Antibacterial potential of non-volatile constituents of Rosmarinus officinalis against 37 clinical isolates of multidrug-resistant bacteria / Actividad antibacteriana de constituyentes no volátiles de Rosmarinus officinalis contra 37 aislamientos clínicos de bacterias multirresistentes

In this paper we investigated the antibacterial activity of a methanolic extract of Rosmarinus officinalis L. and their main constituents, carnosic acid and rosmarinic acid, against 37 nosocomial strains of multidrug-resistant bacteria. Results obtained showed that both the rosemary extract and carnosic acid inhibited all clinical isolates of Staphylococcus aureus methicillin-resistant and Enterococcus faecalis gentamicin and streptomycin-resistant bacteria examined (MICs 60 ug/mL vs. 200 ug/mL, respectively). Rosemary extract showed MIC values between 400 and 1600 ug/ml against the Gram-negative multidrug-resistant bacteria: Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Pseudomonas aeruginosa, Morganella morganii and Providencia stuartii, while carnosic acid showed MIC of 120 to 240 ug/mL. Bactericidal effect of carnosic acid against S. aureus and E. faecalis was observed at their MIC value, while 2 x MIC to 4 x MIC were needed to kill Gram-negative bacteria. Rosmarinic acid showed a narrow spectrum of action against a few Gram-negative clinical isolates. Our findings suggest that carnosic acid would be a good lead candidate useful in counteracting drug-resistant infections.

En este trabajo evaluamos la actividad antibacteriana de un extracto metanólico de Rosmarinus officinalis L. y sus principales componentes el ácido carnósico y ácido rosmarínico, contra 37 cepas de bacterias multirresistentes nosocomiales. Los resultados muestran que el extracto de romero y el ácido carnósico, inhibieron las bacterias Gram-positivas Staphylococcus aureus resistentes a meticilina y Enterococcus faecalis resistentes a gentamicina y estreptomicina (CIM 200 ug/mL y 60 ug/mL, respectivamente). El extracto de romero inhibió los Gram negativos multirresistentes: Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Pseudomonas aeruginosa, Morganella morganii y Providencia stuartii (CIM 400 a 1600 ug/mL), mientras que el ácido carnósico mostró valores de CIM entre 120 a 240 ug/mL. El ácido carnósico mostró actividad bactericida contra S. aureus y E. faecalis a su CIM, mientras que 2 a 4 X CIM se requirieron para matar las bacterias Gram-negativas. El ácido rosmarínico mostró inhibió unos pocos aislados clínicos Gram-negativos. Estos hallazgos sugieren que el ácido carnósico puede ser de utilidad contra infecciones bacterianas multirresistentes a antibióticos.

Antioxidant/antibacterial activities of a topical phytopharmaceutical formulation containing a standardized extract of Baccharis incarum, an extremophile plant species from Argentine Puna.

The antimicrobial and antioxidant activities of standardized extracts of Baccharis incarum in 60° and 80° ethanol and of a phytopharmaceutical formulation obtained from them were measured. Baccharis tinctures showed antimicrobial activity against clinically isolated antibiotic resistant Staphylococcus aureus and Enterococcus faecalis, with MIC values of 40-80 µg GAE/mL. Both tinctures exhibited ABTS(●+) scavenging activity with SC(50) values between 1.6 and 4.0 µg GAE/mL. The tinctures were not genotoxic in the Salmonella assay. For this reason, the tincture in 60° ethanol was incorporated into a topical pharmaceutical formulation (Hydrogel/ Carbopol® 934). The phytopharmaceutical formulation also showed antibacterial and antioxidant activities in the in vitro assays. The hydrogel showed microbiological, chemical, physical and functional stability during storage at room temperature. Studies that measure drug release as a determination of bioavailability were also carried out using the Franz diffusion cell (FC). The results demonstrated the release of two bioactive compounds (chlorogenic acid and 4',5-dihydroxy-3',3,6,7,8-pentamethoxyflavone) from the phytotherapic preparation in HPLC studies of FC receptor solution. In consequence, the phytopreparation applied topically could be used to treat skin and soft tissue infection produced by methicillin-resistant Staphylococcus aureus (MRSA) or Enterococcus faecalis and opens new opportunities for the use of active natural ingredients in the cosmeceutical field as antiacne and antioxidant.

A colorimetric method to quantify endo-polygalacturonase activity.

