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- Sweet Cherries as Health Promoters: Valuable Red Fruits with Nutritive and Functional PropertiesPublication . Gonçalves, Ana Carolina Almeida ; Silva, Luís Manuel Lopes Rodrigues da; Alves, Gilberto Lourenço; Ferreira, Amílcar Celta Falcão RamosCurrently, it is widely recognized that consuming fruits and vegetables effectively reduces the risk of morbidity and mortality caused by cardiovascular and cancerous diseases, among others, being widely recommended a daily intake of 400 g of fruits and vegetables. In fact, the potential of fruits and vegetables to treat various ailments and alleviate symptoms, such as migraines, metabolic syndrome, intestinal problems, physical pain, certain cancer types, rheumatoid arthritis, dizziness, colds, fever, psychological fatigue, and symptoms derived from rheumatoid arthritis, among others, has been known since ancient times. Nowadays, the trend is increasing, being accompanied by an emphasis on different communities. This interest is essentially since it is believed that, unlike synthetic pharmaceutics that cause undesirable side effects, natural products have few, or no side effects, and are easy to obtain and economical. Natural products have been the subject of many studies and an important topic of discussion among the medical and scientific communities. Indeed, the biological potential of a wide range of products has already been recognized, being already incorporated in many pharmaceutical drugs. These beneficial activities are directly related to their nutritional constituents, namely due to the presence of vitamins, minerals, carotenoids and fiber, as well as phenolic compounds. The combination of all these compounds is beneficial and capable of promoting the good functioning of human organisms, and hence, promoting the normalization of several parameters to basal levels and contributing to general well-being. Among the various compounds, special emphasis has been given to phenolic compounds. As far as we know, phenolic compounds derive from the secondary metabolism of plants and their main function is to protect them against abiotic (water, sunlight, and temperature) and biotic (attacks by microorganisms) factors. These are commonly divided into 2 large subclasses: (i) non-coloured phenolic compounds (e.g., hydroxybenzoic and hydroxycinnamic acids, flavan-3-ols, flavonols, among others) and (ii) coloured compounds (anthocyanins, which are largely responsible by the colors exhibited by various natural products). Recent research has demonstrated that their chemical structure gives them a remarkable ability to reduce levels of oxidative stress and interact with proinflammatory cascades, thus restoring basal levels and consequently reducing the risk of occurrence of many diseases, or alleviating your symptoms, and consequently, contributing to a better quality of life. The human body naturally possesses intracellular antioxidant enzymes (superoxide dismutase, glutathione peroxidase and catalase) that are considered essential for the survival and health of the population, once, under physiological conditions, they are capable of balancing the levels of free radicals. However, given genetic factors, unexpected decompensation, severe discomfort, and lifestyle choices, such as sedentary habits, consumption of alcohol and tobacco, and/or intake of foods very rich in fats and calories, the aforementioned enzymes become insufficient to guarantee basal levels, leading to proteins, organs and cells injury. This damage is caused by high levels of free radicals and the appearance of exacerbated inflammatory responses in the human body. Consequently, this decompensation “triggers” for the appearance and progression of many diseases whose prevalence is increasing worldwide. These data are corroborated by many studies, which describe that free radicals and pro-inflammatory species are largely related to oxidative stress, and the appearance of various types of cancer, autoimmune diseases, such as rheumatoid arthritis, and syndromes, including the metabolic one. Given the mentioned facts, it is not surprising that many studies involving cherries consumption are being carried out, since it is urgent to find accessible and effective therapies for the entire population, and preferably natural based, in order to improve their quality of life. Cherry (Prunus avium Linnaeus) is a fruit highly appreciated by consumers, not only due to its organoleptic characteristics, but also due to its nutritional value and health benefits. In fact, it has been shown that this fruit is an excellent source of macro and micronutrients, and phytochemicals. In fact, this last class of compounds has been the subject of many studies, being a topic of discussion among various communities given their various positive effects on health. Among the different nutritional classes found, cherries have high contents of phenolic compounds, highlighting the presence, until now, of anthocyanins, flavan-3-ols, flavonols and hydroxycinnamic acids. Particularly, the main compounds identified are cyanidin 3-O-rutinoside and -glucoside, quercetin, rutin, kaempferol, catechin, and ρcoumaroylquinic and chlorogenic acids. Therefore, the richness of cherries in bioactive compounds has been one of the reasons for the growing interest of the scientific community in exploring the beneficial healthpromoting effects associated with their intake. Furthermore, consumers are increasingly well-informed and, once, they are looking for healthy products, such as