title: Antibacterial activity of Limonium brasiliense (Baicuru) against multidrug-resistant bacteria using a statistical mixture design.
abstract: BACKGROUND
  Limonium brasiliense (Boiss.) Kuntze (Plumbaginaceae) is commonly known as "baicuru" or "guaicuru" and preparations of its dried rhizomes have been popularly used in the treatment of premenstrual syndrome and menstrual disorder, and as an antiseptic in genito-urinary infections. This study evaluated the potential antibacterial activity of rhizome extracts against multidrug-resistant bacterial strains using statistical mixture design.
  
  
  METHODS
  The statistical design of four components (water, methanol, acetone, and ethanol) produced 15 different extracts and also a confirmatory experiment, which was performed using water:acetone (3:7, v/v). The crude extracts and their ethyl-acetate fractions were tested against vancomycin-resistant Enterococcus faecium (VREfm), methicillin-resistant Staphylococcus aureus (MRSA) and Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae, all of which have been implicated in hospital and community-acquired infections. The dry residue, total polyphenol, gallocatechin and epigallocatechin contents of the extracts were also tested and statistical analysis was applied in order to define the fit models to predict the result of each parameter for any mixture of components. The principal component and hierarchical clustering analyses (PCA and HCA) of chromatographic data, as well as mass spectrometry (MS) analysis were performanced to determine the main compounds present in the extracts.
  
  
  RESULTS
  The Gram-positive bacteria were susceptible to inhibition of bacterial growth, in special the ethyl-acetate fraction of ternary extracts from water:acetone:ethanol and methanol:acetone:ethanol against, respectively, VREfm (MIC=19µg/mL) and MRSA (MIC=39µg/mL). On the other hand, moderate activity of the ethyl-acetate fractions from primary (except water), secondary and ternary extracts (MIC=625µg/mL) was noted against KPC. The quadratic and special cubic models were significant for polyphenols and gallocatechin contents, respectively. Fit models to dry residue and epigallocatechin contents were not possible. PCA and HCA of the chromatographic fingerprints were disturbed by displacement retention time of some peaks, but the ultraviolet spectra indicated the homogeneous presence of flavan-3-ols characteristic of tannins. The MS confirmed the presence of gallic acid, gallocatechin, and epigallocatechin in extracts, and suggested the presence of monomers and dimers of B- and A-type prodelphinidins gallate, as well as a methyl gallate.
  
  
  CONCLUSIONS
  Our results showed the antibacterial potential of L. brasiliense extracts against multidrug-resistant Gram-positive bacteria, such as VREfm and MRSA. The statistical design was a important tool to evaluate the biological activity by optimized form. The presence of some phenolic compounds was also demonstrated in extracts.
authors.affiliation: Programa de Pós-graduação em Ciências Farmacêuticas, Universidade Estadual de Maringá, Avenida Colombo, 5790, BR-87020-900 Maringá, PR, Brazil.
authors.forename: Andressa
authors.initials: A
authors.lastname: Blainski
authors.affiliation: Laboratório de Ecologia Microbiana, Departamento de Microbiologia, Universidade Estadual de Londrina, PR, Brazil.
authors.forename: Barbara
authors.initials: B
authors.lastname: Gionco
authors.affiliation: Laboratório de Ecologia Microbiana, Departamento de Microbiologia, Universidade Estadual de Londrina, PR, Brazil.
authors.forename: Admilton G
authors.initials: AG
authors.lastname: Oliveira
authors.affiliation: Laboratório de Ecologia Microbiana, Departamento de Microbiologia, Universidade Estadual de Londrina, PR, Brazil.
authors.forename: Galdino
authors.initials: G
authors.lastname: Andrade
authors.affiliation: Laboratório de Quimiometria em Ciências Naturais, Departamento de Química, Universidade Estadual de Londrina, PR, Brazil.
authors.forename: Ieda S
authors.initials: IS
authors.lastname: Scarminio
authors.affiliation: Laboratórios de Produtos Naturais e Espectrometria de Massas (LAPNEM), Universidade Federal de Mato Grosso do Sul, MS, Brazil.
authors.forename: Denise B
authors.initials: DB
authors.lastname: Silva
authors.affiliation: Núcleo de Pesquisa em Produtos Naturais e Sintéticos, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil.
authors.forename: Norberto P
authors.initials: NP
authors.lastname: Lopes
authors.affiliation: Programa de Pós-graduação em Ciências Farmacêuticas, Universidade Estadual de Maringá, Avenida Colombo, 5790, BR-87020-900 Maringá, PR, Brazil. Electronic address: mello@uem.br.
authors.forename: João C P
authors.initials: JCP
authors.lastname: Mello
mesh_terms: D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000494:pharmacology*; D024901:Drug Resistance, Multiple, Bacterial; D006094:Gram-Positive Bacteria / Q000187:drug effects*; D013058:Mass Spectrometry / Q000379:methods; D008826:Microbial Sensitivity Tests; D010936:Plant Extracts / Q000494:pharmacology*; D029606:Plumbaginaceae / Q000737:chemistry*; D025341:Principal Component Analysis; D027343:Rhizome
chemical_list: D000900:Anti-Bacterial Agents; D010936:Plant Extracts
keywords: Epigallocatechin (PubChem CID: 72277); Gallic acid (PubChem CID: 370); Gallocatechin (PubChem CID: 65084); Limonium brasiliense; Mass spectrometry; Multidrug–resistant bacteria; Prodelphinidins; Simplex centroid