In vitro fungitoxic activity of Larrea divaricata cav. extracts.

AIMS: To evaluate the fungitoxic activity of Larrea divaricata Cav. extract and one of its components against yeasts and fungi. This activity was compared with the action of ketoconazole, a known synthetic antimycotic. METHODS AND RESULTS: Antifungal activity of Larrea divaricata extract and of a fraction (Fr. B) purified by thin layer chromatography, was investigated using different methodologies. Both exhibited strong activity against the majority of the assayed fungi. Only Fusarium oxysporum and Schizophyllum commune growth was not affected with the assayed conditions. The fungitoxic and cytotoxic activity of the ethanolic extract and ketoconazole were compared. CONCLUSIONS: Ethanolic extracts of L. divaricata Cav. produce growth inhibition of several fungi. One of its constituents with the same activity was purified and identified as a glycoside of a flavanone. A comparison with the action of ketoconazole, which is currently used as antimycotic and can cause adverse health effects was made. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest that L. divaricata extract contains, at least, one compound of phenolic nature, with fungitoxic potency against yeasts and fungi.

Proteinaceous inhibitor versus fructose as modulators of Pteris deflexa invertase activity.

An acid invertase from the fern Pteris deflexa Link was purified and the effect of reaction products on enzyme activity was studied. Fructose and glucose were competitive and non-competitive inhibitors of the enzyme, respectively. Since proteins suppressed glucose and fructose inhibition of the enzyme, an invertase modulation by reaction products is unlikely; nevertheless, an invertase proteinaceous inhibitor previously reported could have a role in this respect. The purified enzyme was an heterodimer Mr 90,000 Daltons composed of subunits of 66,000 and 30,000 Daltons. The enzyme had beta-fructofuranosidase activity and hydrolyzed mainly sucrose but also raffinose and stachyose, with Km of 3.22, 10.80 and 38.50 mM, respectively. Invertase activity with an optimum pH at 5.0 was present in almost every leaf fern tissue. Pinnas (sporophyll leaflets) had the higher enzyme levels. Invertase histochemical and immunochemical localization studies showed the enzyme mainly in phloem cells. Epidermis, collenchyma and parenchyma cells also showed invertase protein.

An N-acetylglucosamine oligomer binding agglutinin (lectin) from ripe Cyphomandra betacea Sendt. fruits.

A new agglutinin (lectin), called CBL3, was purified from the juice of ripe Cyphomandra betacea Sendt. fruits until electrophoretically pure to homogeneity. The lectin is a homodimer of M(r)=50800 with subunits of 26200 bound by disulfide linkages with a pI of 4.9. The agglutinating capacity of the lectin is only inhibited by oligomers of N-acetylglucosamine in the following order of potency: tetrasaccharide>trisaccharide>disaccharide. CBL3 is not inhibited by N-acetylglucosamine, the same as all known lectins of the Solanaceae family. The human blood group recognition is non-specific. The lectin is a glycoprotein with 13.6% (w/w) of carbohydrates. The agglutinating activity is not affected by EDTA nor by cations. Mitogenic activity was not detected. Heat and pH stability, amino acid composition, N-terminal amino acid sequence and immunological properties show substantial differences to the reported lectins isolated from the Solanaceae family.

Antioxidant activity of Argentine propolis extracts.

Propolis is used in Argentine folk medicine. We have examined its possible protective action against oxidative modification of lipid in unfractionated serum. The kinetics of copper-induced oxidation was continuously monitored by measuring the formation of conjugated dienes, as the increase in the absorbance at 234 nm. According to the kinetics of oxidation, the propolis were classified in three different groups. Group I (CE, CO, BO, MO, BE) inhibited lipid oxidation during the initiation and propagation phases even at low concentrations. Group II (SP, CA, AM) increased the lag-phase for conjugated diene formation. All propolis in groups I and II diminished the maximal rate of diene production and the maximal amount of dienes produced. Group III (PA, RA, FE, VR, TV) had no effect on the lipid oxidation. The extent of lipoprotein oxidation was measured by the thiobarbituric acid reactive substance assay. Generation of malondialdehyde-like substances was inhibited and delayed by the presence of propolis extracts from group I and II. Our results justify the use of propolis (groups I and II) as a source of natural antioxidants.

Screening antifungal activities of selected medicinal plants.

Plants synthesise a vast array of secondary metabolites that are gaining importance for their biotechnological applications. The antifungal activity of the ethanolic extracts of ten Argentinean plants used in native medicine is reported. Antifungal assays included radial growth inhibition, disk and well diffusion assays and growth inhibition by broth dilution tests. The chosen test fungi were yeasts, microfungi and wood-rot causing Basidiomycetes. Extracts of Larrea divaricata, Zuccagnia punctata and Larrea cuneifolia displayed remarkable activity in the assays against the majority of the test fungi. In addition to the former plants, Prosopanche americana also inhibited yeast growth.