cherries, there is verified an increase in their demand. Faced with this, the market responds by increasing their global production, especially of the cultivars that most attract consumers. To date, several scientific studies carried out on animals and humans suggest that consuming cherries reduces the risk of several inflammatory and chronic diseases, such as rheumatoid arthritis, cardiovascular diseases, diabetes and cancer. Clinical evidence has already demonstrated that consuming cherries can reduce the scale of pain caused by arthritis, gout and inflammation, possibly due to their ability to increase superoxide dismutase levels, and reduce inflammatory mediators (TNF-α, MDA and PGE-2) and serum levels of C-reactive protein levels, as well as inhibiting cyclooxygenase (COX)-2, which is one of the main proteins responsible for enhancing the pro-inflammatory response. Furthermore, cherries have also shown to be effective in reducing muscle pain, accelerating recovery and improving the performance of recreational exercisers and high-competition athletes, diminishing risk factors associated with the onset of diabetes and cardiovascular diseases, and associated with stress and anxiety, thus improving sleep and mood, memory and cognitive functions. More recently, it has been reported that the consumption of cherries can also alleviate hepatic steatosis and inhibit the activity of the α-glucosidase enzyme, thus delaying the conversion of starch and disaccharides into glucose. Furthermore, cherries also show potential to protect human erythrocytes against free radicals and to inhibit the proliferation of cancer cells. These health-promoting activities are closely related to cherry phenolic content, which is the main responsible for offering this berry, notable antioxidant and antiinflammatory capabilities. This fact is supported by several studies and correlations already performed. In recent years, the production of this fruit has increased considerably worldwide, including in Portugal. Our country produces around 20,000 tonnes per year. A large part of this production occurs in the Fundão region, and hence, it is not surprising that sweet cherries present a distinguished economic impact in this region. The availability of detailed information about their health-promoting properties could lead to an increase in consumer demand, raising their consumption and use of pharmaceutical and nutraceutical products, contributing to the valorisation of the region and the fixation of people and industrial companies. Therefore, with this doctoral project, the aim was to extensively characterize, for the first time, the quality parameters and phytochemical and mineral composition of the best-known cultivars in the region, with the intention of helping in the selection of the most promising cultivars. In total, 23 cultivars were characterized. The results obtained revealed that, among the cultivars studied, there are significant differences in the physicochemical characteristics and in the phenolic, mineral and volatile profiles, showing the verified variability between the various cherry cultivars is mainly influenced by the genotype of the cultivar. In general, Black star and Starkrimson cultivars had the highest soluble solids content, while the highest acidity value was found in Sweetheart cultivar. On the other hand, Cristalina, Kordia and Santina cultivars were those that exhibited the most intense/dark color, while the lightest ones were Sunburst and Sweetheart. Relatively to phenolic compounds, 46 phenolic compounds were identified by HPLC-DAD-ESI/MSn, including 9 hydroxycinnamic acids, 2 hydroxybenzoic acids, 13 flavonols, 5 flavan-3-ols, 2 flavanones, 1 flavanonol and 4 anthocyanins. Among the compounds, chlorogenic acids were the majority noncoloured phenolic compounds, while cyanidin 3-O-rutinoside was the most predominant coloured phenolic compound. Regarding their levels, Sunburst cultivar had the highest amounts of unstained compounds, while the Tavora, Garnet and 4-84 cultivars had the highest concentration of anthocyanins. With regard to mineral content, 27 were identified by ICP-MS and flame atomic absorption spectrometry, namely 12 essential and 15 non-essential. The element potassium (K) was the most abundant element detected in all cultivars, while Thallium (Tl) was the least abundant. On the other hand, the analysis of volatile organic compounds by SPME/GC-MS showed that cherries have a wide variety of these, having been detected a total of 66 volatiles from 8 different families, including 16 aldehydes, 23 alcohols, 6 ketones, 6 esters, 8 monoterpenes, 3 norisoprenoids, 2 hydrocarbons and 2 acids. Among the compounds, benzaldehyde, hexanal, nonanal, benzyl alcohol, (E)-2-hexen-1-ol, 1- hexanol, (Z)-2-hexen -1-ol, 2-ethyl-1-hexanol, linalool, α-terpineol and α-ionone were the main found. Based on the obtained results, from a commercial point of view, Cristalina, Saco, Tavora, 4-84, Bigalise, Celeste and Satin cultivars might be considered some of the most interesting cultivars, since they offer a better flavor and a higher percentage of edible fruit, and consequently, major intake of phytochemicals, mainly due to their size, weight, and phenolic, mineral and volatile contents. The microbial ecology of the Saco cultivar was also explored for the first time. In total, 22 different bacteria and 33 fungi were isolated. The genera of Pseudomonas spp. (27.273%) and Ralstonia spp. (18.182%) were the most dominant bacteria, followed by Bacillus spp., Staphylococcus spp., Erwinia