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 ß-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.

Acid invertase localization in leaves of the fern Pteris deflexa link.

The localization of invertase, a key enzyme in plant carbohydrate metabolism, has been established in several higher plants, but there are no reports of it in ferns. The aim of the present work was to establish the localization of the previously reported acid invertase activity of Pteris deflexa in fronds tissues and to compare the findings with invertase localization in higher plants. Acid invertase, localized by immuno-histochemical and histochemical techniques on fresh tissues, was evident in vascular tissue, mainly in phloem. It was also detected in parenchymatic, sclerenchymatic and epidermic cells of petiole, rachis and rachis branches as well as in veins of leaf blades. Our results demonstrate that P. deflexa acid invertase localization is the same to that of higher plants. Hence, potential roles of the fern enzyme in relation to the storage and utilization of sucrose and to control carbon flux could be the same of those proposed to higher plants.

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 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.

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.