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.

Magnetic carbon xerogels for the catalytic wet peroxide oxidation of sulfamethoxazole in environmentally relevant water matrices

Novel magnetic carbon xerogels consisting of interconnected carbon microspheres with iron and/or cobalt microparticles embedded in their structure were developed by a simple route. As inferred from the characterization data, materials with distinctive properties may be directly obtained upon inclusion of iron and/or cobalt precursors during the sol-gel polymerization of resorcinol and formaldehyde, followed by thermal annealing. The unique properties of these magnetic carbon xerogels were explored in the catalytic wet peroxide oxidation (CWPO) of an antimicrobial agent typically found throughout the urban water cycle – sulfamethoxazole (SMX). A clear synergistic effect arises from the inclusion of cobalt and iron in carbon xerogels (CX/CoFe),the resulting magnetic material revealing a better performance in the CWPO of SMX at the ppb level(500 microg L−1) when compared to that of monometallic carbon xerogels containing only iron or cobalt.This effect was ascribed to the increased accessibility of highly active iron species promoted by the simultaneous incorporation of cobalt.The performance of the CWPO process in the presence of CX/CoFe was also evaluated in environmentally relevant water matrices, namely in drinking water and secondary treated wastewater, considered in addition to ultrapure water. It was found that the performance decreases when applied to more complex water and wastewater samples. Nevertheless, the ability of the CWPO technology for the elimination of SMX in secondary treated wastewater was unequivocally shown, with 96.8% of its initial content being removed after 6 h of reaction in the presence of CX/CoFe, at atmospheric pressure, room temperature(T = 25◦C), pH = 3, [H2O2]0= 500 mg L−1and catalyst load = 80 mg L−1. A similar performance (97.8% SMX removal) is obtained in 30 min when the reaction temperature is slightly increased up to 60◦C in an ultra-pure water matrix. Synthetic water containing humic acid, bicarbonate, sulphate or chloride, was also tested. The results suggest the scavenging effect of the different anions considered, as well as the negative impact of dissolved organic matter typically found in secondary treated wastewater, as simulated by the presence of humic acid.An in-situ magnetic separation procedure was applied for catalyst recovery and re-use during reusability cycles performed to mimic real-scale applications. CWPO runs performed with increased SMX concentration (10 mg L−1), under a water treatment process intensification approach, allowed to evalu-ate the mineralization levels obtained, the antimicrobial activity of the treated water, and to propose adegradation mechanism for the CWPO of SMX.

Effects of electron-beam irradiation on chemical and antioxidation parameters of wild Macrolepiota procera dried samples

Mushrooms are very perishable foods due to their high susceptibility to moisture loss, changes in color and texture, or microbiological spoilage. Drying is considered as the most appropriate method to prevent these alterations, but it has some limitations, such as shrinkage, enzymatic and non-enzymatic browning reactions, and oxidation of lipids and vitamins. Irradiation might effectively attenuate the undesirable changes caused by drying process, ensuring also higher shelf-life of mushrooms and their decontamination [I]. In the present work, the combined effects of electron-beam irradiation (at 0, 0.5, 1 and 6 kGy doses) and storage time (at 0, 6 and 12 months) were evaluated and compared. Macrolepiota procera (Scop.) Singer wild samples were obtained in Tnis-os-Montes, in the Northeast of Portugal, and dried at 30 •c in an oven. Subsequently, the samples were divided in four groups: control (non-irradiated, 0 kGy); sample 1 (0.5 kGy); sample 2 (1 kGy) and sample 3 (6 kGy). The irradiation was performed at the lNCTInstitute of Nuclear Chemistry and Technology (lNCT), in Warsaw, Poland. Moisture, protein, fat, carbohydrates and ash were determined following standard procedures. Free sugars and tocopherols were determined by high performance liquid chromatography coupled to a refraction index detector (HPLC-RI) and a fluorescence detector, respectively; fatty acids were determined by gas-liquid chromatography with flame ionization detection (GC-FID). Antioxidant activity was evaluated in the methanolic extracts by in vitro assays measuring DPPH (1,1-diphenyl-2- picrylhydrazyl) radical scavenging activity, reducing power, inhibition of ~-carotene bleaching and inhibition oflipid peroxidation using thiobarbituric acid reactive substances (TBARS) assay. Total phenolics were also determined by the Folin-Ciocalteu assay. All the parameters showed a decrease tendency with storage time. Trehalose and y-tocopherol were preserved with 1 kGy dose. Electron-beam irradiation did not impart additional changes to most of the chemical and antioxidant parameters of M. procera dried samples. This is a very promising result, since electron-beam irradiation might attenuate most unwanted changes caused by drying, maintaining its long-term effectiveness.

