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- Blackberries and Mulberries from Covilhã as Health Promotors: Physical and Phytochemical Characterization and Biological PotentialPublication . Martins, Mariana Sofia Morgado; Alves, Gilberto Lourenço; Silva, Luís Rodrigues da; Gonçalves, Ana CarolinaFruits and vegetables are increasingly the focus of scientific studies due to their relevance in terms of healthy eating, well-being, and medical and therapeutic importance. This highlight is closely related to the various bioactive compounds present in them, which in turn, gives them notable antioxidant, anti-inflammatory, anti-cancer and anti-aging properties, as well as protection against cardiovascular, neurodegenerative and metabolic diseases (diabetes, hypertension, among others). Among the fruits, blackberries (Rubus and Morus) are one of the summer fruits most appreciated by consumers, due to their sweet flavor and pleasant aroma, their colour, high percentage of water, and low fat and calorie content, as well as due to its richness in several bioactive compounds that have already demonstrated the ability to produce notable physiological and metabolic effects that are important in the prevention of some pathologies, such as cardiovascular, neurodegenerative diseases and cancer. This research project carried out the characterization of 3 similar red fruits highly appreciated by consumers, which were 2 species of shrub blackberries (R. fruticosus and R. ulmifolius) and 1 species of tree mulberry (M. nigra). Taking into account the increase in their interest and in their production, which translates into an increase in their economic impact, it was decided to study their physicochemical characteristics, namely their weight, firmness, acidity, total soluble solids, among other characteristics, and their nutritional values in order to select the one with the greatest commercial value and biological potential. Considering the physicochemical data, the length and width varied between 14.34-20.42 mm and 14.31-25.58 mm, respectively, with the species R. fruticosus having the greatest length and the species R. ulmifolius the greatest width. The weight varied between 2.16 g and 7.75 g, with the species R. fruticosus presenting the highest value. On the other hand, firmness varied from 5.5 N (R. ulmifolius) to 5.6 N (R. fruticosus), while moisture and ash contents varied between 75.8% (M. nigra) and 79.1% R. fruticosus) and between 97.7% (R. ulmifolius) and 99.5% (R. fruticosus), respectively. The species R. fruticosus was the one that exhibited the darkest color. The phenolic profile was identified by HPLC-DAD-ESI/MSn and quantified by HPLC-DAD. In total, 20 phenolic compounds were determined, including 8 anthocyanins and 12 unstained phenolic compounds. On the other hand, through ICP-MS and FAAS, a total of 27 minerals were determined, including 13 essential and 14 non-essential elements. Ca and Mg were the most abundant in all species, while Pb was found in smaller quantities. A total of 68 volatiles were identified using SPME/GC-MS, namely 17 aldehydes, 13 alcohols, 3 ketones, 20 esters, 2 furans, 8 hydrocarbons and 5 acids. As expected, qualitative and quantitative differences were observed between species. All families described influence the nutritional potential, aroma and consumer interest. The initial characterization of blackberries allowed us to conclude that blackberries from Covilhã region have a good nutritional and bioactive composition, which could be an added value for the development of new dietary, nutraceutical, pharmaceutical and other products. Furthermore, qualitative and quantitative differences were found in the parameters evaluated between the different species. Comparing the values obtained, R. fruticosus appears to be the species with the most appealing characteristics, both for the consumer and for the products and market. Then, using the concentrated phenolic extracts of the varieties, obtained by solid phase extraction, their antioxidant potential was also evaluated, as well as the potential arising from their combination with the positive control used, which was ascorbic acid. The objective of this last test was to detect the existence of a possible synergistic interaction, enhancing the activity of both. In fact, it is common knowledge that cellular metabolism generates nitrogen and oxygen free radicals necessary for the survival and activity of cells, whose levels are mostly controlled by intracellular antioxidant enzymes, namely catalase, glutathione peroxide and superoxide dismutase. However, age, a sedentary lifestyle, smoking and drinking habits, among others, contribute to an abnormal increase in these species, which can consequently react with proteins, lipids and DNA of healthy cells, damaging them, thus contributing for the acceleration of aging and the development of a wide range of diseases, such as cancer, neurodegenerative and cardiovascular diseases, diabetes, among others. Therefore, to reduce the excessive formation of free radicals and their harmful effects on the human body, it is essential to adopt a diet rich in antioxidant foods. In fact, a daily intake of 400 g of fruits and vegetables is highly recommended. Therefore, the antioxidant activity of blackberry extracts was evaluated through in-house tests against the radicals 1,1-diphenyl-2- picrylhydrazyl (DPPH?), nitric oxide (?NO) and superoxide (O2 ?-). The species R. fruticosus proved to be the most active against DPPH? (IC50=34.29 ± 0.55 µg/mL) while the species R. ulmifolius was the one that revealed the greatest potential in capturing ?NO (IC50=59.49 ± 0.81 µg /mL). On the other hand, in the O2?- test, the species M. nigra and R. fruticosus showed the best results (IC25=14.26 ± 0.47 µg/mL and 14.70 ± 0.58 µg/mL, respectively). Regarding its combination with the positive control, and among the mixtures made, the combination of the species M. nigra with ascorbic acid (25:75) was the mixture that showed the greatest activity against DPPH?, revealing an IC50 of 3.14 ± 0.18 µg/mL, presenting synergistic potential. On the other hand, in terms of activity against ?NO, the mixture M. nigra + ascorbic acid (75:25) was the most promising (IC50=16.97 ± 0.88 µg/mL). On the other hand, in the O2?- uptake assay, the combination of the species R. ulmifolius + ascorbic acid (25:75) was the one that proved to be the most promising, presenting an IC25 value of 7.73 ± 0.33 µg/mL. The data obtained through this study supports the evidence already reported that blackberries have a promising antioxidant capacity, being able to capture free radicals, and most likely, protect cells from the damage caused by them. Finally, the cytotoxicity effects of the extracts were tested, first on normal human dermal fibroblast (NHDF) cells and then on human Caco-2 colon carcinoma cells. The first evaluation was to select non-toxic concentrations for non-cancerous cells. With the selected concentrations, the effect of the extracts on Caco-2 cells was tested. Carrying out this test made it possible to verify that, among the 3 extracts studied, that of the cultivated species R. fruticosus is the one that has the most notable antiproliferative effect. Next, the most promising extract was combined with the anti-tumor drug 5- fluorouracil (5-FU) in order to understand whether the combination of both would enhance the effects of inhibiting the growth of Caco-2 cells. With this last test carried out, it was possible to see that the 50:50 combination between the minimum concentration of 5-FU studied (0.65 µg/mL) together with the highest concentration of the promising extract tested (800 µg/mL) presents potential synergistic and highly notable against this studied tumor line. These results may also be highly promising, encouraging the combination of blackberry and mulberry extracts with new therapeutic, pharmacological and nutraceutical approaches, as well as their incorporation into dietary supplements.