Study of antioxidant, antiproliferative and apoptosis-inducing properties of wild mushrooms from the Northeast of Portugal

Mushrooms are known as a powerful source of bioactive compounds including antioxidants, inhibitors of human tumour cell lines growth, inducers of apoptosis and enhancers of immunity. Indeed, many pre-clinical studies have been conducted in human tumour cell lines and in some cases a number of compounds isolated from mushrooms have followed to clinical trials. The Northeast of Portugal is one of the European regions with higher wild mushrooms diversity. However, to our knowledge, no studies had been conducted so far to verify their bioactivities. The main aim of this work was the evaluation of the bioactive properties (antioxidant properties and growth inhibitory potential on human tumour cell lines) of wild edible mushrooms collected in the Northeast of Portugal. Once properly identified, methanolic, ethanolic and boiling water extracts were prepared from thirty eight wild mushroom species collected in that region. Chemical characterization was obtained by high performance liquid chromatography (HPLC) coupled to a photodiode array detector (DAD) or to a refraction index detector (RI). Antioxidant activity assays were carried out in those extracts, including evaluation of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging capacity, reducing power and inhibition of β-carotene bleaching. Extract-induced cell growth inhibition was assessed with the sulforhodamine B assay in four human tumour cell lines (NCI-H460 - lung cancer, MCF-7 -breast cancer, HCT-15 -colon cancer and AGS - gastric cancer). The effects on cell cycle profile and apoptosis were evaluated by flow cytometry and the effect on the expression levels of proteins related to cell cycle and apoptosis was further investigated by Western blotting. Three wild edible mushroom species revealed growth inhibitory activity in the studied human tumour cell lines: Clitocybe alexandri ethanolic extract, Lepista inversa methanolic extract and Suillus collinitus methanolic extract. C. alexandri ethanolic extract induced an S-phase cell cycle arrest and increased the percentage of apoptotic cells, in the NCI-H460 cell line. The analysed mushroom species also provided interesting antioxidant potential, mainly the boiling water extract of L. inversa which showed the highest DPPH radical scavenging activity, reducing power and β-carotene bleaching inhibition. S. collinitus methanolic extract induced a slight increase in the number of cells in G1, with a concomitant decrease in the percentage of cells in the S phase of the cell cycle and an increase in the percentage of apoptotic cells, in the MCF-7 cell line. The combined use of the S. collinitus methanolic extract and etoposide caused a greater decrease in the percentage of cell growth, when compared to either of them used individually, indicating the potential benefit of this combination. The tested extracts were chemically characterized and protocatechuic, p-hydroxybenzoic, p-coumaric and cinnamic acids were the main compounds identified on the phenolic (methanolic and ethanolic) extracts, while mannitol, trehalose and arabinose were the main sugars found in the polysaccharidic (boiling water) extracts after hydrolysis. The individual compounds identified in the extracts were submitted to a screening of tumour cells growth inhibitory activity, but only the phenolic acids and a related compound, cinnamic acid, presented activity. This compound was found to be the most potent one regarding cell growth inhibition in the NCI-H460 cell line. The effect of the individual and combined treatment with the identified compounds was also evaluated. Cinnamic and protochatequic acids caused a statistically significantly reduction in the number of viable cells. In addition, p-hydroxybenzoic acid did not show any significantly reduction in the viable cell number. Nevertheless, it was verified that the concomitant use of the three compounds provided the strongest decrease in the viable cell number, suggesting a possible concomitant effect of those compounds. Overall, the present work has contributed to further understand the bioactive potential of wild edible mushrooms from the Northeast of Portugal. This study allowed to identify some species with antioxidant or tumour cell growth inhibitory potential.

Investigating mushroom LMW compounds as potential Bcl-2 inhibitors: docking studies using AutoDock4