We report a new colorimetric assay to quantify endo-polygalacturonase activity, which hydrolyzes polygalacturonic acid to produce smaller chains of galacturonate. Some of the reported polygalacturonase assays measure the activity by detecting the appearance of reducing ends such as the Somogyi-Nelson method. As a result of being general towards reducing groups, the Somogyi-Nelson method is not appropriate when studying polygalacturonase and polygalacturonase inhibitors in plant crude extracts, which often have a strong reducing power. Ruthenium Red is an inorganic dye that binds polygalacturonic acid and causes its precipitation. In the presence of polygalacturonase, polygalacturonic acid is hydrolyzed bringing about a corresponding gain in soluble Ruthenium Red. The described assay utilizes Ruthenium Red as the detection reagent which has been used previously in plate-based assays but not in liquid medium reactions. The new method measures the disappearance of the substrate polygalacturonic acid and is compared to the Somogyi-Nelson assay. The experimental results using lemon peel, a fern fronds and castor leaf crude extracts demonstrate that the new method provides a way to the quickly screening of polygalacturonase activity and polygalacturonase inhibitors in plant crude extracts containing high amounts of reducing power. On the other hand, the Ruthenium Red assay is not able to determine the activity of an exo-polygalacturonase as initial velocity and thus would allow the differentiation between endo- and exo-polygalacturonase activities.

Evaluation of antioxidant activity and genotoxicity of alcoholic and aqueous beverages and pomace derived from ripe fruits of Cyphomandra betacea Sendt.

Cyphomandra betacea ripe fruits can be a source of value-added byproducts and products such as antioxidant supplements, ingredients for food processing or alternative medical products. The aims of the present study were to obtain different preparations of C. betacea fruits, such as juice, decoction, and maceration and to characterize them in terms of microbiological stability, sensorial and chemical parameters, antioxidant potential (DPPH and ABTS*+ radical scavenging, beta-carotene bleaching, nitrite scavenging activities), capacity to prevent oxidative stress-induced cell death, and genotoxicity. The best antioxidant activity was found in C. betacea fruit maceration, probably as a consequence of the high flavonoid and anthocyanin content. Nevertheless, all preparations analyzed proved to be good as free radical scavengers (SC50 values between 1.88 and 44 microg/mL) and exerted protection against beta-carotene oxidation. Total phenolic compounds and flavonoids showed a better correlation than anthocyanins with the free radical scavenging effect of the assayed foods. The insoluble matters (pomace) obtained after juice preparation showed antioxidant activity by quenching free radicals. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction assay showed that C. betacea preparations prevent oxidative stress-induced cell death in HepG2 cells in a dose-dependent manner. Salmonella microsome assays show no mutagenic effect. The data presented in this study demonstrate that C. betacea ripe fruits, aqueous and ethanolic preparations, and pomace may be a good source of antioxidant compounds in nutraceutical or functional-food products.

Design and quality control of a pharmaceutical formulation containing natural products with antibacterial, antifungal and antioxidant properties.

The aims of the present study were to determine the antibacterial and antifungal activity as well as mutagenicity of Sechium edule fluid extract and to obtain a pharmaceutical formulation with them. The extract exhibited antimicrobial activity against Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Morganella morganii, Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Candida spp. and Aspergillus spp. isolated from clinical samples from two hospitals of Tucuman, Argentina. Non-toxicity and mutagenicity on both Salmonella typhimurium TA98 and TA 100 strains until 100 microg/plate were observed. A hydrogel with carbopol acrylic acid polymer containing S. edule fluid extract as antibacterial, antimycotic and antioxidant agent was obtained. Microbiological, physical and functional stability of pharmaceutical formulation conserved at room temperature for 1 year were determined. Addition of antioxidant preservatives to store the pharmaceutical formulation was not necessary. The semisolid system showed antimicrobial activity against all gram positive and gram negative bacteria and fungi assayed. The minimal inhibitory concentration (MIC) values ranged from 20 to 800 microg/mL. Its activity was compared with a pharmaceutical formulation containing commercial antibiotic and antifungal. A pseudoplastic behavior and positive thixotropy were observed. Our current finding shows an antimicrobial activity of hydrogel containing S. edule extract on a large range of gram negative and gram positive multi-resistant bacteria and fungi. This topical formulation may be used as antimycotic and as antibacterial in cutaneous infections.

Singlet oxygen quenching and radical scavenging capacities of structurally-related flavonoids present in Zuccagnia punctata Cav.

The singlet oxygen (1O2) quenching and free radical (DPPH(*), ABTS(* +) and O2(* -)) scavenging ability of three structurally-related flavonoids (7-hydroxyflavanone HF, 2',4'-dihydroxychalcone DHC and 3,7-dihydroxyflavone DHF) present in the Argentinean native shrub Zuccagnia punctata Cav. were studied in solution by combining electrochemical and kinetic measurements, mass spectroscopy, end-point antioxidant assays and computational calculations. The results showed that the antioxidant properties of these flavonoids depend on several factors, such as their electron- and hydrogen atom donor capacity, the ionization degree of the more acidic group, solvatation effects and electrostatic interactions with the oxidant species. The theoretical calculations for both the gas and solution phases at the B3LYP level of theory for the Osanger reaction field model agreed with the experimental findings, thus supporting the characterization of the antioxidant mechanism of the Z. punctata flavonoids.

