Diamine Oxidase as a Therapeutic Enzyme: Study of Germination from Vegetal Sources and Investigation of the Presence of ß-N-Oxalyl-L-α,ß-diaminopropionic Acid (ß-ODAP) Using LC-MS/MS.

Vegetal diamine oxidase (vDAO), an enzyme proposed to relieve symptoms of histaminosis, shows better reactivity with histamine and aliphatic diamines, as well as higher enzymatic activity than DAO of animal origin. The objective of this study was to evaluate the enzyme activity of vDAO from germinating grains from Lathyrus sativus (grass pea) and Pisum sativum (pea), and to verify the presence of a neurotoxin, ß-N-Oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), in the crude extract obtained from their seedlings. A targeted liquid chromatography-multiple-reaction monitoring mass spectrometry method was developed and used to quantify ß-ODAP in the analysed extracts. An optimized sample preparation procedure, involving protein precipitation with acetonitrile followed by mixed-anion exchange solid-phase extraction, allowed for high sensitivity and good peak shape for ß-ODAP detection. The Lathyrus sativus extract exhibited the highest vDAO enzyme activity of the extracts, followed by the extract from pea cultivar Amarillo from the Crop Development Centre (CDC). The results have also shown that even though ß-ODAP was present in the crude extract from L. sativus, its content was far below the toxicity threshold (300 mg of ß-ODAP/kg body/day). CDC Amarillo showed 5000-fold less ß-ODAP than the undialysed L. sativus extract. It was concluded that both species can be considered as convenient sources of vDAO for potential therapeutic use.

Enhanced Stability of Vegetal Diamine Oxidase with Trehalose and Sucrose as Cryoprotectants: Mechanistic Insights.

Enteric dysfunctions are common for various histamine-related intestinal disorders. Vegetal diamine oxidase (vDAO), an enzyme able to decompose histamine and thus alleviate histamine-related dysfunctions, was formulated in gastro-resistant tablet forms for oral administration as a food supplement and possible therapeutic agent. A major challenge for the use of proteins in the pharmaceutical field is their poor stability. In this study, vDAO was freeze-dried in the absence or in the presence of sucrose or trehalose as cryoprotectants and then formulated as tablets by direct compression. The stability of the obtained preparations was followed during storage at 4 °C and -20 °C for 18 months. In vitro dissolution tests with the vDAO powders formulated as tablets were performed in simulated gastric and in simulated intestinal fluids. The tablets obtained with the powder of the vDAO lyophilized with sucrose or trehalose cryoprotectants offered better protection for enzyme activity. Furthermore, the release of the vDAO lyophilized with the cryoprotectants was around 80% of the total loaded activity (enzyme units) compared to 20% for the control (vDAO powder prepared without cryoprotectants). This report revealed the potential of sucrose and trehalose as cryoprotectants to protect vDAO from freeze-drying stress and during storage, and also to markedly improve the vDAO release performance of tablets obtained with vDAO powders.

Synthesis of Metal-Loaded Carboxylated Biopolymers with Antibacterial Activity through Metal Subnanoparticle Incorporation.

Carboxymethyl starch (CMS) and carboxymethyl cellulose (CMC) loaded by highly dispersed metal subnanoparticles (MSNPs) showed antibacterial activity against E. coli and B. subtilis strains. Copper and silver were found to act in both cationic and zero-valence forms. The antibacterial activity depends on the metal species content but only up to a certain level. Silver cation (Ag+) showed higher antibacterial activity as compared to Ag0, which was, however, more effective than Cu0, due to weaker retention. The number of carboxyl groups of the biopolymers was found to govern the material dispersion in aqueous media, the metal retention strength and dispersion in the host-matrices. Cation and metal retention in both biopolymers was found to involve interactions with the oxygen atoms of both hydroxyl and carboxyl groups. There exists a ternary interdependence between the Zeta potential (ZP), pH induced by the biocidal agent and its particle size (PS). This interdependence is a key factor in the exchange processes with the surrounding species, including bacteria. Clay mineral incorporation was found to mitigate material dispersion, due to detrimental competitive clay:polymer interaction. This knowledge advancement opens promising prospects for manufacturing metal-loaded materials for biomedical applications.

Insights into the metal retention role in the antibacterial behavior of montmorillonite and cellulose tissue-supported copper and silver nanoparticles.

The role of the retention strength of Cu0 and Ag0 nanoparticles on the induced antibacterial properties of montmorillonite and cellulose-supported polyol dendrimer was comparatively investigated. An unprecedented approach involving X-ray photoelectron spectroscopy, thermal analyses, and surface charge measurements allowed correlating the host-matrix features to the different antibacterial activities of Cu0 and Ag0 nanoparticles against both the bacterial strains. Optimal metal-matrix interactions appear to favor high dispersion of both metal particles and material grains, thereby improving the contact surface with the cultivation media. This was explained in terms of hydrophilic character and judicious compromise between the metal retention by the host-matrix and release in the impregnating media. Competitive Lewis acid-base interactions appear to occur between MNP, solid surface and liquid media. These findings are of great importance, providing a deeper understanding of the antibacterial activity of metal-loaded materials. This opens promising prospects for vegetal fibers and clay-supported drugs to treat dermatological and gastro-intestinal infections.