The B cell CLL/lymphoma-2 (Bcl-2) family is functionally classified as either anti-apoptotic or pro-apoptotic, and the regulation of its interactions dictates survival or commitment to apoptosis. Bcl-2 family is also implicated in a wide range of diseases. In some types of cancers, including lymphomas and epithelial cancers, protein overexpression of anti-apoptotic Bcl-2 family, such as the Bcl-2 protein is indicative of cancer in an advanced stage, with a poor prognosis and resistant to chemotherapy [1]. Several reports indicate that mushrooms have the ability to promote apoptosis in tumour cell lines, but the mechanism of action is not fully understood. Inhibition of the interaction between Bcl-2 (anti-apoptotic protein) and proapoptotic proteins could be an important step in the mechanism of mushroom induced apoptosis. Therefore, the discovery of compounds with the capacity to inhibit Bcl-2 is an ongoing research topic on cancer therapy. In this work, docking studies were performed using a dataset of 40 low molecular weight (LMW) compounds present in mushrooms. The docking software AutoDock 4 was used and docking studies were performed using 5 selected Bcl-2 crystal structures as targets. Compounds with the lowest predicted binding energy (predΔG) are expected to be the more potent inhibitors. Among the tested compounds, steroids presented the lowest predΔG with several exhibiting values below -9 kcal/mol. The results are corroborated by several reports that state that steroids induce apoptosis in several tumor cells. It is thus feasible that they might act by preventing Bcl-2 from forming complexes with the respective proapoptotic protein interaction partners, namely Bak, Bax, and Bim. Moreover, previous studies on our research group demonstrated that 48 h treatment of MCF-7 cells (breast carcinoma) with Suillus collinitus methanolic extract caused a decrease in Bcl-2, highlighting the antitumor potential of this mushroom species [2]. In conclusion, the process of apoptosis promoted by mushroom extracts may be related to the inhibition of Bcl-2 by the steroid derivatives herein studied. However, further studies are needed to confirm this hypothesis.

Chromatographic techniques to obtain the biomolecules profile of wild Suillus granulates

In recent years the interest in naturally occurring compounds has been increasing worldwide. Indeed, many of the bioactive compounds currently used as medicines have been synthesized based on the structure of natural compounds [1]. In order to obtain bioactive fractions and subsequently isolated compounds derived from natural matrices, several procedures have been carried out. One of these is to separate and assess the concentration of the active compound(s) present in the samples, a step in which the chromatographic techniques stand out [2]. In the present work the mushroom Sui/Ius granulatus (L.) Roussel was chemically characterized by chromatographic techniques coupled to different detectors, in order to evaluate the presence of nutritional and/or bioactive molecules. Some hydrophilic compounds, namely free sugars, were identified by high performance liquid chromatography coupled to a refraction index detector (HPLC-RI), and organic and phenolic acids were assessed by HPLC coupled to a photodiode array detector (HPLC-PDA). Regarding lipophilic compounds, fatty acids weredetermined by gas chromatography with a flame ionization detector (GC-FID) and tocopherols by HPLC-fluorescence detection. Mannitol and trehalose were the main free sugars detected. Different organic acids were also identified (i.e. oxalic, quinic and fumaric acids), as well as phenolic acids (i.e. gallic and p-hydroxybenzoic acids) and the related compound cinnamic acid. Mono- and polyunsaturated fatty acids were the prevailing fatty acids and a-, ~- and ~-tocopherol were the isoforms of vitamin E detected in the samples. Since this species proved to be a source of biologically active compounds, the antioxidant and antimicrobial properties were evaluated. The antioxidant activity was measured through the reducing power, free radical's scavenging activity and lipid peroxidation inhibition of its methanolic extract, and the antimicrobial activity was also tested in Gram positive and Gram negative bacteria and iri different fungi. S. granulatus presented antioxidant properties in all the performed assays, and proved to inhibit the growth of different bacterial and fungal strains. This study is a first step for classifying S. granulatus as a functional food, highlighting the potential of mushrooms as a source of nutraceutical and biologically active compounds.

Nutritional and biochemical profiling of Leucopaxillus candidus (Bres.) singer wild mushroom

The wild mushroom Leucopaxillus candidus (Bres.) Singer was studied for the first time to obtain information about its chemical composition, nutritional value and bioactivity. Free sugars, fatty acids, tocopherols, organic and phenolic acids were analysed by chromatographic techniques coupled to different detectors. L. candidus methanolic extract was tested regarding antioxidant potential (reducing power, radical scavenging activity and lipid peroxidation inhibition). L. candidus was shown to be an interesting species in terms of nutritional value, with high content in proteins and carbohydrates, but low fat levels, with the prevalence of polyunsaturated fatty acids. Mannitol was the most abundant free sugar and β-tocopherol was the main tocopherol isoform. Other compounds detected were oxalic and fumaric acids, p-hydroxybenzoic and cinnamic acids. The methanolic extract revealed antioxidant activity and did not show hepatoxicity in porcine liver primary cells. The present study provides new information about L. candidus.

