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.

Flowers of Gomphrena globosa L. as an alternatve source of betacyanins: optimization of the extraction using response surface methodology

Betacyanins are betalain pigments that display a red-violet colour which have been reported to be three times stronger than the red-violet dye produced by anthocyanins [1]. The applications of betacyanins cover a wide range of matrices, mainly as additives or ingredients in the food industry, cosmetics, pharmaceuticals and livestock feed. Although, being less commonly used than anthocyanins and carotenoids, betacyanins are stable between pH 3 to 7 and suitable for colouring in low acid matrices. In addition, betacyanins have been reported to display interesting medicinal character as powerful antioxidant and chemopreventive compounds either in vitro or in vivo models [2]. Betacyanins are obtained mainly from the red beet of Beta vulgaris plant (between I 0 to 20 mg per I 00 g pulp) but alternative primary sources are needed [3]. In addition, independently of the source used, the effect of the variables that affect the extraction of betacyanins have not been properly described and quantified. Therefore, the aim of this study was to identifY and optimize the conditions that maximize betacyanins extraction using the tepals of Gomphrena globosa L. flowers as an alternative source. Assisted by the statistical technique of response surface methodology, an experimental design was developed for testing the significant explanatory variables of the extraction (time, temperature, solid-liquid ratio and ethanolwater ratio). The identification was performed using high-performance liquid chromatography coupled with a photodiode array detector and mass spectrometry with electron spray ionization (HPLC-PDAMS/ ESI) and the response was measured by the quantification of these compounds using HPLC-PDA. Afterwards, a response surface analysis was performed to evaluate the results. The major betacyanin compounds identified were gomphrenin 11 and Ill and isogomphrenin IJ and Ill. The highest total betacyanins content was obtained by using the following conditions: 45 min of extraction. time, 35•c, 35 g/L of solid-liquid ratio and 25% of ethanol. These values would not be found without optimizing the conditions of the betacyanins extraction, which moreover showed contrary trends to what it has been described in the scientific bibliography. More specifically, concerning the time and temperature variables, an increase of both values (from the common ones used in the bibliography) showed a considerable improvement on the betacyanins extraction yield without displaying any type of degradation patterns.

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.

Postharvest changes in profiles of sugars, organic acids and tocopherols in leafy vegetables monitored by chromatographic techniques

After harvest, plants remain living organisms with the capacity to carry out metabolic processes. Thus, from the moment they are detached from the source of nutrients, they become entirely dependent on their own organic reserves [1]. Postharvest changes cannot be stopped, but they can be slowed within certain limits. Therefore, this study was conducted to evaluate the effects induced by storage in the profiles of sugars, organic acids and tocopherols of two leafy vegetables. Wild samples of watercress (Nasturtium officinale R. Br.) and buckler sorrel (Rumex induratus Boiss. & Reut.), from the Northeastern region of Portugal, were analyzed after harvest (control) and after storage in sterilized packages (using the passive modification mode) at 4ºC for 7 or 12 days, respectively. Analyses were performed by high-performance liquid chromatography (HPLC) using different detectors, i.e., a refraction index detector (RID) for free sugars, a photodiode array detector (PDA) for organic acids, and a fluorescence (FP) detector for tocopherols. The storage time decreased the levels of fructose, glucose and total sugars in both leafy vegetables and increased the total organic acids content. The decrease of these sugars can be related to its use by the plant to produce the required energy. Ascorbic acid was detected in buckler sorrel and decreased with storage; while the amount of malic acid increased in both species. Curiously, all the tocopherol isoforms increased in watercress, while buckler sorrel just present higher values of γ- and δ- tocopherols. In fact, the de novo synthesis of these bioactives compounds can be a plant strategy to fight against the reactive species that are produced during storage. The knowledge of the behavior of these compounds during storage that was achieved with this study [2] may contribute to the development of more effective preservation strategies for leafy vegetables.

Quality control of gamma irradiated dwarf mallow (Malva neglecta Wallr.) based on color, organic acids, total phenolics and antioxidant parameters

This study addresses the effects of gamma irradiation (1, 5 and 8 kGy) on color, organic acids, total phenolics, total flavonoids, and antioxidant activity of dwarf mallow (Malva neglecta Wallr.). Organic acids were analyzed by ultra fast liquid chromatography (UFLC) coupled to a photodiode array (PDA) detector. Total phenolics and flavonoids were measured by the Folin-Ciocalteu and aluminium chloride colorimetric methods, respectively. The antioxidant activity was evaluated based on the DPPH(•) scavenging activity, reducing power, β-carotene bleaching inhibition and thiobarbituric acid reactive substances (TBARS) formation inhibition. Analyses were performed in the non-irradiated and irradiated plant material, as well as in decoctions obtained from the same samples. The total amounts of organic acids and phenolics recorded in decocted extracts were always higher than those found in the plant material or hydromethanolic extracts, respectively. The DPPH(•) scavenging activity and reducing power were also higher in decocted extracts. The assayed irradiation doses affected differently the organic acids profile. The levels of total phenolics and flavonoids were lower in the hydromethanolic extracts prepared from samples irradiated at 1 kGy (dose that induced color changes) and in decocted extracts prepared from those irradiated at 8 kGy. The last samples also showed a lower antioxidant activity. In turn, irradiation at 5 kGy favored the amounts of total phenolics and flavonoids. Overall, this study contributes to the understanding of the effects of irradiation in indicators of dwarf mallow quality, and highlighted the decoctions for its antioxidant properties.