Natural products as a powerful source of antioxidants

Synthetic antioxidants could be replaced by the valid bio-based alternatives. These renewable and low cost resources are available and offers a market opportunity for a green supply of raw materials for different industrial and health products. The aim of the present PhD project was to analyse the antioxidant activity of extracts obtained from different natural sources or microorganisms, in order to study their beneficial effects against oxidative stress injury and to characterize their chemical composition to obtain molecules to be used in food or cosmetic industry. Therefore, fruits and vegetables were considered a good starting point, as it is widely known that they are rich in molecules with antioxidant activity. As a first goal, the methanol extracts from two tomato varieties and from açai berries were analysed. These products are among the most consumed in Mediterranean and Brazilian diet, respectively. The beneficial effects of the extracts were demonstrated on a cell-based model in which oxidative stress was induced and the molecules responsible for the antioxidant activity were determined. Thereafter, to avoid the toxicity and high costs of solvent extractions, the attention was focused on extracts obtained without the need of solvents. In particular, the antioxidant activity of a water extract from the cladodes of Opuntia ficus-indica was analysed. The whole extract, as well as its active molecules, showed a good ability to protect human keratinocytes from UVA induced oxidative stress. Plants and their fruits are not the only source of antioxidants, as these are metabolites produced by microbes. From a biotechnological and economical point of view, the production of antioxidants by microorganisms is very convenient and provides an alternative to chemical synthesis. For this reason, two bacterial strains were used in different ways. In the first case, a lipophilic extract from Novosphingobium sp. PP1Y was obtained and tested, as the characterization of this recently isolated microorganism showed the presence of a great abundance of genes encoding for carotenoids. PP1Y extract was able to counteract oxidative stress in an in vitro system on cells as well as in an in vivo system on C. elegans. In the second case, the whole spore from the bacterium Bacillus subtilis was used as antioxidant. The results showed that incubation with spores induced the activation in the treated cells of Nrf-2 pathway, which induced the transcription of anti-stress response genes. Finally, a waste fraction from microalgae Chlorella sorokiniana Shihiraet Krauss was tested for its antioxidant activity. This fraction was discarded after starch purification (PhD project of Dr. Imma Gifuni). Therefore, in order to minimize the generation of waste, the recovery of high-value products was attempted. It was demonstrated that the cascade approach was a good strategy to produce starch and active antioxidants. In conclusion, in the present PhD thesis, different antioxidants from natural sources have been isolated and tested, and extracts from these sources showed an antioxidant power similar or higher than the isolated molecules. This opens the way to the use of the whole extract in food or cosmetic industries, avoiding the high costs for the purification of single compounds, in accordance with the increasing demand of natural products endowed with pro-healthy effects

Alternative use of Bacillus subtilis spores: Protection against environmental oxidative stress in human normal keratinocytes

Inorganic trivalent arsenic is a major environmental pollutant and exposure to human results in many pathologies, including keratosis and carcinoma. Here, we analyzed the effects of B. subtilis spores on human normal keratinocytes in the presence of sodium arsenite oxidative stress. Pre-treatment of cells with spores before inducing oxidative stress was able to keep normal levels of intracellular ROS, GSH and lipid peroxidation, as well as to inhibit the activation of the MAPK cascade. Moreover, spores showed a positive effect on cell proliferation, probably due to their binding on the cell surface and the activation of intracellular catalases. We found that spores exert their protective effect by the nuclear translocation of Nrf-2, involved in the activation of stress response genes. This, in turn, resulted in a protective effect against sodium arsenite stress injury, as oxidative stress markers were reported to physiological levels when cells were stressed before incubating them with spores. Therefore, B. subtilis spores can be considered as a new agent to counteract oxidative stress on normal human keratinocytes

Isolation of Myricitrin and 3,5-di-O-Methyl Gossypetin from Syzygium samarangense and Evaluation of their Involvement in Protecting Keratinocytes against Oxidative Stress via Activation of the Nrf-2 Pathway

The wax apple (Syzygium samarangense) is traditionally employed as an antibacterial and immunostimulant drug in traditional medicine. This plant is rich in different flavonoids and tannins. In this study, we isolated two compounds from S. samarangense leaves: myricitrin and 3,5-di-O-methyl gossypetin. Then, we investigated the mechanisms of action of the two compounds against oxidative stress (induced by sodium arsenite) and inflammation (induced by UV light) on human keratinocytes. We could clearly demonstrate that the pre-treatment of cells with both compounds was able to mitigate the negative effects induced by oxidative stress, as no alteration in reactive oxygen species (ROS) production, glutathione (GSH) level, or protein oxidation was observed. Additionally, both compounds were able to modulate mitogen-activated protein kinase (MAPK) signaling pathways to counteract oxidative stress activation. Finally, we showed that 3,5-di-O-methyl gossypetin exerted its antioxidant activity through the nuclear transcription factor-2 (Nrf-2) pathway, stimulating the expression of antioxidant proteins, such as HO-1 and Mn-SOD-3

An ascorbic acid-enriched tomato genotype to fight UVA-induced oxidative stress in normal human keratinocytes

