Euterpe oleracea pulp extract: Chemical analyses, antibiofilm activity against Staphylococcus aureus, cytotoxicity and interference on the activity of antimicrobial drugs
Graphical abstract
Introduction
Staphylococcus aureus is a bacterial species that is both part of the human skin microbiota and a facultative intracellular opportunistic pathogen, which causes diseases such as folliculitis, necrotizing fasciitis, bacteremia, septic arthritis and endocarditis [1]. This species is able to evade the host immune system due to events that include biofilm formation and production of toxins, what extend the inflammatory process and delay wound healing [2]. Biofilm formation by S. aureus is critical concerning issues such as hospital infections and food safety: hospital infections by S. aureus are generally hard to control and treat due to poor susceptibility to biocides and antimicrobials, and are responsible for several deaths worldwide; furthermore, staphylococcal contamination of food and water, one of the leading causes of foodborne illnesses, is associated to biofilm formation and production of heat-stable enterotoxins [3], [4], [5].
Biofilms are structurally complex microcolonies that grow on extracellular polymeric substances (EPS) matrix, which is generally composed of carbohydrates, extracellular DNA, proteins and lipids [5]. The biochemical composition of the EPS may undergo variations triggered by factors such as type and availability of nutrients, pH and temperature variations, and the diversity of the microbial community [6]. Most of the known bacterial species live as biofilms (sessile cells), and less than 1% lives as free (planktonic) cells. In the environment, microorganisms attached to biofilms are shielded against environmental factors such as radiation, temperature extremes and lack of nutrients, and in living organisms, against the immune system and antimicrobial drugs, in order to guarantee their existence [6].
Bacterial resistance to antimicrobial drugs is a current public health concern, and a growing resistance picture of S. aureus is observed worldwide [3]. Infectious diseases can quickly become technically difficult to treat with synthetic drugs due to resistance mechanisms such as biofilm formation and efflux pumps. The market of antimicrobial drugs is financially not attractive to pharmaceutical companies, and the discovery of novel drugs is far of providing solutions for this eminent problem [7]. Thus, exploring the antimicrobial potential of natural products of vegetable sources becomes an important alternative in this context. Curiously, plant food often largely used for culinary purposes due to their benefits to health are not so investigated for this objective as other (generally) non-edible parts like stem barks and leafs.
Euterpe oleracea Mart. (Arecaceae) is a palm tree broadly distributed in the northern zone of Brazil (Amazon forest region), and its dark violet berries (Açaí fruit) are used in the preparation of different food products such as juices and other beverages, creams and ice creams. In Brazil, the appreciation of the exotic flavor of the berries was mostly concentrated in the northern zone of the country at first. In the latest years, açaí commercialization as a functional food increased considerably. Açaí extracts are also explored for the development of nutraceutical, dietary supplements and cosmetics, reaching markets worldwide [8], [9]. Açaí berries and extracts are among the most important food products of the Brazilian Amazon region. Different pharmacological activities have been described for the açaí pulp, such as improvement of serum lipid profile and cardiovascular functions, immunomodulation, anti-inflammatory, antioxidant, and more recently, anticonvulsant properties [8], [9], [10], [11], [12], [13], [14], [15], [16]. Conversely, the antimicrobial potential of the pulp remains poorly explored.
Here, to the best of our knowledge, we report for the first time the antimicrobial activity of the methanolic extract of the Brazilian açaí pulp (MEAP) against clinical isolates of S. aureus, including biofilms. The antiproliferative potential of the MEAP was investigated in vitro against hepatocellular carcinoma cells. We investigated biochemical and physicochemical characteristics of the pulp and conducted UPLC and GC-MS analyses for polyphenols and volatiles, respectively. Also, we combined the MEAP to antimicrobial drugs, and observed a statistically significant synergism with most of the tested antimicrobial drugs. Considering the scarcity of antimicrobial studies with this plant and its potential for clinical use in a near future, our results become even more relevant.
Section snippets
Açaí samples
A pure açaí pulp was purchased from the local artisanal market in Belém (capital of Pará state, eastern Brazilian Amazon), a city where the plant is widely available. Fruits were harvested at the fully mature stage, which coincided with the highest anthocyanin concentration [17]. The pulp was prepared by macerating the berries in lukewarm potable water, grinding them to crush the softened pulp. The resulting juice pulp was briefly filtered for removing large-sized particles like fragments of
Chemical analyses
The correlation of flavonoids and colorimetric analysis for qualitative studies is mostly based on the molecule characteristics such as the number and position of substituents moieties. In this work, two preliminary approaches were explored to infer the presence of flavonoids in the açaí sample. Shinoda test was positive for the pulp extract, in which a red color was observed. In the TLC analysis, the pulp extract presented two bands: a yellow-orange band, suggestive of the presence of aurones
Discussion
A growing resistance picture of S. aureus is observed worldwide [3]. Here, to our knowledge, we show for the first time that the MEAP is effective against both planktonic cells and biofilms of S. aureus. A previous work explored the antimicrobial activity of Brazilian açaí pulp extracts prepared in aqueous solutions of 0.1% hydrochloric acid and 0.1% sodium bisulfate [29]. Both extracts failed to kill strains of S. aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli and
Conclusion
The MEAP showed antimicrobial activity against S. aureus planktonic cells, and biofilm eradication by this extract was also demonstrated. Synergic interactions of the MEAP and some synthetic antimicrobial drugs were observed, suggesting that the extract may provide positive contributions to drug therapy in this context. Moreover, the extract was effective against HepG2 cells. However, a limitation of the method is the necessity of confirmatory studies of the synergic effect in vivo, as well as
Acknowledgments
The authors are thankful to Gustavo Costa (PharmBSc, Centro Universitário de Caratinga, MG, Brazil), for the excellent support on the experiments. This study was partially was partially funded by grants provided from the Santo Agostinho Institute to FJBF. MVDS is supported by grants from Fundação de Amparo à Pesquisa do Estado de Minas Gerais and RMDS is recipient of a Masters scholarship from CNPq.
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