Jorge E. Sayago

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.

Flour from Prosopis alba cotyledons: A natural source of nutrient and bioactive phytochemicals

The Prosopis alba seed is a waste material in the process to produce pod flour. To suggest a potential use of these seeds it is necessary to determine the nutritional, phytochemical and functional quality of cotyledon flour from Prosopis alba. This flour showed high level of proteins (62%), low content of total carbohydrate and fat. Free polyphenol (1150±20mg GAE/100g flour) and carotenoids (10.55±0.05mg β-CE/100g flour) compounds were the dominant compounds. The main identified constituents in the polyphenolic extracts were C- glycosyl flavones, including schaftoside, isoschaftoside, vicenin II, vitexin and isovitexin. The extract enriched in polyphenolic compounds exhibited ABTS(+) reducing capacity and scavenging activity of H2O2; and was able to inhibit phospholipase, lipoxygenase and cyclooxygenase, three pro-inflammatory enzymes. According to our results, the P. alba cotyledon flour could be considered as a new alternative in the formulation of functional foods or food supplements.

Chemical and functional characterization of seed, pulp and skin powder from chilto (Solanum betaceum), an Argentine native fruit. Phenolic fractions affect key enzymes involved in metabolic syndrome and oxidative stress

The aim of this work was to assess the nutritional and functional components of powder obtained by lyophilization of whole fruits, seeds, pulp and skin from chilto (Solanum betaceum Cav) cultivated in the ecoregion of Yungas, Argentina. The powders have low carbohydrate and sodium content and are a source of vitamin C, carotenoid, phenolics, potassium and fiber. The HPLC-ESI-MS/MS analysis of the fractions enriched in phenolics allowed the identification of 12 caffeic acid derivatives and related phenolics, 10 rosmarinic acid derivatives and 7 flavonoids. The polyphenols enriched extracts before and after simulated gastroduodenal digestion inhibited enzymes associated with metabolic syndrome, including α-glucosidase, amylase and lipase and exhibited antioxidant activity by different mechanisms. None of the analyzed fruit powders showed acute toxicity or genotoxicity. The powders from the three parts of S. betaceum fruit may be a potential functional food and the polyphenol enriched extract of seed and skin may have nutraceutical properties.

Tetraglochin andina Ciald.: A medicinal plant from the Argentinean highlands with potential use in vaginal candidiasis

ETHNOPHARMACOLOGICAL RELEVANCE The Argentinean medicinal plant Tetraglochin andina Ciald, formerly classified as T. cristatum (Britton) Rothm is used in traditional medicine by inhabitants from Argentinean northwestern highlands (Puna) to treat candidiasis and as anti-inflammatory. AIM OF THE STUDY To assess the potential of the crude drug as an anti-Candida agent with anti-inflammatory properties. The bioactivity and phytochemical composition of a dry extract of the plant was investigated. MATERIAL AND METHODS The pharmacognostic description of the crude drug is carried out for the first time, including macroscopic and microscopic examinations of the different organs, physicochemical and extractive values (petroleum ether-, ethanol- and water-soluble). The dry extract from T. andina was evaluated as antifungal against pathogenic Candida sp. and Saccharomyces cerevisiae isolated from vaginal infections and reference strains, by the macrodilution and microdilution assays. The normal vaginal microbiome in women is characterized by the dominance of lactic acid-producing bacteria, mainly Lactobacillus spp. The effect of T. andina extract on Lactobacillus strains was also assayed. The inhibitory effect on proinflammatory enzymes (cyclooxygenase, lipoxygenase and phospholipase A2) and antioxidant capacity was studied. The chemical profile was analyzed by HPLC-ESI-MS. RESULTS The hydroalcoholic extract inhibited the growth of all yeasts with Minimal Inhibitory Concentration (MIC) values between 12.5 and 400 µg GAE/mL and the MIC values on Lactobacillus were higher than the MIC values against Candida isolates ( > 400 µg GAE/mL). These results indicate that the hydroalcoholic extract could be used without affecting the normal microbiota of vaginal fluid. The extract showed antioxidant activity and could modulate the inflammatory process by three pathways (sPLA2, COX-2, LOX). The plant extract contained high total phenolic levels (386.9±1.7 mg GAE/g dry extract) and flavonoid levels (260.4±2.7 mg GAE/g dry extract). Fifty phenolic compounds were identified by HPLC-ESI-MS. They were mainly hydrolysable and condensed tannins. The dry extract was chemically and biologically stable during one year at room temperature or 4 °C. CONCLUSIONS The presence of anti-Candida and anti-inflammatory activities in Tetraglochin andina extracts give support to their traditional use for treating conditions associated with microorganism infections and inflammatory process in humans. This plant preparation could be used to design phytopharmaceutical preparations to inhibit yeast growth and moderate the inflammatory and oxidative process.

Control of citrus pathogens by protein extracts from Solanum tuberosum tubers

A national program for citrus certification was started in Argentina in 2005 in order to provide healthy fruits free of toxic residues. In line with this goal, the aim of this study was to evaluate the efficacy of natural products, protein extracts obtained from potato tubers for the control of fungi responsible for disease in post-harvest citrus fruits. Different protein fractions were obtained from Solanum tuberosum tubers (IF25, IF50, SF25 and SF50) and their effect were evaluated on Penicillium digitatum and Geotrichum candidum, two citrus-pathogenic fungi. All fractions showed antifungal activity against both fungi species, the intensity of this activity being dependent on the type of fungus and extract. The fraction IF25 was the most active as an antifungal agent: it inhibited the mycelia growth of both pathogens, the elongation of the germ tube of P. digitatum and the conidial isotropic growth of G. candidum as well as its polygalacturonase activity. None of the IF25 concentrations were mutagenic in Salmonella typhimurium TA98 or TA100 strains. The efficacy of protein extracts to control P. digitatum and G. candidum growth was tested in artificially inoculated citrus fruits. Extracts were tested built-in to the wax used in citrus industry to coat and protect the fruit. The IF25 extract was effective in inhibiting the growth and development of P.digitatum and G. candidum. Consequently, the IF25 extract plus the wax could be used preventively in controlling fungal infection on post-harvest citrus.

Electrosprayed chitosan microcapsules as delivery vehicles for vaginal phytoformulations

The design of novel delivery systems to treat vaginal fungal infections is a topic of high interest. Chitosan, being itself antimicrobial and having good mucoadhesive properties, is an excellent candidate as a delivery matrix for active compounds. In this work, chitosan microcapsules containing dry extracts of Argentinean medicinal plants with proved biological properties (Larrea divaricata, L. cuneifolia, L. nitida, Zuccagnia punctata and Tetraglochin andina) were developed through electrospraying and compared with conventionally used tablets containing the same extracts. Total phenolics, loading efficacy, physical properties, morphology and particle size, molecular organization, water sorption capacity, release of bioactive compounds and biological properties were assessed. The encapsulation process or the inclusion in tablets did not degrade the bioactive compounds of the extracts. The release of phenolic compounds from chitosan microcapsules was faster than from tablets. The fingerprint of released phenolic compounds from microcapsules and tablets was similar to that from the dry extracts and the antioxidant and antifungal capacity remained unchanged. The FT-IR analysis suggested interactions between the chitosan and the extracts, which explained why the microcapsules kept the integrity in slightly acidic media. Increased solubility of the extracts when incorporated in the microcapsules was seen in simulated vaginal fluid, potentially increasing the bioavailability of bioactive compounds in the vaginal environment. This work highlights the potential of the chitosan-based delivery systems for phytomedicines with antifungal and antioxidant activity to be used in vulvovaginal candidiasis.