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- Cytotoxity induced by extracts of Pisolithustinctorius spores on human cancer and normal cell lines – evaluation of the anticancer potentialPublication . Alves, Ricardo; Preto, Marco; Oliveira, Rui S.; Martins, RosárioCancer is one of the most important causes of death and morbidity worldwide. According to the World Health Organization 8.2 million people have died from cancer in 2012. The treatment of this fast growing disease is regarded as a major challenge in health for the XXI century. Conventional anti-cancer therapy is usually aggressive to the patients due to systemic toxicity caused by lack of specificity of the chemotherapy drugs (Kidd, 2000). This has triggered the interest of the scientific community to search for new and more effective therapies, less aggressive than conventional treatments. Used since antiquity in oriental civilizations, fungi, in particular mushrooms have been shown to limit or reverse tumor growth (Kidd, 2000). Pisolithustinctorius (Basidiomycota) is a soil fungus with world-wide distribution that it known to form ectomycorrhizal symbioses in forest ecosystems (Kope & Fortin, 1990). The sporocarps of this fungus contain Pisosterol, a triterpene that has been shown to have antitumor activity against some cancer cell lines (Montenegro et al., 2004). Nevertheless, no studies have focused on the anticancer potential of other structures such as spores and thus the anticancer potential of P. tinctorius , remains largely unknown.
- Cyanobacteria for skin care and cosmeceutical formulationsPublication . Melo, Luísa; Morone, Janaína; Silva, Margarida; Ferraz, Ricardo; Martins, RosárioSkin acts as a barrier from external stimulus such as pollutants and ultraviolet radiation. Disturbs in the skin are involved in skin aging, which mostly results on a thinner epidermis, dryness, wrinkles, and loss of elasticity. Synthetic ingredients in cosmetics are known to be more toxic and to cause negative impact on the environment. In a society increasingly worried with human and environment health, natural skin care products gain special attention and boosted the search for natural sources. Due to the production of bioactive compounds, cyanobacteria emerged as an excellent source for cosmetic ingredients. Cyanobacteria strains of CIIMAR culture collection (LEGE-CC) have already proven to be interesting for cosmetic purposes [1,2]. In this project we aimed to evaluate the potential of four LEGE-CC cyanobacteria strains for skin care purposes. The cytotoxicity of acetonic and aqueous extracts was evaluated in keratinocytes (HaCAT), fibroblasts (3T3L1) and endothelial cells (hCMEC/D3) along with the total phenolic content and antioxidant properties. Cytotoxic assays revealed toxicity of acetonic extracts to keratinocytes in the higher concentrations tested. Aqueous extracts revealed low or non-cytotoxicity. Mainly in low concentrations of extracts, cellular proliferation was registered. Aqueous extracts from strain JM/RS021A and acetone extracts from strain JM/RS035B revealed higher values for total phenolic compounds. The acetone extracts of strain JM/RS021A showed the greatest scavenging activity values on the DPPH assay.
- Stalling the course of neurodegenerative diseases: could cyanobacteria constitute a new approach toward therapy?Publication . Ramos, Vitória; Reis, Mariana; Ferreira, Leonor; Silva, Ana Margarida; Ferraz, Ricardo; Vieira, Mónica; Vasconcelos, Vitor; Martins, RosárioNeurodegenerative diseases (NDs) are characterized by progressive and irreversible neuronal loss, accompanied by a range of pathological pathways, including aberrant protein aggregation, altered energy metabolism, excitotoxicity, inflammation, and oxidative stress. Some of the most common NDs include Alzheimer’s Disease (AD), Parkinson’s Disease (PD), Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS), and Huntington’s Disease (HD). There are currently no available cures; there are only therapeutic approaches that ameliorate the progression of symptoms, which makes the search for new drugs and therapeutic targets a constant battle. Cyanobacteria are ancient prokaryotic oxygenic phototrophs whose long evolutionary history has resulted in the production of a plethora of biomedically relevant compounds with anti-inflammatory, antioxidant, immunomodulatory, and neuroprotective properties, that can be valuable in this field. This review summarizes the major NDs and their pathophysiology, with a focus on the anti-neurodegenerative properties of cyanobacterial compounds and their main effects.