Ethanolic extracts from three commercial edibe mushrooms: determination of ergosterol contente and evaluation of bioactives properties

Mushrooms are rich in several bioactive metabolites among them are phenolic compounds, terpenoids, polysaccharides, lectins, and steroids including mycosterols, namely ergosterol [1]. Ethanolic extracts prepared by maceration of several mushroom species have been recently described as having antiinflammatory properties [2]. In the present work, ethanolic extracts of Agaricus bisporus L., Lentinus edodes (Berk.) Pegler and Pleurotus ostreatus (Jacq. ex Fr.) P.Kumm., purchased from a local supermarket in the Northeast of Portugal, were obtained by Soxhlet and chemically characterized in terms of ergosterol content by HPLC-UV. The antioxidant properties of these extracts were evaluated through DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity (RSA), reducing power (RP), p. carotene bleaching inhibition (CBI) and lipid peroxidation inhibition in TBARS (thiobarbituric acid reactive substances) assay (LPI); the antioxidant activity of ergosterol was also evaluated by the DPPH assay. The anti-inflammatory activity of the same extracts and ergosterol was evaluated in LPS (lipopolysaccharide) stimulated RAW 264.7 macrophages, through the inhibition of NO production. A. bisporus revealed the highest content in ergosterol (44.8 ± 0.4 mg/ g extract) followed by P. ostreatus (34 ± 3 mg/ g extract) and finally L. edodes (8.9 ± 0.1 mg/ g extract). A. bisporus showed the highest RSA, RP and CBI (EC50 values= 7.0 ± 0.8, 2.3 ± 0.1 and 1.4 ± 0.1 mg/mL, respectively), while L. edodes presented the highest LPI (2.5 ± 0.1 mg/mL ); ergosterol revealed higher RSA (0.46±0. 0 I mg/mL) than the extracts. Concerning the anti-inflammatory potential, the most efficient species was L. edodes (lC50 value = 164 ± 16 J.lg/mL), followed by A. bisporus (185 ± 16 J.lg/mL) and finally P. ostreatus (290 ± 10 J.lg/mL). However, ergosterol presented lower activity (338 ± 23 J.lg/mL) due to its low solubility in the culture medium. The higher antioxidant properties displayed by A. bisporus can be related with its higher ergosterol content, while in the anti-inflammatory activity this relation cannot be established also due to the low solubility of ergosterol in the cells culture medium, decreasing the ergosterol availability. More studies are being conducted regarding the ergosterol solubility. Several compounds have been implicated in the bioactivity of mushrooms and in this study we have found that ergosterol can give an important contribution.

