Solubility study and intensification of extraction of phenolic and anthocyanin compounds from Oryza sativa L. 'Violet Nori'.

Oryza sativa L. 'Violet Nori' is an Italian cultivar of spontaneous growing aromatic purple rice which is particularly rich in polyphenolic compounds, especially anthocyanins, conferring it an excellent antioxidant capacity. The present study aimed at increasing the extraction yields of its antioxidant compounds with green strategies and it is divided into two steps. The first step concerned a solubility study of the targeted polyphenols in different ethanol:water mixtures by means of a theoretical prediction method, using the simulation program COSMO-RS, and the subsequently confirmation of the computational results by practical experiments. Once the best extraction mixture was identified, the second step of the work was performed, with the purpose of intensifying the extraction yield. Therefore, various innovative green extraction techniques, including ultrasound, using both the probe system and the ultrasonic bath, bead milling, microwave and accelerated solvent extractions were tested and compared to conventional maceration. Results, expressed in terms of total phenolic and total monomeric anthocyanin contents, showed that the best extracting solvent for 'Violet Nori' rice was the mixture ethanol:water (60:40 v/v), being COSMO-RS computational predictions in good correlation with the experimental results. Moreover, the most efficient techniques to extract the antioxidant compounds resulted to be both ultrasound-assisted extraction probe and bead milling, that in only 5 min got the same extractive efficiency obtained after 3 h of conventional maceration.

Phytochemical fingerprint and chemometrics for natural food preparation pattern recognition: an innovative technique in food supplement quality control.

Recently, the fingerprint approach using chromatography has become one of the most effective tools for quality assessment of herbal medicines and food supplements: due to the complexity of the chromatographic fingerprint and the irreproducibility of chromatographic instruments and experimental conditions, chemometric approach is employed to deal with the chromatographic fingerprint. The study was aimed at developing new analytical methods for the multivariate phytochemical fingerprinting of bioactive compounds in eight tree-species bud-preparations, commonly used in phytotherapy. Methods was used to identify and quantify the main bioactive compounds (polyphenols, organic acids and vitamins), and obtain a specific botanical profile in order to assess the contribution of each single bioactive class to the total bud preparation phytocomplex. A chemometric approach was used to distinguish among different genotypes assuring the identity, safety and quality of the botanical raw materials. The established protocol was simple, sensitive and reliable and it could be used for the evaluation and quality control of bud-extracts and natural food supplements: the proposed method was successfully applied to the characterization of commercial bud-preparations, demonstrating to be an effective tool for the fingerprinting of this plant material. The new approach developed in this study represents a good alternative for improving the classification results of herbal materials with complex chromatograms. It should be necessary to develop a "multivariate chromatographic fingerprint", in order to differentiate the herbal preparations according to their genotype, avoiding substitutions, changes or adulterations with other species or synthetic drugs.

7-ketocholesterol in human and adapted milk formulas.

In the last few years, a variety of experimental and clinical studies concerning the formation, metabolism, and cellular effects of cholesterol oxidation products (COPs) have been carried out. Nevertheless, a substantial lack of knowledge exists regarding the possible intake of these compounds by the newborn through human and/or adapted formula milk. As far as the pathological role of COPs is concerned, exhaustive studies have shown that since dietary COPs are cytotoxic and atherotoxic, they may lead to adverse effects on health. The aim of this study was to investigate the possible development of cholesterol oxidation in adapted formula and in human milk by comparing the main cholesterol oxidation biomarker (7-ketocholesterol) concentration in both. To do so, the total (bonded and free) 7-ketocholesterol content was measured in ten fresh human mature milk samples and in ten milk adapted formula samples by high performance liquid chromatography and diode array detection. The 7-ketocholesterol human milk content (0.7+/-0.3) was often below the quantifiable limit (0.5 microg/g of extracted lipids) while 7-ketocholesterol adapted milk concentrations were often above (3.6+/-4.0) this limit. The 7-ketocholesterol content of adapted formula samples was significantly higher as compared to human milk samples (P<0.05). This is the first study to provide data concerning the concentrations of cholesterol oxides in human milk and in formula milk. Our results clearly suggest that the manufacturing technologies employed and the nutrient extractive sources play a crucial role in the development of cholesterol oxides in the end product. Careful surveillance has to be paid in order to avoid alteration of bioactive properties of nutrients and/or development of potentially toxic derivative compounds.