Effect of seasonal variations and collection form on antioxidant activity of propolis from San Juan, Argentina.

Propolis was included in the Argentine Food Code as a functional food. The chemical parameters and antioxidant properties of propolis samples from the same colonies of Apis mellifera in San Juan (Cuyo region, Western Argentine) were compared every month for 1 year using two collection methods. Chemical parameters were analyzed by the spectrophotometric method and fingerprinting using high-performance liquid chromatography with ultraviolet detection. The antioxidant activities of propolis samples were measured using model systems including the analysis of the scavenging activities for 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and the beta-carotene bleaching assay. The results showed that propolis had a higher free radical scavenging and lipid peroxidation inhibitory capacity than butylated hydroxytoluene and quercetin, antioxidants used in the pharmaceutical and food industries. The concentration required to scavenge 50% of free radicals (SC(50)) values differed depending on the sample collection month. Samples collected in November had the highest antioxidant capacity. In all cases, SC(50) values of propolis samples obtained by scraping were similar to those collected from a wire mesh (5 microg/mL for ABTS and 20-30 microg/mL for DPPH radicals). A significant positive correlation was found between the antioxidant capacity and flavonoid content of each analyzed extract. The chemical profiles were very similar. Galangin (3,5,7-trihydroxyflavone), an antioxidant compound, was detected in all samples as a major compound. The chromatographic profile suggests that of Baccharis sp., which would be one of the botanical sources of propolis from western Argentina, and the content of galangin can be used as a parameter for evaluating propolis quality. Our results suggest that Argentine propolis from Cuyo is a promising source of bioactive compounds as ingredients for developing functional foods with a beneficial impact on oxidative stress.

Antimicrobial activity of glycosidase inhibitory protein isolated from Cyphomandra betacea Sendt. fruit.

Broad-spectrum antimicrobial activity of an invertase inhibitory protein (IIP) isolated from Cyphomandra betacea ripe fruits is documented. Minimal inhibitory concentration (MIC) values were determined by agar macrodilution and broth microdilution assays. This IIP inhibited the growth of xylophagous and phytopatogenic fungi (Ganoderma applanatum, Schizophyllum commune, Lenzites elegans, Pycnoporus sanguineous, Penicillium notatum, Aspergillus niger, Phomopsis sojae and Fusarium mango) and phytopathogenic bacteria (Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae pv. syringae and Erwinia carotovora var carotovora). The IIP concentration required to completely inhibit the growth of all studied fungi ranged from 7.8 to 62.5 microg/ml. Phytopatogenic bacteria were the most sensitive, with MIC values between 7.8 and 31.25 microg/ml. Antifungal and antibacterial activities can be associated with their ability to inhibit hydrolytic enzymes. Our results indicate the possible participation of IIP in the plant defense mechanism and its potential application as a biocontrol agent against phytopathogenic fungi and bacteria.

Evaluation of the cytotoxicity, genotoxicity, mutagenicity, and antimutagenicity of propolis from Tucuman, Argentina.

This study evaluates the toxic, genotoxic/mutagenic, and antimutagenic effects of propolis extract from Amaicha del Valle, Tucumán, Argentina. The cytotoxicity assays carried out with the lethality test of Artemia salina revealed that the LD50 was around 100 microg/mL. Propolis extracts showed no toxicity to Salmonella typhimurium TA98 and TA100 strains and Allium cepa at concentrations that have antibiotic and antioxidant activities. Otherwise, for the testing doses, neither genotoxicity nor mutagenicity was found in any sample. The propolis extracts were able to inhibit the mutagenesis of isoquinoline (IQ) and 4-nitro o-phenylenediamine (NPD) with ID50 values of 40 and 20 microg/plate, respectively. From this result, the studied propolis may be inferred to contain some chemical compounds capable of inhibiting the mutagenicity of direct-acting and indirect-acting mutagens. A compound isolated from Amaicha del Valle propolis, 2',4'-dihydroxychalcone, showed cytotoxic activity (LC50 values of 0.5 microg/mL) but was not genotoxic or mutagenic. Furthermore, this compound was able to inhibit the mutagenicity of IQ (ID50 values of 1 microg/plate) but was unable to inhibit the mutagenicity of NPD. Our results suggest a potential anticarcinogenic activity of Amaicha del Valle propolis and the chalcone isolated from it.