Gamma radiation preserves chemical and bioactive properties of Boletus edulis wild mushrooms

Boletus edulis Bull: Fr. is an edible mushroom quite appreciated for its organoleptic and nutritional properties. However, the seasonality and perishability cause some difficulties in its distribution and marketing in fresh form; losses associated with this type of food during marketing can reach 40% [1]. Irradiation is recognized as a safe and effective method for food preservation, being used worldwide to increase shelf life of fresh and dehydrated products (e.g. fruits, vegetables and spices) [2]. In particular, gamma irradiation has already been applied to cultivated mushrooms (especially Agaricus, Lentinula and Pleurotus Genus) and proved to be an interesting conservation technology [3]. However, the studies with added-value wild species are scarce. In this work, the effects of gamma irradiation on chemical and antioxidant properties of wild B. edulis, were evaluated. Fruiting bodies were obtained in Trás-os-Montes, in the Northeast of Portugal, in November 2012. The irradiation was performed in experimental equipment with 60Co sources at 1 and 2 kGy. All the results were compared with nonirradiated samples (control). Macronutrients and energy value were determined following official procedures of food analysis; fatty acids were analyzed by gas-chromatography coupled to flame ionization detection (GC-FID), while sugars and tocopherols were determined by high performance liquid chromatography (HPLC) coupled to refraction index (RI) and fluorescence detectors, respectively. Antioxidant activity was evaluated in the methanolic extracts by in vitro assays measuring DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity, reducing power, inhibition of β- carotene bleaching and inhibition of lipid peroxidation using thiobarbituric acid reactive substances (TBARS) assay. Total phenolics were also determined by the Folin-Ciocalteu assay. The nutritional profiles were not affected in high extension. Fatty acids and sugars were slightly affected, decreasing with the increasing doses. The performed assays for antioxidant activity, indicate that irradiated samples tended to have lower scavenging activity and reducing power, but higher lipid peroxidation inhibition. Despite the detected differences in individual compounds, the results of nutritional parameters, the most relevant in terms of mushroom acceptability by consumers, were less affected, indicating an interesting potential of gamma-irradiation to be used as an effective conservation technology for the studied mushrooms.

Bioactivity and chromatographic analysis of nutrients and non-nutrients of two Leccinum species

Nowadays the rising cost of health care and pharmaceutical products, the increase in life expectancy as well as the demand for an improved quality of life, has led to an increased concern about food intake and an emergence of new concepts of nutrition [1]. Mushrooms have been pointed out as an excellent option to include in a healthy diet, due to their nutritional value [2] associated with their bioactive properties [3]. The current study presents the chemical profile of two edible species, Leccinum molle (Ban) Ban and Leccinum vulpinum Watling, harvested in the outskirts of Bragan9a (Northeastern Portugal), regarding their content in nutrients and nonnutrients. Individual profiles of sugars and fatty acids were obtained by HPLC-RI and GC-FID, respectively. Tocopherols were analysed by HPLC-fluorescence, and the non-nutrients (i.e., phenolic and other organic acids) by HPLC-PDA. The antioxidant activity of the methanolic extracts obtained from both species was assessed with different assays (e.g. reducing power, radical scavenging activity and lipid peroxidation inhibition) and their hepatotoxicity was evaluated in primary cell cultures obtained from porcine liver, PLP2. Generally, both Leccinum species revealed similar nutrient profiles, with low fat levels, fructose, mannitol and trehalose as the foremost free sugars, and higher percentages of mono- and polyunsaturated fatty acids in comparison with saturated fatty acids. The presence of bioactive compounds was also detected, namely phenolic (e.g., gallic, protocatechuic and p-hydroxybenzoic acids) and organic acids (e.g., citric and fumaric acids). Both species presented antioxidant properties, being L. vulpinum the species which showed the most promising results (higher contents of total phenolic acids and lower ECso values in all the performed assays). Neither of the extracts presented toxicity against the liver primary cells PLP2, up to maximal concentration tested (Giso > 400 μg/ml).

