Roxana Mabel Ordóñez

Industrial effluents and surface waters genotoxicity and mutagenicity evaluation of a river of Tucuman, Argentina.

Assessment of water pollution and its effect upon river biotic communities and human health is indispensable to develop control and management strategies. The aim of this work was to ascertain the biotoxicity of water pollution in samples from industrial effluent discharge areas of Tucumán, Argentina by means of biological tests. Chemical characterization of the water pollution was verified by measuring dissolved oxygen concentration or levels of suspended matter and salts. Genotoxic/mutagenic potential was determined using Allium anaphase-telophase and Ames/Salmonella tests. All samples were phytotoxic and genotoxic for Allium roots. Micronucleus and anaphase aberrations were observed, but they did not show mutagenic effects on Salmonella typhimurium, TA98 and TA100 strains with and without metabolic activation (S9). Our results show the importance of testing industrial effluents by chemical methods and complementary biological tests to optimize the control policy on these environmental samples.

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.

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.

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.

Invertase activity associated with the walls of Solanum tuberosum tubers

Three fractions with invertase activity (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) were isolated from mature Solanum tuberosum tubers: acid soluble invertase, invertase I and invertase II. The first two invertases were purified until electrophoretic homogeneity. They are made by two subunits with an apparent M(r) value of 35,000 and their optimal pH is 4.5. Invertase I was eluted from cell walls with ionic strength while invertase II remained tightly bound to cell walls after this treatment. This invertase was solubilized by enzymatic cell wall degradation (solubilized invertase II). Their K(m)s are 28, 20, 133 and 128 mM for acid soluble invertase, invertase I, invertase II and solubilized invertase II, respectively. Glucose is a non-competitive inhibitor of invertase activities and fructose produces a two site competitive inhibition with interaction between the sites. Bovine serum albumin produces activation of the acid soluble invertase and invertase I while a similar inhibition by lectins and endogenous proteinaceous inhibitor from mature S. tuberosum tubers was found. Invertase II (tightly bound to the cell walls) shows a different inhibition pattern. The test for reassociation of the acid soluble invertase or invertase I on cell wall, free of invertase activity, caused the reappearance of all invertase forms with their respective solubilization characteristics and molecular and kinetic properties. The invertase elution pattern, the recovery of cell wall firmly bound invertase and the coincidence in the immunological recognition, suggest that all three invertases may be originated from the same enzyme. The difference in some properties of invertase II and solubilized invertase II from the other two enzymes would be a consequence of the enzyme microenvironment in the cell wall or the result of its wall binding.

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.

Autographic assay for the rapid detection of antioxidant capacity of liquid and semi-solid pharmaceutical formulations using ABTS•+ immobilized by gel entrapment.

An autographic assay suitable for the detection of antioxidant compounds in a complex matrix (liquid and semi-solid pharmaceutical formulations) or in isolated compounds was described. The pre-formed radical monocation of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) was generated by oxidation of ABTS with potassium persulfate and reduced in the presence of hydrogen-donating antioxidants. For a further comparative estimation of its applicability and sensitivity, different medicinal plant extracts, hydrogels and antioxidant compounds were dot seeded or chromatographed on silica gel (TLC) and revealed with ABTS•+ solution (System I) or ABTS•+ immobilized by gel entrapment (System II). Both systems were effective and able to detect antioxidant activity in a micromolar range in seconds. System II was more sensitive and reproducible than System I. This micromethod is quick, inexpensive, and particularly helpful whether it works with numerous samples or on a small scale.

Polyphenolic compounds and anthocyanin content of Prosopis nigra and Prosopis alba pods flour and their antioxidant and anti-inflammatory capacities

The aim of this study was to determine the content of total free and bound phenolics, free and bound flavonoids, anthocyanins, and alkaloids and the profile of polyphenols of edible ripe pods of Prosopis alba and Prosopis nigra. P. alba flour showed significantly higher total (sum of Free- and Bound) phenolic content and total flavonoid compounds than P. nigra (p<0.05) while P. nigra had higher concentrations of anthocyanins than P. alba (p<0.05). The P. nigra flour shows a pattern characterized by the occurrence of anthocyanins as well as 14 flavonoid glycosides, with higher chemical diversity than P. alba, which shows 8 flavonoid glycosides as relevant constituents. The main compounds were quercetin O-glycosides and apigenin-based C-glycosides. The phenolic composition of two South American algarrobo pod flour is presented for the first time. P. nigra pods having higher content of anthocyanins are darker (purple) than those of P. alba (light brown). Furthermore, the sugar-free polyphenolic extracts of P. nigra and P. alba as well as anthocyanins enriched extracts from P. nigra showed antioxidant activity. P. nigra and P. alba polyphenolic extracts showed activity against a pro-inflammatory enzyme. In conclusion, algarrobo pods meal contained biologically active polyphenols, with a positive impact on human health.

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 SC50 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. Copyright