UVA radiations contribute up to 95% of the total UV exposure and are known to induce cell damage, leading to apoptosis. Since the benefic effects of ascorbic acid on human health are well known, a new tomato genotype (namedDHO4), highly rich in ascorbic acid, has been recently obtained. Here,we compared the effects of ascorbic acid and hydrophilic DHO4 extracts in protecting human keratinocytes exposed to UVA stress. Keratinocytes were pre-incubated with ascorbic acid or with extracts from the ascorbic acid enriched tomato genotype and irradiated with UVA light. Then, ROS production, intracellular GSH and lipid peroxidation levels were quantified. Western blots were carried out to evaluate mitogen-activated protein kinases cascade, activation of caspase-3 and inflammation levels. We demonstrated that ROS, GSH and lipid peroxidation levels were not altered in cell exposed to UVA stress when cellswere pre-treatedwith ascorbic acid or with tomato extracts. In addition, no evidence of apoptosis and inflammationwere observed in irradiated pre-treated cells. Altogether,we demonstrated the ability of an ascorbic acid enriched tomato genotype to counteract UVA-oxidative stress on human keratinocytes. This protective effect is due to the high concentration of vitamin C that acts as free radical scavenger. This novel tomato genotype may be used as genetic material in breeding schemes to produce improved varieties with higher antioxidant levels

Gold-based drug encapsulation within a ferritin nanocage: X-ray structure and biological evaluation as a potential anticancer agent of the Auoxo3-loaded protein

Auoxo3, a cytotoxic gold(iii) compound, was encapsulated within a ferritin nanocage. Inductively coupled plasma mass spectrometry, circular dichroism, UV-Vis absorption spectroscopy and X-ray crystallography confirm the potential-drug encapsulation. The structure shows that naked Au(i) ions bind to the side chains of Cys48, His49, His114, His114 and Cys126, Cys126, His132, His147. The gold-encapsulated nanocarrier has a cytotoxic effect on different aggressive human cancer cells, whereas it is significantly less cytotoxic for non-tumorigenic cells

Malvidin and cyanidin derivatives from açai fruit (Euterpe oleracea Mart.) counteract UV-A-induced oxidative stress in immortalized fibroblasts

UV-A radiations are known to induce cellular oxidative stress, leading to premature skin aging. Consumption of açai fruit (Euterpe oleracea Martius) is known to have many health benefits due to its high level of antioxidants. Herein, we analyzed the ability of phenolic compounds extracted from this fruit to attenuate UV-A-induced oxidative stress in immortalized fibroblast. A methanol/water açai extract was fractionated by HPLC and each fraction tested for anti-oxidant stress activity. Immortalized fibroblasts were pre-incubated with açai fractions and then exposed to UV-A radiations. Açai extract was found to be able to strongly protect cells from oxidative stress. In particular, reactive oxygen species (ROS) production, GSH depletion, lipid peroxidation and no increase in the phosphorylation levels of proteins involved in the oxidative stress pathway was observed in cells pre-incubated with the extract and then irradiated by UV-A. Mass spectrometry analyses of HPLC fractionated extract led us to the identification of malvidin and cyanidin derivatives as the most active molecules able to counteract the negative effects induced by UV-A irradiation. Our results indicate, for the first time, that açai fruit is a valuable natural source for malvidin and cyanidin to be used as anti-stress molecules and represent good candidates for dietary intervention in the prevention of age related skin damage

Protective effect of Opuntia ficus-indica L. cladodes against UVA-induced oxidative stress in normal human keratinocytes

Opuntia ficus-indica L. is known for its beneficial effects on human health, but still little is known on cladodes as a potent source of antioxidants. Here, a direct, economic and safe method was set up to obtain water extracts from Opuntia ficus-indica cladodes rich in antioxidant compounds. When human keratinocytes were pre-treated with the extract before being exposed to UVA radiations, a clear protective effect against UVA-induced stress was evidenced, as indicated by the inhibition of stress-induced processes, such as free radicals production, lipid peroxidation and GSH depletion. Moreover, a clear protective effect against apoptosis in pre-treated irradiated cells was evidenced. We found that eucomic and piscidic acids were responsible for the anti-oxidative stress action of cladode extract. In conclusion, a bioactive, safe, low-cost and high value-added extract from Opuntia cladodes was obtained to be used for skin health/protection

Bioactive compounds in brassicaceae vegetables with a role in the prevention of chronic diseases

The beneficial role of the Mediterranean diet in the prevention of chronic diseases, including cardiovascular diseases, diabetes, and obesity, is well-recognized. In this context, Brassicaceae are considered important vegetables due to several evidences of their health promoting effects that are associated to bioactive compounds present in the edible parts of the plants. In this review, the mechanisms of action and the factors regulating the levels of the bioactive compounds in Brassicaceae have been discussed. In addition, the impact of industrial and domestic processing on the amount of these compounds have been considered, in order to identify the best conditions that are able to preserve the functional properties of the Brassicaceae products before consumption. Finally, the main strategies used to increase the content of health-promoting metabolites in Brassica plants through biofortification have been analyzed

Ferritin nanocages loaded with gold ions induce oxidative stress and apoptosis in MCF-7 human breast cancer cells

Two anticancer gold(III) compounds, Au2phen and Auoxo4, were encapsulated within a ferritin nanocage. The gold-compound loaded proteins were characterized by UV-Vis spectroscopy, inductively coupled plasma mass spectrometry and circular dichroism. X-ray crystallography shows that the compounds degrade upon encapsulation and gold(I) ions bind Ft within the cage, close to the side chains of Cys126. The gold-encapsulated nanocarriers are cytotoxic to human cancer cells. Au(I)-loaded Ft, obtained upon the encapsulation of Au2phen within the cage, induces oxidative stress activation, which finally leads to apoptosis in MCF-7 cell