Antioxidant potential of two Apiaceae plant extracts: a comparative study focused on the phenolic composition

The present study aimed to characterize the extracts prepared from Pimpinella anisum L. (anise) and Coriandrum sativum L. (coriander) (Apiaceae plants) seeds in terms of phenolic composition, and to correlate the obtained profiles with the antioxidant activity. Anise gave the highest abundance in phenolic compounds (42.09± 0.11 mg/g extract), mainly flavonoids (28.08±0.17 mg/g extract) and phenolic acids (14.01±0.06 mg/g extract), and also the highest antioxidant potential, measured by the ability to inhibit lipid peroxidation and β-carotene bleaching, the reducing power and the free radical scavenging activity. Apigenin and luteolin derivatives, as also caffeoylquinic acid derivatives seem to be directly related with the higher in vitro antioxidant potential of the anise extract. In contrast, the lower antioxidant potential of coriander seems to be due to its lower abundance in phenolic compounds (2.24±0.01 mg/g extract). Further studies are necessary to evaluate the in vivo antioxidant potential of the tested extracts, but the in vitro experiments already performed highlight them as potential health promoters.

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.

Polyporus squamosus (Huds.) Fr from different origins: chemical characterization, screening of the bioactive properties and specific antimicrobial effects against Pseudomonas aeruginosa

Wild mushrooms are mainly collected during the rainy season and valued as a nutritious food and sources of natural medicines and nutraceuticals. The aim of this study was to determine the chemical composition and bioactive properties (antioxidant, antimicrobial and cytotoxicity) of Polyporus squamosus from two different origins, Portugal and Serbia. The sample from Portugal showed higher contents of as protein (17.14 g/100 g), fat (2.69 g/100 g), ash (3.15 g/100 g) and carbohydrates (77.02 g/100 g); the same sample gave the highest antioxidant activity: highest reducing power, DPPH radical scavenging activity, and lipid peroxidation inhibition in both β-carotene/linoleate and TBARS assay. These results could be related to its higher content in total tocopherols (1968.65 μg/100 g) and phenolic compounds (1.29 mg/100 g). Both extracts exhibited antibacterial activity against all the tested organisms. The samples from Serbia gave higher overall antibacterial activity and showed excellent antibiofilm activity (88.30 %). Overall, P. squamosus methanolic extracts possessed antioxidant, antimicrobial, antibiofilm and anti-quorum sensing activity, and without toxicity for liver cells. This investigation highlights alternatives to be explored for the treatment of bacterial infections, in particular against Pseudomonas aeruginosa. This study provides important results for the chemical and bioactive properties, especially antimicrobial activity of the mushroom P. squamosus. Moreover, to the authors’ knowledge this is the first report on sugars, organic acids, and individual phenolic compounds in P. squamosus.

Novel chromone and xanthone derivatives: Synthesis and ROS/RNS scavenging activities

Chromones and xanthones are oxygen-containing heterocyclic compounds acknowledged by their antioxidant properties. In an effort to develop novel agents with improved activity, a series of compounds belonging to these chemical classes were prepared. Their syntheses involve the condensation of appropriate 2-methyl-4H-chromen-4-ones, obtained via Baker-Venkataraman rearrangement, with (E)-3-(3,4-dimethoxyphenyl)acrylaldehyde to provide the corresponding 2-[(1E,3E)-4-(3,4-dimethoxyphenyl)buta-1,3-dien-1-yl]-4H-chromen-4-ones. Subsequent electrocyclization and oxidation of these compounds led to the synthesis of 1-aryl-9H-xanthen-9-ones. After cleavage of the protecting groups, hydroxylated chromones and xanthones were assessed as scavenging agents against both reactive oxygen species (ROS) [superoxide radical (O2(•-)), hydrogen peroxide (H2O2), hypochlorous acid (HOCl), singlet oxygen ((1)O2), and peroxyl radical (ROO(•))] and reactive nitrogen species (RNS) [nitric oxide ((•)NO) and peroxynitrite anion (ONOO(-))]. Generally, all the tested new hydroxylated chromones and xanthones exhibited scavenger effects dependent on the concentration, with IC50 values found in the micromolar range. Some of them were shown to have improved scavenging activity when compared with previously reported analogues, allowing the inference of preliminary conclusions on the structure-activity relationship.