3-Acetyl-5-acylpyridin-2(1H)-ones and 3-acetyl-7,8-dihydro-2,5(1H,6H)-quinolinediones: synthesis, cardiotonic activity and computational studies.

A series of milrinone analogues, namely 6-substituted 3-acetyl-5-acylpyridin-2(1H)-ones 4a-c, e, f and 7-substituted or unsubstituted 3-acetyl-7,8-dihydro-2,5(1H,6H)-quinolinediones 4g-j, in which the cyano group was replaced by the acetyl function, was prepared. In a preliminary pharmacological investigation on spontaneously beating atria from reserpine-treated guinea-pigs, all new compounds did not induce any inotropic effect equivalent or higher than that of the milrinone chosen as the reference compound. In order to rationalise how the structure modifications influence the activity and the selectivity of the title compounds, a computational study has been performed. The important role of the substituents in positions-3 and -6 on the pyridone nucleus has been highlighted.

Toward the identification of the cardiac cGMP inhibited-phosphodiesterase catalytic site.

Cyclic nucleotide phosphodiesteras (PDEs) comprise a complex group of enzymes; five major PDE families or classes with distinctive properties have been identified. Among these a great deal of interest has recently been focused on the so called cGMP-inhibited low K(m) cAMP phosphodiesterase (cGI PDE) or PDE III. A number of positive inotropic agents, including the well-known milrinone, display a specific inhibition of PDE III as primary mechanism of action. Recent studies have been carried out to develop a pharmacophore model of the PDE III active site. We therefore performed molecular modelling and 3D-SAR studies so as to better define structural requirements for potent and selective enzymatic inhibition. The DISCO (DIStance COmparison) strategy has been applied on a set of compounds taken from literature and a milrinone analogue previously synthesized by us, all of which are characterized by a marked inotropic effect but with varying degrees of enzyme selectivity. A common pharmacophoric model was derived, validated and considered as starting point to perform a 3D-SAR study using the GRID force field and PCA (Principal Component Analysis) with the aim of rationally designing more selective inhibitors. This paper presents the results of this theoretical approach.

Inotropic agents. Synthesis and structure-activity relationships of new milrinone related cAMP PDE III inhibitors.

The synthesis of 6-substituted 5-acyl-1,2-dihydro-2-oxo-3-pyridinecarbonitriles 1b,c, 1,2,5,6,7,8-hexahydro-2,5-dioxo-3-quinolinecarbonitriles 1d-g and esters of 5-cyano-1,6-dihydro-2-methyl-6-oxo-3-pyridinecarboxylic acid 2b-e is described. In the case of 1e and 1f, a careful elucidation of the reaction mechanism is discussed. As milrinone analogues, the above compounds were tested on contractile activity and frequency rate of spontaneously beating atria from reserpine-treated guinea pigs. The methyl and the benzyl esters 2b and 2e showed an appreciable positive inotropic activity when compared to milrinone. A fitting study with the DISCO (Distance Comparison) model has been carried out on 2e. This modeling approach allowed for the improvement of the pharmacophoric requirements for a better interaction with the cAMP-specific PDE (PDE III), thought to be the final biological target of these cardiotonic agents.

Zupan's descriptors in QSAR applied to the study of a new class of cardiotonic agents.

Recently a new class of molecular descriptors has been proposed and used in QSAR with simulated data and with regression performed by neural networks. In the present paper these descriptors (Zups, from the name of their author, Juri Zupan) have been slightly modified and then applied to a real data set with the aim of studying the structure-activity relationships of a new class of cardiotonics. Forty-one molecules (thirty-seven milrinone analogues, the two lead compounds amrinone and milrinone, and two commercial products) have been studied using classical chemometrical techniques such as PCA (Principal Components Analysis) and PLS (Partial Least Squares regression). Zups describe essentially the local geometry of the molecules. They show promising performances, as compared with other classical geometrical descriptors (as molecular volume, etc.), both in that regards the overall performances, measured by the C.V. Explained variance and in the interpretability of the regression equation. However they have not all the requirements of a good structure representation. Moreover some selectable parameters seem to have a great importance, so that the refinement of the regression model requires time and the evaluation step must be performed in condition of full-validation, because predictive optimisation is used in the selection of parameters, and the final model must be checked on molecules never used to refine the model or, in this case, the parameters of the structure representation.