spp., Tatumella spp. and Dermacoccus spp. (each with 9.091%). Regarding fungi, Metschnikowia spp. (39.394%) was the most abundant genus, followed by Aureobasidium spp. (27.273%) and Hanseniaspora spp. (18.182%). In the initial stages of fruit development, Erwinia Tasmaniensis, Peudomonas viridiflava and Pseudomonas syringae bacteria are the first to emerge, while Ralstonia pickettii, Bacillus altitudinis, Enterococcus Rotai, Tatumellla terrea, Pseudomonas qingdaonensis, Pseudomonas gramininis, Dermacoccus nishinomiyaensis and Buttiauxella ferragutiae appear in the final stages of fruit ripening. Regarding fungi, Metschnikowia spp. (39.39%) was the most abundant genus, followed by Aureobasidium spp. (27.27%) and Hanseniaspora spp. (18.18%). The majority of fungi were detected in the final stages of fruit ripening, particularly the fungi Hanseniaspora uvarum, Metschnikowia pulcherrima, Hanseniaspora pseudoguilliermondii, Penicillium crustosum, Hanseniaspora meyeri, Aureobasidium proteae and Aureobasidium pullulans. The study of the microbial ecology of fruits and vegetables is vital because they can be a potential vector of foodborne pathogenic diseases and/or an important reservoir of microorganisms capable of improving the quality, characteristics and nutritional value of foods, and exert positive effects on human health. The isolation of certain microorganisms can also be an added value at an industrial level. Additionally, the in vitro biological properties of Saco cultivar were also evaluated, namely its potential to reduce free radicals and pro-inflammatory levels, as well as its potential to protect human blood samples against hemolysis and hemoglobin oxidation, interfere with the growth of cancer cells and with the activity of P-glycoprotein (P-gp), one of the main proteins related to resistance seen against several drugs, as well of αglucosidase enzyme. It was also observed that both fractions and the total extract can also inhibit the activity of α-glucosidase enzyme. Saco cultivar was chosen to carry out these assays, since it already showed to possess considerable biological properties and also for being one of the most produced cultivars in Portugal, possessing inclusive, protected geographical indication. Therefore, with the intention of increasing knowledge of the biological potential of the various phenolic compounds, and for the first time, 2 fractions rich in phenolic compounds were extracted using a solid phase C18 column, a fraction I, rich in noncoloured phenolic compounds, and fraction II, rich in anthocyanins. For comparison purposes, a total extract (III) rich in both phenolic subclasses was also tested and the results were further compared between them and with positive controls. In general, the three extracts showed a remarkable ability to capture free radicals and ferric species, as well as to interfere with the activity of proteins related to inflammation (iNOS and COX-2), and with the transmembrane transport protein, P-gp, as well as with the activity of α-glucosidase enzyme, in dose-dependent manner. It was also possible to verify that the three extracts demonstrated effectiveness in inhibiting the proliferation of cancer cells, namely colon, stomach and liver cancer cells, causing necrosis at the highest concentration (800 µg/mL for colon and stomach cells) and 100 µg/mL for liver cells). Both fractions and the total extract also showed the ability to reduce induced-oxidative stress in cancer cells, as well as in neuroblastoma model cells. From the obtained data, it is important to highlight the biological potential of the fraction rich in anthocyanins, which is in accordance with the literature. In fact, anthocyanins have been a target of many studies, due to their chemical structure. Indeed, anthocyanins are composed of several hydroxyl groups, which gives them a remarkable biological potential, namely, to reduce levels of free radicals and inflammation. Furthermore, it was also verified that the interaction between different phenolic compounds, which was observed in the total extract, was an added value in the majority of the assays done. In order to deepen the results obtained, the antioxidant activity of the main individual phenolic compounds present in cherries was evaluated against DPPH, nitric oxide and superoxide radicals. The obtained values for the DPPH radical revealed that anthocyanins, (-)-epicatechin and kaempferol 3-O-rutinoside were the most active phenolic compounds against this radical, while isorhamnetin 3-O-glucoside was the least. On the other hand, anthocyanins, (-)-epicatechin, quercetin 3-O-glucoside and caffeic acid proved to be the most effective in scavenging nitric oxide radicals, while ρhydroxybenzoic acid was the least efficient. In relation to the superoxide radical, quercetin and its derivatives showed the highest capacity, while cyanidin aglycone did not show the potential to intercept this radical at the concentrations tested. Additionally, molecular docking and absorption, distribution, metabolism, and excretion (ADME) studies were carried out, and it was observed that compounds with lower molecular masses, such as kaempferol, can easily interact with proteins related to oxidative stress, interfering in their activity, and thus, contributing to lower the concentration of free radicals to basal levels. The results obtained are highly promising and encourage translation into clinical trials, as well as the incorporation of cherries and/or their extracts into new medicines, cosmetic products, food supplements and nutraceuticals.