- Evaluation of the potential of marine cyanobacteria towards Alzheimer’s diseasePublication . Moreira, Ana Rita; Silva, Ana Margarida; Vieira, Mónica; Martins, RosárioAlzheimer’s disease (AD) is one of the most prevalent neurodegenerative diseases (NDs) and the major cause of dementia. Despite the constant efforts to achieve cure, no treatment is currently effective and new approaches are urgently needed. Marine biotechnology has been at the frontline for the bioprospection of natural sources to treat ND. Several marine compounds have already provided evidence for anti-AD drugs by acting in mechanisms such as the inhibition of acetylcholinesterase (AChE). Among marine organisms, cyanobacteria have been recognized as a major source of bioactive compounds and the potential towards AD has been highlighted, namely AChE inhibitors. This project is included in a screening program that aims to evaluate the potential of cyanobacteria from the Culture Collection of CIIMAR -LEGE-CC against AD. In this work, 80 fractions of marine cyanobacteria were screened for the potential to inhibit AChE since this is a proven enzyme target to ameliorate the loss of cholinergic signal and to induce cytotoxicity in the neuroblastoma cell line SHY5Y, in the normal fibroblasts cell line 3T3L1, and in the endothelial cells from central nervous system capillaries hCMEC/D3. Results considering the enzymatic assays and cytotoxic assays revealed that the fractions are not toxic or with low toxicity. The strains Phormidium sp; an unidentified filamentous Synechococcales and Nodosilinea nodulosa revealed moderate toxicity towards AChE.
- Exploring the bioactive potential of Pisolithus (Basidiomycota): comprehensive insights into antimicrobial, anticancer, and antioxidant properties for innovative applicationsPublication . S. Oliveira, Rui; Preto, Marco; Santos, Germana; Silva, Ana Margarida; Vasconcelos, Vitor; Martins, RosárioAddressing pressing health concerns, modern medical research seeks to identify new antimicrobials to combat drug resistance, novel molecules for cancer treatment, and antioxidants for inflammation-related diseases. Pisolithus (Basidiomycota) is a ubiquitous and widely distributed fungal genus in forest ecosystems, known for establishing ectomycorrhizal associations with a range of host plants, enhancing their growth, and conferring protection against biotic and abiotic stresses. Beyond ecological applications, Pisolithus yields bioactive compounds with medicinal potential. This comprehensive review explores the transversal biological activity of Pisolithus fungi, aiming to provide a thorough overview of their antimicrobial, anticancer, and antioxidant potential. The focus is on elucidating bioactive compounds within Pisolithus to trigger further research for innovative applications. Compounds from Pisolithus displayed antimicrobial activity against a broad spectrum of microorganisms, including antibiotic-resistant bacteria. The efficacy of Pisolithus-derived compounds matched established medications, emphasizing their therapeutic potential. In anticancer research, the triterpene pisosterol stood out with documented cytotoxicity against various cancer cell lines, showcasing promise for novel anticancer therapies. Pisolithus was also recognized as a potential source of antioxidants, with basidiocarps exhibiting high antioxidant activity. In vivo validation and comprehensive studies on a broader range of compounds, together with mechanistic insights into the mode of action of Pisolithus-derived compounds, are compelling areas for future research.
