljujući sposobnosti eradikacije patogenih mikroorganizama, ekstrakti makromiceta su važni pri formulaciji novih antimikrobnih agenasa. Upotreba makromiceta ima značajan potencijal u unapređivanju zdravlja, dugovečnosti i kvaliteta života i predstavlja primer jedinstva tradicije i nauke. Dalja proučavanja makromiceta ukazuju na pravce za njihov napredak od funkcionalne hrane do dostupnih medicinskih preparata, a kako su neke od testiranih vrsta uspešno kultivisane, veliki su izgledi da se ovako nešto zaista i dogodi u bliskoj budućnosti.

Macromycetes are fungi from subdivisions Ascomycotina and Basidiomycotina that are large enough to be seen with naked eye and to be picked by hand. In this thesis, selected species from the genera: Agrocybe, Laetiporus, Pleurotus and Polyporus were harvested in Serbia and chemically characterized. Their nutrients profile and selected metabolites as well as their biological potential was evaluated. Quantification of macronutrients (carbohydrates, proteins, fat and ash), selected sugars, tocopherols, fatty, phenolic and organic acids was determined by means of HPLC coupled with various detectors, depending on the type of desired compound. Regarding the evaluation of biological potential, detailed assesments of antioxidant, antimicrobial, anti-quorum sensing and cytotoxic activities were performed. A lectin was extracted from the fruiting body of Laetiporus sulphureus 1 as a compound of interest, and was evaluated for its antibacterial activity. Results of the macronutrient profile suggest that macromycetes are a rich source of carbohydrates and proteins, with low fat content which makes them excellent candidates for food. Sugar analysis revealed the presence of: rhamnose, fructose, mannitol, glucose and trehalose which was the most abundant sugar with 16,50 g/100 g dry weight in Pleurotus ostreatus K and 32,60 g/100 g dry weight in P. ostreatus S. Four isoforms of tocopherols (α-, β-, γ- and δ- tocopherol) were quantified in samples, while the fatty acid analysis revealed the presence of 27 fatty acids with linoleic acid as the most abundant polyunsatturated fatty acid in sample Agrocybe aegerita 1 (78,40%). Amongst simple organic acids, the following were detected in the samples: oxalic, quinic, malic, citric and fumaric acid. Phenolic group containing organic acids analysis indicated the presence of: protocatechuic, p-hydroxybenzoic, p-coumaric and cinnamic acid. Isolation and characterization of lectin from the L. sulphureus 1 basidiocarp revealed that lectin is a multimer with pI value of ~ 4,6. Results of antioxidant activity which was evaluated using four protocols (reducing power, DPPH· scavenging activity, β-caroten/linoleic acid and TBARS test) indicate that selected macromycetes are potent antioxidant agents that showed ability to neutralize free radicals, inhibit lipid peroxidation and reducing power (EC50/EC0.5 is in range of 0,25 – 23,78 mg/mL). Ethanolic and methanolic extracts of mushroom fruiting bodies revealed potent antimicrobial potential against the tested pathogenic microorganisms (compared to the aqueous extracts), while lectin didn't show antibacterial activity at the tested concentrations, which supports the claim that natural sourced substances achieve their full biological potential thanks to the additive and synergistic interactions of all the present compounds. Methanolic extracts of the selected macromycetes were tested at subinhibitory level for their ability to interfere with bacterial cell-cell communication using Pseudomonas aeruginosa PAO1 as a model system. Obtained results suggest that mushroom extract very efficiently reduced the diameter of bacterial colony, structures that enable its dispersion, as well as the synthesis of pigment pyocyanin – its virulent factor. The best antibiofilm effect was observed for the methanolic extract of Polyporus squamosus (88,30%). Moderate cytotoxic potential was exclusive for polysaccharide extract of L. sulphureus 1 against the following carcinogenic cell lines: HeLa, NCI-H460 and MCF-7. In addition, results of the preliminary hepatotoxicity analysis indicate the extracts are non-toxic for the porcine primary cells in tested concentrations, providing the opportunity for safe human consumption of these mushrooms. All the obtained results indicate that the selected macromycetes have potential as functional foods thanks to the production of biologically active compounds which have beneficial health effects and in addition, given their ability to eradicate pathogenic microorganisms, mushrooms are important in the formulation of new available antimicrobial agents. Macromycetes are a true example of tradition and novelty unity and they have significant potential in maintaining and improving health, longevity and quality of life. Their current status and future prospects open up new avenues for improving their status from functional foods to proper medicinal products. Since the tested mushrooms have been successfully cultivated, something like this may actually happen in near future.