Evidence and Perspectives on the Use of Phlorotannins as Novel Antibiotics and Therapeutic Natural Molecules †

: Multiple drug-resistant bacterial strains are showing new different mechanisms to overcome the antimicrobial action which reduces the efficacy of conventional antibiotics [1]. Therefore, drug discovery research has focused on developing fast, effective and safe alternatives to prevent this multiresistance. Phlorotannins are a diverse class of polyphenols, secondary metabolites described in brown algae, that are mainly constituted of polymers of phloroglucinol and depending on their linkage and structure can be classified mainly as fucols, fucophlorethols, eckols and phlo-roethols [2]. These polyphenols have been described in both macro-and microalgae, suggesting that they can be recovered from a great variety of sources [2]. Phlorotannins have been extensively described to possess several biological properties, foremost as antioxidant and antimicrobial compounds. Several in vitro reports have described that phlorotannins showed growth inhibition and bactericidal effects against Gram + (e.g., Bacillus cereus , Streptococcus epidermidis , Staphylococcus aureus ) and Gram ‒ bacteria ( e.g., Salmonella sp., Campylobacter jejuni , Pseudomonas aeruginosa ), also including antibiotic-resistant strains like MRSA [3]. Although the mechanisms of action of this group of compounds has not been fully elucidated, tannins could interact with membrane proteins and key metabolic enzymes, impeding bacterial growth and resulting in membrane lysis [3,4]. Moreover, different phlorotannins were able to inhibit bacterial biofilm formation, production of quorum-sensing molecules, and also viral replication ( e


Introduction
Antibiotic resistance has become the topmost threat to public health in the 21st century, with more than 40 countries shared reports on antimicrobial resistance, which signifies criticality [8]. As such, there is a need for alternative compounds and treatments that may function as antibiotics in order to deal with this issue. In the last decade, research in natural compounds from plant and algae sources has rapidly increased, with a great number of natural molecules characterized and described. In this context, marine algae, a traditional food and medicinal East-Asian ingredient, have proven to be an excellent source of natural molecule with numerous potential and effective applications in human health [1,3]. In specific, brown algae possess some unique natural compounds with various bioactive properties, such as polysaccharides (laminaran, fucoidan, alginate), lectins, alkaloids, or polyphenols, such as phlorotannins (PT) [1,9]. PT are composed of polymeric units of phloroglucinol (1,3,5-trihydroxybenzene), with molecular weights ranging between 126-650 kDa. They are classified into six major groups, according to the type of linkages between phloroglucinol units and the number of hydroxyl groups (Error! Reference source not found.) [2,10] eckols, which possess at least one three-ring moiety with a dibenzodioxin element substituted by a phenoxyl group at C-4.
Due to their polymeric structure and number of hydroxyl groups, PT are potent freeradical scavengers and can modulate proteins and chelate metals. These capacities explain the wide range of cellular and ecological roles of phlorotannins in seaweeds. Moreover, PT are herbivore deterrents and protect against desiccation, high UVB radiation, and toxic heavy metals, acting as chelators [6,7]. These compounds have been classified as generally regarded as safe (GRAS) substances by both the Food and Drug Administration (FDA) and the European Food Safety Agency (EFSA) [11,12]. In the EU, phlorotannin-rich extracts from Ecklonia cava are approved as a "novel food" and considered safe to consume, due to an great number of studies supporting their safety [12]. Applications of PT, however, are currently limited as antioxidant cosmetic ingredients, or nutraceuticals for metabolic modulation, as there is a growing body of evidence supported by regulators, suggesting that these marine polyphenols are safe and non-toxic. In addition, several reports suggest their antimicrobial potential. Herein, the potential use of PT as antibiotic compounds based on scientific evidence is briefly reviewed.

Scientific Studies on Antimicrobial and Antiviral Effects of Phlorotannins
PT antimicrobial potential has mainly been studied through in vitro assays, for which screening studies made on brown algae usually employ representative nosocomial Gram + and Gram-bacteria, such as Staphylococcus aureus, or Pseudomonas aeruginosa, which usually present antibiotic resistance (Table 1). PT extracts from Sargassum species have demonstrated varying microbial growth inhibition on P. aeruginosa, S. aureus, and also in other pathogenic species such as Vibrio parahaemolyticus and the facultative pathogen Escherichia coli [6,13,14]. The brown algae Ecklonia cava and Eisenia bicyclis are also some of the most studied algae as source of PT, since these contain eckols, such as dieckol or phlorofucofuroeckol-A. These purified compounds were reported to display inhibitory and bactericidal effects on relevant gastrointestinal pathogenic bacteria such as Campylobacter jejuni, Salmonella typhimurium, or S. typhi at concentrations below 200 μg/mL [15,16]. Moreover, these eckols have been studied to reduce or nullify antibiotic resistance in some bacterial strains. For example, phlorofucofuroeckol-A from E. bicyclis downregulated the Penicillinbinding protein 2a gene, related to methicillin resistance in S. aureus exhibiting a minimum inhibitory concentration (MIC) of 16 μg/mL [17]. Likewise, fucofuroeckol-A from the same brown alga reduced biofilm formation and virulence of Streptomycin-resistant Listeria monocytogenes, with a MIC of 16 μg/mL [18]. In addition to these results, a few reports indicate that PT may also act as antiviral agents, as phlorofucofuroeckol-A from E. cava was described to greatly inhibit various strains of influenza [5], and dieckol to almost nullify human immunodeficiency virus (HIV) replication [19].

Discussion and Future Perspectives
Evidence regarding PT as antioxidants and metabolic and inflammatory modulators is vast, with a great number of studies confirming these properties by in vivo studies and clinical trials [10,27]. However, research on their potential applications as antibiotics is still limited, mainly due to the scope of reported studies (number of PT and/or microorganisms tested) and the fact that many of such works have been focused on in vitro studies, which unfortunately may not be translated to effective results in animal models or clinical trials. Nevertheless, the reported inhibitory and bactericidal effects of these molecules, specially purified compounds (e.g., dieckol, phlorofucofuroeckol-A) should be considered.are effective at low concentrations. Since PT have shown antioxidant, photoprotective and anti-inflammatory activities, these could be excellent ingredients for topical antibiotic formulations and cosmetics. Moreover, PT are reported to inhibit quorum sensing and limit biofilm formation, which suggests that these compounds could contributed to reduce topical or gut infections [6,15].
Two main factors should be considered regarding potential effectiveness of PT: (1) their generally poor bioavailability and (2) their binding effectiveness to microbial pathogens. Polyphenols and specially highly-polymerized have shown very poor bioavailability (around 5-10% are absorbed), and thus, encapsulation strategies are usually developed [28]. Notably, it has also been described that PT may act as prebiotics but research on this still appears limited, and microbiota alterations based on gut infections and changes due to PT treatment should be further explored [29]. On the other hand, it has been suggested that PTs, similarly to other polyphenols, could interact with cell membrane proteins or metabolic enzymes, precipitating them and disrupting the membrane integrity [3]. This fact was reported as the main factor liable of membrane lysis in MRSA, which also acted in synergy with methicillin [25]. Altogether, despite much needed further research, there is founding evidence suggesting the potential of PT as an effective antimicrobial and antiviral compounds, among other demonstrated properties.