An invertase inhibitory protein from Pteris deflexa link fronds.

Plant invertases play important roles in sucrose metabolism. Cell wall invertase was reported to participate in phloem loading and unloading. Soluble invertases would be involved in hexose level regulation in mature tissues and in stored sucrose utilization within vacuoles. Invertase inhibitory proteins were described as one of the possible mechanisms for invertase activity regulation in some plant species; nevertheless, these proteins were found only in sink tissues, suggesting that this mechanism would not be relevant in the sucrose turnover of leaves. This report describes the purification of invertase from Pteris deflexa fronds and the occurrence of an invertase inhibitory protein in this fern organ, as well as its purification and invertase-inhibitor interactions. The Mr of the invertase and of its inhibitory protein were 90,000 and 18,000, respectively. SDS-PAGE in the presence of 2-mercaptoetanol gave two subunits for the enzyme (Mr=66,000 and 30,000) and only one for the inhibitor. The inhibitor protein is a glycoprotein (12% w/w of neutral sugars) that did not show agglutinating activity like some others, and also showed a high heat stability at pH 5.0. The optimum pH of invertase activity is 5.0, while invertase inhibitory protein caused maximal inhibition at the same pH value. Invertase-inhibitor complex formation occurs in an immediate manner and a protease activity was discarded. The inhibition is non-competitive (Ki=1.5 x 10(-6) M) without interactions among the binding sites. The complex is slightly dissociable and sucrose was able to partially reduce the inhibitory effect. Up to the present, invertase inhibitory proteins have been found solely in heterotrophic tissues. In this work we demonstrate that this protein is also present in an autotrophic tissue of a lower vascular plant.

Comparison of the free radical-scavenging activity of propolis from several regions of Argentina.

Propolis is extensively used in Argentine folk medicine. Alcoholic extracts of propolis from different regions of Argentina were prepared. The extracts were analysed for the determination of total flavonoid content (from 13.3 to 42.6 mg/g of propolis) by using the aluminum nitrate method, UV spectrophotometry and thin layer chromatography. All of them contained high total flavonoid content. It was also observed that all samples of ethanolic extracts of propolis showed free radical-scavenging activity in terms of scavenging of the radical DPPH but the highest activities were found for samples from Tucumán and Santiago del Estero. In all cases with 20 microg/ml of soluble principles, the percentage of DPPH degradation was different (Banda Oeste: 67.5%; Verónica: 45%; Forres: 35%; Saenz Peña: 20% and Juan José Castelli: 55%). These results may justify their use as a source of natural antioxidants.

Invertase proteinaceous inhibitor of Cyphomandra betacea Sendt fruits.

This work describes a new invertase proteinaceous inhibitor from Cyphomandra betacea Sendt. (tomate de arbol) fruits. The proteinaceous inhibitor was isolated and purified from a cell wall preparation. The pH stability, kinetics of the inhibition of the C. betacea invertase, inhibition of several higher plant invertases and lectin nature of the inhibitor were studied. The inhibitor structure involves a single polypeptide (Mr = 19000), as shown by gel filtration and SDS-PAGE determinations. N-terminal aminoacid sequence was determined. The properties and some structural features of the inhibitor are compared with the proteinaceous inhibitors from several plant species (Beta vulgaris L., Ipomoea batatas L. and Lycopersicon esculentum Mill.). All these inhibitors share lectinic properties, some common epitopes, some aminoacid sequences and a certain lack of specificity towards invertases of different species, genera and even plant family. In consequence, the inhibitors appear to belong to the same lectin family. It is now known that some lectins are part of the defence mechanism of higher plants against fungi and bacteria and this is a probable role of the proteinaceous inhibitors.

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.

Screening of antibacterial activity of Amaicha del Valle (Tucumán, Argentina) propolis.

Propolis is extensively used in Argentine folk medicine. Alcoholic extracts of propolis from four localities of Amaicha del Valle (El Paraiso, La Banda Este, La Banda Oeste and El Molino), Province of Tucumán and from Cerrillos, Province of Santiago del Estero, Argentina were prepared. All showed antibacterial activity against Gram positive bacteria, the propolis from La Banda Este being the most active (MIC = 7.8 microg/ml) against Streptococcus piogenes, an antibiotic resistant bacterium. Thin layer chromatographic (TLC) separation profiles of propolis from Amaicha del Valle region were similar but differ from the alcoholic extract of the propolis from Cerrillos, another phytogeographical region of Argentina (provincia chaqueña). Bioautographic assays of the TLC profiles showed that several separated compounds of the Amaicha del Valle propolis have antibacterial activity. The difference in composition between Amaicha del Valle and Cerrillos propolis coincides with a different phytogeographical formation.

Hydrolysis of sucrose within isolated vacuoles from Solanum tuberosum L. tubers.