- Cyanobacteria secondary metabolites as biotechnological ingredients in natural anti-aging cosmetics: potential to overcome hyperpigmentation, loss of skin density and UV radiation-deleterious effectsPublication . Favas, Rita; Morone, Janaína; Martins, Rosário; Vasconcelos, Vitor; Lopes, GracilianaThe loss of density and elasticity, the appearance of wrinkles and hyperpigmentation are among the first noticeable signs of skin aging. Beyond UV radiation and oxidative stress, matrix metalloproteinases (MMPs) assume a preponderant role in the process, since their deregulation results in the degradation of most extracellular matrix components. In this survey, four cyanobacteria strains were explored for their capacity to produce secondary metabolites with biotechnological potential for use in anti-aging formulations. Leptolyngbya boryana LEGE 15486 and Cephalothrix lacustris LEGE 15493 from freshwater ecosystems, and Leptolyngbya cf. ectocarpi LEGE 11479 and Nodosilinea nodulosa LEGE 06104 from marine habitats were sequentially extracted with acetone and water, and extracts were analyzed for their toxicity in cell lines with key roles in the skin context (HaCAT, 3T3L1, and hCMEC). The non-toxic extracts were chemically characterized in terms of proteins, carotenoids, phenols, and chlorophyll a, and their anti-aging potential was explored through their ability to scavenge the physiological free radical superoxide anion radical (O2•−), to reduce the activity of the MMPs elastase and hyaluronidase, to inhibit tyrosinase and thus avoid melanin production, and to block UV-B radiation (sun protection factor, SPF). Leptolyngbya species stood out for anti-aging purposes: L. boryana LEGE 15486 presented a remarkable SPF of 19 (at 200 µg/mL), being among the best species regarding O2•− scavenging, (IC50 = 99.50 µg/mL) and also being able to inhibit tyrosinase (IC25 = 784 µg/mL), proving to be promising against UV-induced skin-aging; L. ectocarpi LEGE 11479 was more efficient in inhibiting MMPs (hyaluronidase, IC50 = 863 µg/mL; elastase, IC50 = 391 µg/mL), thus being the choice to retard dermal density loss. Principal component analysis (PCA) of the data allowed the grouping of extracts into three groups, according to their chemical composition; the correlation of carotenoids and chlorophyll a with MMPs activity (p < 0.01), O2•− scavenging with phenolic compounds (p < 0.01), and phycocyanin and allophycocyanin with SPF, pointing to these compounds in particular as responsible for UV-B blockage. This original survey explores, for the first time, the biotechnological potential of these cyanobacteria strains in the field of skin aging, demonstrating the promising, innovative, and multifactorial nature of these microorganisms.
- Elucidating the mechanisms Involved in the cytotoxicity induced by marine cyanobacteria strains against the RKO colon carcinoma cell linePublication . Freitas, S.; Urbatzka, R.; Osório, H.; Costa, M.; Barros, Piedade; Vasconcelos, V.; Martins, RosárioCyanobacteria are known to synthesize secondary metabolites that may have potential as drugs for the treatment of human diseases such as cancer. Previous studies on marine cyanobacteria isolated from the Portuguese coast revealed strains of the picoplanktonic genera Cyanobium and Synechocystis as potential sources of anticancer compounds. The ethyl acetate fraction of the strains Cyanobium sp. LEGE06113 and the Synechocystis salina LEGE06155 was found to reduce cell viability of cancer cell lines. This work aimed to elucidate the mechanisms involved in the cytotoxicity of this strains in the colon adenocarcinoma cell line RKO by employing real-time PCR (RT-PCR) for genes involved in cell cycle and apoptosis, by flow cytometry for cell cycle and by two-dimensional gel electrophoresis for protein expression. RT-PCR results revealed differences in mRNA expression of genes CCNB1 (cell cycle) and BCL-2 (apoptosis). Flow cytometry results revealed a decrease in the G0/G1 and S phase and increased its number in the G2/M phase, which is in accordance with the lower expression of CCNB1. The proteomic results demonstrated different protein patterns comparing treatment groups with control. Proteins differentially regulated in exposed RKO cells were involved in cell cycle regulation, apoptosis, cell structure, protein regulation and cell metabolism. Although several of these proteins were identified in cells exposed to both cyanobacterial extracts, the data provides an indication that the cytotoxicity is induced by different toxic mechanisms in LEGE06113 and LEGE06155 fractions.
