In Vitro Antimicrobial Potential of Portuguese Propolis Extracts from Gerês against Pathogenic Microorganisms.

Antimicrobial resistance (AMR) is one of humanity's main health problems today. Despite all the breakthroughs and research over the past few years, the number of microbial illnesses that are resistant to the available antibiotics is increasing at an alarming rate. In this article, we estimated the biomedical potential of Portuguese propolis harvested from the Gerês apiary over five years, evaluating the in vitro antimicrobial effect of five hydroalcoholic extracts prepared from five single propolis samples and of a hydroalcoholic extract obtained from the mixture of all samples. The antimicrobial potential was firstly assessed by determining the minimum inhibitory concentration (MIC) of these extracts against a panel of three Gram-positive (Bacillus subtilis, methicillin-sensitive Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus) and one Gram-negative bacteria (Escherichia coli), as well as two yeasts (Candida albicans and Saccharomyces cerevisiae). As MIC values against each bacterium were consistent across all the evaluated propolis extracts, we decided to further conduct a disk diffusion assay, which included three commercial antibiotics-erythromycin, vancomycin, and amoxicillin/clavulanic acid-for comparison purposes. In addition to displaying a concentration-dependent antibacterial effect, the hydroalcoholic extracts prepared with 70% ethanol exhibited stronger antimicrobial capacity than vancomycin against B. subtilis (% of increase ranged between 26 and 59%) and methicillin-sensitive S. aureus (% of increase ranged between 63 and 77%). Moreover, methicillin-resistant S. aureus (MRSA) showed susceptibility to the activity of the same extracts and resistance to all tested antibiotics. These findings support that propolis from Gerês is a promising natural product with promising antimicrobial activity, representing a very stimulating result considering the actual problem with AMR.

Examination of Raw Samples and Ethanol Extracts of Gerês Propolis Collected in Different Years.

Propolis, a natural resin created by bees, has garnered significant attention from both the scientific community and industry due to an impressive range of bioactivities. Nonetheless, the intrinsic variability in its chemical composition and bioactive profiles has been hindering propolis' full potential use. We previously showed that ethanol extracts (EEs) of a Portuguese propolis sample (Gerês) collected over four consecutive years displayed similar chemical and biological profiles, a constancy never documented before. However, the characteristics of the unprocessed samples of Gerês propolis were never described. Hence, the central objective of this study is to assess the quality parameters of unprocessed propolis samples collected from Gerês (G), over a four-year period (2019-2022), alongside the analysis of the chemical composition and bioactivities of the EEs prepared with the same raw samples. The ash, wax, balsam and water contents of the unprocessed samples-G19 to G22-showed minor fluctuations, likely attributed to uncontrollable natural events impacting the propolis source and collection process. On the other hand, the antimicrobial and antioxidant activities of all the four ethanol extracts (G19.EE-G22.EE) consistently align with prior studies. Furthermore, the Gerês propolis extracts showed remarkable uniformity in chemical composition parameters too, particularly concerning total polyphenol, flavonoid and ortho-diphenol contents. In summary, our research reinforces the beneficial properties of propolis and show that extracts' bioactivities remain within the reference ranges for Gerês propolis, despite minor differences in unprocessed samples, suggesting a consistent action over time. Thus, this work could be instrumental towards the establishment of standard parameters for propolis applications, offering valuable insights to this field of propolis research.

Phenolic Compounds Contribution to Portuguese Propolis Anti-Melanoma Activity.

Melanoma is the deadliest type of skin cancer, with about 61,000 deaths annually worldwide. Late diagnosis increases mortality rates due to melanoma's capacity to metastasise rapidly and patients' resistance to the available conventional therapies. Consequently, the interest in natural products as a strategy for drug discovery has been emerging. Propolis, a natural product produced by bees, has several biological properties, including anticancer effects. Propolis from Gerês is one of the most studied Portuguese propolis. Our group has previously demonstrated that an ethanol extract of Gerês propolis collected in 2018 (G18.EE) and its fractions (n-hexane, ethyl acetate, and n-butanol) decrease melanoma cell viability. Out of all the fractions, G18.EE-n-BuOH showed the highest potential as a melanoma pharmacological therapy. Thus, in this work, G18.EE-n-BuOH was fractioned into 17 subfractions whose effect was evaluated in A375 BRAF-mutated melanoma cells. The subfractions with the highest cytotoxic activity were analysed by UPLC-DAD-ESI/MSn in an attempt to understand which phenolic compounds could account for the anti-melanoma activity. The compounds identified are typical of the Gerês propolis, and some of them have already been linked with antitumor effectiveness. These results reaffirm that propolis compounds can be a source of new drugs and the isolation of compounds could allow its use in traditional medicine.

An Insight on the Biomedical Potential of Portuguese Propolis from Gerês.

Osteoarthritis (OA), a progressive degenerative disease of weight-bearing joints, is the second leading cause of disability in the world. Despite all the advances and research over the last years, none of the proposed strategies has been effective in generating functional and long-lasting tissue. Due to the high prevalence of OA and the urgent need for an effective and successful treatment, interest in natural products as anti-inflammatory agents, such as propolis and its components, has emerged. In this work, we estimate the biomedical potential of Portuguese propolis, evaluating the in vitro antioxidant and anti-inflammatory effects of single hydroalcoholic extracts prepared with propolis from Gerês sampled over a five-year period (2011-2015) (G.EE70 and G.EE35). The in vivo and in vitro anti-inflammatory potential of the hydroalcoholic extract of mixtures of the same samples (mG.EE70 and mG.EE35) was evaluated for the first time too. DPPH• radical scavenging and superoxide anion scavenging assays showed the strong antioxidant potential of both hydroalcoholic extracts, either prepared from single propolis samples or from the mixtures of the same samples. Results also revealed an anti-inflammatory effect of mG.EE35, both in vitro by inhibiting BSA denaturation and in vivo in the OA-induced model by improving mechanical hyperalgesia as well as the gait pattern parameters. Results further support the use of propolis blends as a better and more efficient approach to take full advantage of the bioactive potential of propolis.

Portuguese Propolis Antitumoral Activity in Melanoma Involves ROS Production and Induction of Apoptosis.

Melanoma is the most aggressive and life-threatening skin cancer type. The melanoma genome is the most frequently mutated, with the BRAF mutation present in 40-60% of melanoma cases. BRAF-mutated melanomas are characterized by a higher aggressiveness and progression. Adjuvant targeted treatments, such as BRAF and MEK inhibitors, are added to surgical excision in BRAF-mutated metastatic melanomas to maximize treatment effectiveness. However, resistance remains the major therapeutic problem. Interest in natural products, like propolis, for therapeutic applications, has increased in the last years. Propolis healing proprieties offer great potential for the development of novel cancer drugs. As the activity of Portuguese propolis has never been studied in melanoma, we evaluated the antitumoral activity of propolis from Gerês (G18.EE) and its fractions (n-hexane, ethyl acetate (EtOAc), and n-butanol) in A375 and WM9 melanoma cell lines. Results from DPPH•/ABTS• radical scavenging assays indicated that the samples had relevant antioxidant activity, however, this was not confirmed in the cell models. G18.EE and its fractions decreased cell viability (SRB assay) and promoted ROS production (DHE/Mitotracker probes by flow cytometry), leading to activation of apoptotic signaling (expression of apoptosis markers). Our results suggest that the n-BuOH fraction has the potential to be explored in the pharmacological therapy of melanoma.