The soluble acid invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) from potato (Solanum tuberosum L. cv. Kennebec) tubers was located in the vacuoles. Although the functionality of this invertase in the vacuoles has been assumed, the activity of the enzyme has never been shown within isolated vacuoles. Vacuoles were prepared by gentle osmotic shock from free protoplasts obtained by enzymic digestion of tuber tissues. The mean volume of these vacuoles, (0.26 +/- 0.05) x 10(-2) microliters, was estimated by optical microscopy. Sucrose, glucose and fructose concentrations were calculated to be 100 mM, 20 mM and 40 mM, respectively, in the vacuoles. Sucrose hydrolysis and the increase in glucose and fructose concentrations within the vacuoles were measured during vacuolar incubations. An almost identical pattern of sucrose hydrolysis by invertase was found by an in-vitro assay reproducing the vacuolar conditions. In view of the determinations of internal vacuolar pH (5.2), the possibility of spontaneous hydrolysis of sucrose was disregarded. Vacuoles were shown to be free from proteinaceous inhibitors, confirming the extravacuolar location of these inhibitors. The vacuolar hydrolytic pattern of sucrose confirms the regulatory role of the reaction products previously proposed for in-vitro assays.

The effects of a selection of alkaloids on the invertase activity of some higher plants.

The effects of several alkaloids on the invertase activities of Ricinus communis, Solanum tuberosum, Oryza sativa and Carica papaya were studied. The following alkaloids: caffeine, berberine, strychnine, morphine, ethyl-narceine and nornicotine, have different inhibitory effects on invertase activity. Kinetic studies of the alkaloid inhibition revealed that different alkaloids exhibited diverse types of inhibition of invertase. It was found that alkaloids form reversible complexes with invertases. However cocaine and potato glycoalkaloids were activators of some invertase. The sugar moiety of potato glycoalkaloids were also activators. It is concluded that alkaloids may be effectors of higher plant invertase activity.

Purification and characterization of the invertase from Pycnoporus sanguineus.

A constitutive invertase (EC 3.2.1.26) was isolated and purified by the first time from Pycnoporus sanguineus. The enzyme is a glycoprotein. Its relative molecular mass is about 84,000 and its structure is dimeric, with two identical subunits (about 41,000). The enzyme is able to attack sucrose, raffinose, stachyose, inulin and levan, being sucrose the preferred substrate (Km 4.89 +/- 0.13 mM). Fructose was a classical competitive inhibitor, but glucose was not an inhibitor of the enzyme. Lectins with specificity toward glucose are inhibitors of the enzyme. Glucose was present in invertase acid hydrolysates. Unlike higher plant invertases, bovine serum albumin is not an effector of the Pycnoporus sanguineus enzyme, and the inhibition by fructose is not suppressed by this protein. The properties of the Pycnoporus sanguineus enzyme are discussed with reference to higher plant invertases.

Biochemical method for molecular weight determinations of proteins and other macromolecules.

The invertase of Ricinus communis complexes with proteins, polyvinylpyrrolidone, polyethylene glycol, heparin and dextran sulfate. This association produces an increase of invertase activity. The minimal concentration of activator giving the maximal activation was attained at the same molarity for a given amount of enzyme for all macromolecules studied. These conditions are used for the molecular weight determination of the activating substance. The method may be used for the molecular weight determination of polymeric substances with a molecular weight in the range from 5000 to 1000,000 Da.

Purification and characterization of Ricinus communis invertase.

An invertase from Ricinus communis leaves was purified 4,400-fold. The preparation was homogeneous by criteria of gel electrophoresis, gel permeation, adsorption, and ionic exchange chromatography. One optimum pH at 3.5 was observed with crude invertase; however, purified preparations showed two optima, at pH 3.5 and 5.5. Addition of bovine serum albumin restored one maximum at pH 3.5 and elicited a 30% activation of the invertase. The effect was caused by many other proteins and by heparin, dextran sulfate, and polyvinylpyrrolidone. Fructose, fructose 1,6-diphosphate, maleic, trans-aconitic, malic, and ascorbic acids were simple competitive inhibitors of the purified enzyme. Glucose was a noncompetitive inhibitor. The activation by proteins suppressed these inhibitory effects. The minimum concentration of activator necessary to reach the maximal activation or "point of optimal activation" was always reached at a concentration of 1 X 10(-6) M, independently of the nature of the activator, when 8.6 X 10(-12) mol of enzyme were used. Apparent molecular weight determinations of the enzyme in the presence and absence of activator and molecular weight determinations based on determinations of the point of optimal activation suggested that the purified enzyme is a heptamer (Mr of 77,900, Stokes radius 32 A, frictional ration f/fo 1.1, partial specific volume 0.749 ml/g) and that the activated form is a trimer consisting of two enzyme subunits and one activator molecule. The activation was lost by dilution of the trimer. The enzyme subunit, as isolated by gel filtration in the presence of sodium dodecyl sulfate (Mr 11,000) was inactive but quickly regained activity upon removal of sodium dodecyl sulfate.