- Nonthermal high-pressure microalgae extracts: A new source of natural ingredients for cosmeticsPublication . Garcia, Mónica P.; Regueiras, Ana; Lopes, Graciliana; Matos, Gabriela; Silva, Lucília P. da; Cerqueira, Mariana T.; Cardoso, Helena; Correia, Nádia; Saraiva, Jorge A.; Silva, Joana L.; Martins, Rosário; Marques, Alexandra P.Microalgae are one of the most prominent sources of ingredients for the cosmetic industry not only due to their diversity but, importantly, due to the low environmental impact in production and extraction. A sustainable extraction process requires the use of effective and environmentally friendly solvents that guarantee an extract with a relevant content and in high yields. In this work, Chlorella vulgaris, Nannochloropsis oceanica, Tetraselmis chui, Chlorococcum amblystomatis and Phaeodactylum tricornutum microalgae species grown in tubular photobioreactors were used to validate a high-pressure extraction (HPE) method. Extraction yields up to 57 % were obtained depending on the used solvent (water, ethanol, or acetone). The extracts were then characterized regarding their phytochemical composition (total phenolic, protein, chlorophylls content, and chlorophyll a). Aqueous extracts showed high protein content (6–51 μg/mg biomass), while ethanolic and acetone extracts showed high amounts of phenolic compounds (0.0007–0.03 μg/mg biomass). The levels of photosynthetic pigments (0.1–11 μg/mg biomass) associated with antioxidant and anti-inflammatory properties were higher when extracted using ethanol and high-pressures. Chlorella vulgaris aqueous extracts presented higher protein content while Nannochloropsis oceanica and Tetraselmis chui ethanolic extracts presented higher amounts of photosynthetic pigments. The effect of the extracts over the metabolic activity of primary human dermal fibroblasts, keratinocytes, melanocytes, and adipocytes was dependent on both microalgae species and cell type. In adipocytes and fibroblasts, extracts presented an IC50 > 500 μg/mL, except for the one of Phaeodactylum tricornutum in fibroblasts (IC50 < 150 μg/mL). In contrast, the IC50 was below 500 μg/mL for most of the extracts in keratinocytes and melanocytes. Overall, extracts of sustainably-grown microalgae obtained by a high-pressure method are a promising source of natural ingredients for cosmeceutical applications.
- Wild mushrooms for Alzheimer’s diseasePublication . Silva, Margarida Lobo; Reis, Filipa S.; Grosso, Clara; Pérez, Diana Valverde; Barros, Lillian; Martins, RosárioAlzheimer’s disease (AD) is among the most incident, debilitating, and widespread forms of dementia. AD causes the decline of cognitive function, making overall day-to-day tasks difficult and even impossible. Despite extensive research, its origin and progression are still unclear, and no efficient treatment is available to prevent disease progression or mitigate symptoms. Due to the low efficiency in the available treatments, natural compounds have arisen as an alternative. These are gaining crescent relevance and have been explored, given their promising results as immunomodulatory, anti-inflammatory, or neuroprotective factors. Mountain natural resources, specifically mushrooms, are among the most studied sources of bioactive molecules. The Montesinho Natural Park, located in northeast Portugal, has incredible mushroom biodiversity, which has been exploited to obtain health-beneficial compounds, namely with anti-inflammatory potential. This work aimed to study different mushroom extracts and isolated compounds for the prevention and progression of AD. So far, our work aimed at bringing insight into the effect of extract administration in neuroblastoma and endothelial cell lines through the MTT viability assay, as well as studying their effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), enzymes known to be involved in AD pathogenesis through a modified Ellman’s method: dry extracts were resuspended in tris-HCl 50mM + 10% DMSO to concentrations between 2 and 0.03125 mg/mL for enzymatic inhibition assays. To be considered promising, extracts must not cause cellular death of over 15-20% and cause enzymatic inhibition. So far, our results using nine mushroom extracts show low-to-no cytotoxicity in concentrations between 100 and 6.25 µg/mL in endothelial and neuroblastoma cell lines. High concentrations of Lycoperdon umbrinum extract caused cytotoxicity levels of approximately 35% in endothelial cells after 24h treatment; different extracts caused cell proliferation in both cell lines, the most evident being 128% in the lowest concentrations of Russula delica and Boletus fragrans. The most promising results were obtained for Boletus aereus, which, while not cytotoxic, inhibited both enzymes by 60%. Around 30% AChE inhibition was achieved for the two highest concentrations of Agaricus silvicola and Boletus fragrans making both extracts good candidates for further studies.
- Isolation of Hierridin B from a culturable Cyanobium sp. strain isolated from the Portuguese coastPublication . Leão, Pedro N.; Pereira, Alban R.; Costa, Margarida; Ramos, Vítor; Martins, Rosário; Gerwick, William H.; Vasconcelos, VitorCyanobacteria are a genetically diverse group of phototrophic prokaryotes. On a secondary metabolite perspective, however, filamentous and benthic forms have been the major sources of compounds isolated from these organisms. Nevertheless, unicellular free-living, planktonic cyanobacteria also contain enzymatic machinery for secondary metabolite biosynthesis.