Wild Roman chamomile extracts and phenolic compounds: enzymatic assays and molecular modelling studies with VEGFR-2 tyrosine kinase

Angiogenesis is a process by which new blood vessels are formed from the pre-existing vasculature, and it is a key process that leads to tumour development. Some studies have recognized phenolic compounds as chemopreventive agents; flavonoids, in particular, seem to suppress the growth of tumor cells modifying the cell cycle. Herein, the antiangiogenic activity of Roman chamomile (Chamaemelum nobile L.) extracts (methanolic extract and infusion) and the main phenolic compounds present (apigenin, apigenin-7-O-glucoside, caffeic acid, chlorogenic acid, luteolin, and luteolin-7-O-glucoside) was evaluated through enzymatic assays using the tyrosine kinase intracellular domain of the Vascular Endothelium Growth Factor Receptor-2 (VEGFR-2), which is a transmembrane receptor expressed fundamentally in endothelial cells involved in angiogenesis, and molecular modelling studies. The methanolic extract showed a lower IC50 value (concentration that provided 50% of VEGFR-2 inhibition) than the infusion, 269 and 301 μg mL(-1), respectively. Regarding phenolic compounds, luteolin and apigenin showed the highest capacity to inhibit the phosphorylation of VEGFR-2, leading us to believe that these compounds are involved in the activity revealed by the methanolic extract.

Candidacidal potential of licorice phenolic extract: emphasis on its mode of action

Medicinal plants bave gained a special attention in the last years, due to its renowned health benefits, such as antimicrobial effects [I]. In fact, several natural matrices bave been increasingly studied, namely for its antifungal activity against opportunistic fungi [2,3]. Candida species, although commensa! microorganisms, have caused severe organic dysfunctions to the host, once current antifungal agents have lost their recognized efficiency [2]. So, numerous studies have been carried out focusing the mechanisms of acquired drug-resistance by Candida species [extracts combination), the discovery of the involved mechanisms of actions, morphological changes and related kinetic parameters are of major importance. Glycyrrhiza glabra L. (licorice) hydromethanolic extract have evidenced promissory candidacidal effects, and therefore, the involved mechanisms of action need to be clarified. Thus, in the present study these modes of action were assessed, by using flow cytometry.Overall, the licorice extract induced significant and irreversible primary damages on Candida cells, being membrane disruption and consequent unviability one of the main targets. In fact, after membrane destabilization, cells lost their proper homeostasis, their metabolic functions were blocked and, consequently cells lost functionality. The relevance and interest of the achieved results open new insights towards the upcorning use of the present phenolic matrix, being important to evaluate its in viva efficacy. Therefore, further studies are necessary to deepen knowledge on this field, aiming not only to establish therapeutic and prophylactic doses, but also to improve the clinical intervention in Candida infections.

Effects of gamma and electron beam irradiation in the antioxidante potential of methanolic extracts and infusions of Arenaria Montana L

Irradiation is a methodology qualified for dry ingredients preservation or decontamination and can be performed using various radiation sources and energy levels in accordance with the objectives to be achieved [1]. Electron beam irradiation is used mainly for food products with low density, while gamma irradiation is mainly used for large volumes [2]. Arenaria Montana L. has a high antioxidant potential and richness in bioactive phytochemicals. It is used in Portuguese traditional medicine, acting therapeutically as an anti-inflammatory and diuretic plant [3]. The aim of this work was to evaluate the effects of gamma and electron beam irradiation at different doses (I and 10 kGy) in the antioxidant activity of A. montana. Free radicals scavenging activity, reducing power and lipid peroxidation inhibition properties of its methanolic extracts and infusions were evaluated. Through a global analysis, it was concluded that the antioxidant activity proved to be higher in methanolic extracts in comparison with the infusions, where it decreased with increasing irradiation dose regardless of the technology used (gamma or electron beam). For methanolic extracts, electron beam resulted in increased antioxidant activity while gamma irradiation caused a decrease in these extracts. Thus, the antioxidant potential is variable depending not only on the type of radiation and the dose applied, but also on the solvent used in the preparation of the extracts (methanol or water).

Ceratonia siliqua L. hydroethanolic extract obtained by ultrasonication: antioxidant activity, phenolic compounds profile and effects in yogurts functionalized with their free and microencapsulated forms

Bioactive extracts were obtained from powdered carob pulp through an ultrasound extraction process and then evaluated in terms of antioxidant activity. Ten minutes of ultrasonication at 375 Hz were the optimal conditions leading to an extract with the highest antioxidant effects. After its chemical characterization, which revealed the preponderance of gallotannins, the extract (free and microencapsulated) was incorporated in yogurts. The microspheres were prepared using an extract/sodium alginate ratio of 100/400 (mg mg(-1)) selected after testing different ratios. The yogurts with the free extract exhibited higher antioxidant activity than the samples added with the encapsulated extracts, showing the preserving role of alginate as a coating material. None of the forms significantly altered the yogurt's nutritional value. This study confirmed the efficiency of microencapsulation to stabilize functional ingredients in food matrices maintaining almost the structural integrity of polyphenols extracted from carob pulp and furthermore improving the antioxidant potency of the final product.