Valorization of legumes, important constituents of the Mediterranean Diet, with specific attention to lentils and their nutraceutical effects

The current thesis has been developed and realized at the University of Camerino, School of Pharmacy in the Food Chemistry laboratories during my Ph.D course. This thesis provides a comprehensive assessment of the state of chemistry, nutrition and beneficial aspects of legumes, mainly lentils, which are an important food from a nutritional point of view. Whereupon, my research has been divided mainly into three parts. First of all, great attention was focused on the development of analytical instrumental methods for the analysis of important bioactive constituents in legumes, especially in lentils, such as soyasaponins and isoflavones, as well as, the fatty acid composition. The second part of this thesis was developed at the University of Valencia, Faculty of Pharmacy in the Toxicology laboratory. In this joint project, antioxidant properties of soyasaponins and lentil extracts were tested in ''in vitro'' cells to determine the cytoprotective action against mycotoxins such as Alternariol. Finally, the third part of the thesis, was focused on the ''in vivo'' study, in collaboration with Pharmacology research group of the University of Camerino, to test hypocholesterolemic action of lentil extracts rich in soyasaponins. Regarding bioactive constituents present in lentils, great attention was focused on soyasaponins that demonstrated to have health-promoting properties including plasma cholesterol-lowering, anti-carcinogenic, antioxidant and hepato-protective. In the first part of my thesis, an innovative and fast analytical method for the quantification of soyasaponins I and βg in lentils was developed. Samples were extracted using 70 % aqueous ethanol at room temperature and then injected into a high-performance liquid chromatography-tandem mass spectrometry system. The correlation coefficients of calibration curves of the analyzed compounds were ≥ 0.9997. The recoveries obtained by spiking the lentil samples with a standard mixture of soyasaponins I and βg at 50 and 100 mg l-1 were in the range of 96 - 101 and 98 - 103 %, respectively. The validated method was applied to the analysis of 30 lentil samples from central Italy. Soyasaponins I and βg were present in these lentils in concentrations that ranged from 54 to 226 mg kg-1 and from 436 to 1272 mg kg-1, respectively. Our data indicated that lentils cultivated in fields at intermediate altitudes (1142 - 1387 m) showed the highest levels of soyasaponins, a result confirmed by principal component analysis. Successively, a new analytical method for determining five isoflavone compounds, three of which are aglycons, namely daidzein, genistein, biochanin A, and two of which, daidzin and genistin, are glycosilated, in lentils and other legumes, by using an effective clean-up system and HPLC-MS/MS (triple quadrupole) method was developed. The recoveries obtained by spiking the lentil samples with a standard mixture of isoflavones at three levels of fortification (5, 25 and 100 μg kg-1) were in the range of 54.4 - 111.1 %, 68.6 - 91.1 %, and 84.4 - 114 %, respectively. The method was then applied to analyse 48 lentil samples from central Italy and other legumes for determining the isoflavone content which in lentils ranged from 1.1 to 95.6 μg kg-1, while chickpeas sample showed the highest isoflavone content (913.8 μg kg-1). After that, the fatty acid composition of 29 legumes (mostly lentils) after the optimization of the extraction method, was studied. The Folch method and liquid-solid extraction with hexane/isopropanol or with hexane/acetone were investigated, as was the effect of previous hydration of samples. Soxhlet extractions were also evaluated with different solvent mixtures. Results on fatty acids composition obtained using the hexane/isopropanol extraction method were the same in terms of fatty acids composition of the Folch method, but the extraction yield was only around 20 - 40 % of that of the Folch method preceded by hydration. Some types of legumes showed particularly interesting values for the ratio of polyunsaturated fatty acids n-6/n-3, in particular lentils, with the value of 4.0, in which PUFAs ranged from 42.0 to 57.4 %. Concerning the second part of my work, the cytotoxicity of Alternariol (AOH), soyasaponin I (SS I) and soyasaponins-rich extract from lentils was investigated, as well as, the cytoprotective effects of SS I and lentil extracts against AOH induced-cytotoxicity on Caco-2 cells. AOH is a mycotoxin produced by Alternaria spp. that has been found in vegetables such as lentils. Cytotoxic and genotoxic effects of AOH have been demonstrated previously in vitro. In our study, cytotoxicity was carried out using MTT and PC assays (AOH: 3.125 - 100 μM, SS I: 3.125 - 50 μM, and lentil extracts: 1:0 - 1:32) during 24 h of exposure in Caco-2 cells. Only AOH showed cytotoxic effect. The reduction in cell proliferation ranged from 25 % to 47 %, while simultaneous combination of SS I with AOH (1:1) increased cell proliferation (35 %) compared to AOH tested alone. Then SS I and lentil extracts showed synergistic cytoprotective effects against cytotoxicity induced by AOH on Caco-2 cells. Thus food commodities, such as lentils, containing soyasaponins could contribute to diminish the toxicological risk that natural contaminant as AOH, can produce to humans. For what concern the third part of my work, an ''in vivo'' study was realized to test the hypocholesterolemic activity of a lentil extract. However, this part of the thesis concerning the ''in vivo'' study are the basis of Patent Application, and no more details can be provided.