Can Phlorotannins Purified Extracts Constitute a Novel Pharmacological Alternative for Microbial Infections with Associated Inflammatory Conditions?

et al. (2012) Can Phlorotannins Purified Extracts Constitute a Novel Pharmacological Alternative for Microbial Infections with Associated Inflammatory Conditions? PLoS ONE 7(2): e31145. doi:10.1371/journal.pone.0031145 Can Phlorotannins Purified Extracts Constitute a Novel Pharmacological Alternative for Microbial Infections with Associated Inflammatory Conditions? Graciliana Lopes 0 Carla Sousa 0 Lus R. Silva 0 Euge nia Pinto 0 Paula B. Andrade 0 Joa o Bernardo 0 Teresa Mouga 0 Patrcia Valenta o 0 Mande Holford, The City University of New York-Graduate Center, United States of America 0 1 REQUIMTE/Laborato rio de Farmacognosia, Departamento de Qu mica, Faculdade de Farma cia, Universidade do Porto , Porto , Portugal , 2 CEQUIMED/Laborato rio de Microbiologia, Departamento de Ciencias Biolo gicas, Faculdade de Farma cia, Universidade do Porto , Porto , Portugal , 3 GIRM - Marine Resources Research Group, School of Tourism and Maritime Technology, Polytechnic Institute of Leiria , Peniche , Portugal Bacterial and fungal infections and the emerging multidrug resistance are driving interest in fighting these microorganisms with natural products, which have generally been considered complementary to pharmacological therapies. Phlorotannins are polyphenols restricted to brown seaweeds, recognized for their biological capacity. This study represents the first research on the antibacterial, antifungal, anti-inflammatory and antioxidant activity of phlorotannins purified extracts, which were obtained from ten dominant brown seaweeds of the occidental Portuguese coast. Phlorotannins content was determined by the specific dimethoxybenzaldehyde (DMBA) method and a yield between 75 and 969 mg/Kg phloroglucinol units (dry matter) was obtained. Fucus spiralis ranked first, followed by three Cystoseira species. The antiinflammatory potential of the purified extracts was assessed via inhibitory effect on nitric oxide (NO) production by lipopolysaccharide-stimulated RAW 264.7 macrophage cells, Cystoseira tamariscifolia being the one showing promising activity for the treatment of inflammation. NO scavenging ability was also addressed in cell free systems, F. spiralis being the species with highest capacity. The antimicrobial potential of the extracts was checked against five Gram-positive and four Gram-negative bacteria and three fungi strains, that commonly colonize skin and mucosa and are responsible for food contamination. The different extracts were more effective against Gram-positive bacteria, Staphylococcus epidermidis being the most susceptible species. Concerning antifungal activity, Trichophyton rubrum was the most sensitive species. Although the molecular mechanisms underlying these properties remain poorly understood, the results obtained turn phlorotannins purified extracts a novel and potent pharmacological alternative for the treatment of a wide range of microbial infections, which usually also present an inflammatory component. In addition to the biological properties demonstrated herein, phlorotannins extracts may also be preferred, in order to avoid side effects and allergic reactions commonly associated with synthetic drugs. - Funding: The authors thank Fundacao para a Ciencia e a Tecnologia (FCT) for grant no. PEst-C/EQB/LA0006/2011. G. Lopes (SFRH/BD/61565/2009) and J. Bernardo (BII) are indebted to FCT for their grants. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Tannins are considered to be one of the most broadly distributed types of plants natural products [1]. These polyphenols are commonly divided into distinct groups according to their structures. They consist of flavonoids or gallic acid polymers in terrestrial plants, while in seaweeds they are composed of phloroglucinol (1,3,5-trihydroxybenzene) units. The last ones, known as phlorotannins, span a wide range of molecular sizes (from 126 Da to 650 kDa) and can be subdivided into six specific groups (fucols, phlorethols, fucophlorethols, fuhalols, isofuhalols and eckols) (Figure 1), characterized by differences in the nature of the structural linkages between phloroglucinol units and the number of hydroxyl groups present [1]. Phlorotannins are restricted to brown seaweeds (Phaeophyceae) and are biosynthesised through the acetate-malonate pathway in Golgi apparatus, in the perinuclear area of the cell. They are stored in vesicles called physodes, appearing as a colourless and refractive acidic fluid [2]. As they are not normally secreted outside the cell, it is necessary for the cells to be damaged before phlorotannins release. Phlorotannins concentration in brown seaweeds can vary among species, being affected by seaweed size, age, tissue type, salinity, season, nutrient levels, intensity of herbivory, light intensity and water temperature. Their concentration can reach the maximum in temperate and tropical Atlantic (up to 20% of brown seaweed dry mass), and the minimum in tropical Pacific and Indo-Pacific regions. Species from the order Fucales are richer in this kind of compounds [1,3]. These compounds have primarily been regarded as defence chemical agents. Due to their protein precipitating capacity, they are capable of deterring grazing by herbivores, especially by fish. Figure 1. Chemical structures of different types of phlorotannins. Phloroglucinol (1), Tetrafucol A (2), Tetraphlorethol B (3), Fucodiphlorethol A (4), Tetrafuhalol A (5), Tetraisofuhalol (6), and Phlorofucofuroeckol (7). doi:10.1371/journal.pone.0031145.g001 They are also important components of the cell wall and are responsible for the absorption of ultraviolet radiation [4]. Like other polyphenolic compounds, phlorotannins have been regarded as potential beneficial for human health [5]. Nowadays, diverse properties of phlorotannins are reported on biological systems, namely anti-inflammatory [6], anti-allergic [7], anti-viral [8], anti-cancer [9], bactericide [10], antioxidant [6], anti-diabetic activities and also radioprotective effects [11]. In particular, the anti-inflammatory potential of phlorotannins has attracted attention. The effect of phlorotannins extracts or isolated compounds from Laminariaceae on the arachidonic dependent pathway inflammatory response was reported [6,12,13]. A few studies regarding the effects of seaweeds extracts on nitric oxide (NO) production by cells involved in the inflammatory response have been performed [6,14]. NO is an important inflammatory mediator synthesized from arginine by nitric oxide synthase (NOS). It is a diffusible free radical with many functions in diverse biological systems. It plays an important role as a vasodilator, neurotransmitter and in the immunological system, as a defence against tumour cells, parasites and bacteria. However, under pathological conditions, and when an oxidative environment takes (...truncated)