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.

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.

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.

Characterisation of PDO olive oil Chianti Classico by non-selective (UV-visible, NIR and MIR spectroscopy) and selective (fatty acid composition) analytical techniques.

An authentication study of the Italian PDO (protected designation of origin) extra virgin olive oil Chianti Classico was performed; UV-visible (UV-vis), Near-Infrared (NIR) and Mid-Infrared (MIR) spectroscopies were applied to a set of samples representative of the whole Chianti Classico production area. The non-selective signals (fingerprints) provided by the three spectroscopic techniques were utilised both individually and jointly, after fusion of the respective profile vectors, in order to build a model for the Chianti Classico PDO olive oil. Moreover, these results were compared with those obtained by the gas chromatographic determination of the fatty acids composition. In order to characterise the olive oils produced in the Chianti Classico PDO area, UNEQ (unequal class models) and SIMCA (soft independent modelling of class analogy) were employed both on the MIR, NIR and UV-vis spectra, individually and jointly, and on the fatty acid composition. Finally, PLS (partial least square) regression was applied on the UV-vis, NIR and MIR spectra, in order to predict the content of oleic and linoleic acids in the extra virgin olive oils. UNEQ, SIMCA and PLS were performed after selection of the relevant predictors, in order to increase the efficiency of both classification and regression models. The non-selective information obtained from UV-vis, NIR and MIR spectroscopy allowed to build reliable models for checking the authenticity of the Italian PDO extra virgin olive oil Chianti Classico.

Sterol oxidation in infant milk formulas and milk cereals.

7-Ketocholesterol and 7-ketositosterol were chosen as reliable markers of the oxidation of cholesterol and phytosterols in infant milk formulas and infant milk cereals. A reversed-phase HPLC method was developed to measure them simultaneously in infant formulas. This method was then tested on a wide range of infant milk formulas and milk cereals on sale in Italy whose lipid composition is representative of the most common commercial formulas. The analytical results revealed no significant differences in the extent of oxidation of cholesterol and sitosterol. As the level of 7-ketocholesterol often followed the cholesterol level, a cholesterol content similar to that of human milk produced amounts of cholesterol oxides with possible negative effects on infant health. In contrast, the low cholesterol content of milk cereals never produced amounts of cholesterol oxides high enough to cause concern. The contents of phytosterols and hence their oxides were always low.

[Essential fatty acids in adipose tissue]. / Gli acidi grassi essenziali nel tessuto adiposo.

Among the numerous causes of lipomobilization of fatty acids from the adipose tissue it is also carbon tetrachloride (CCl4). The research shows that the saturated fatty acids decreased in this tissue of the rats injected with CCl4, even if the linolenic and alpha-linolenic acids are present in the diet.

[Correlation between medium C-chain fatty acids and polyunsaturated fatty acids]. / Correlazioni tra gli acidi grassi a C-catena media e poli-insaturi.

The medium C-chain fatty acids increased in the muscle, lungs, pancreas and adipose tissue (and not in the liver) of the rats injected with CCl4 or nourished with "balanced" diet for the lipids. When CCl4 and balanced diet are furnished together, these acids decrease strongly: the discussion of the results is difficult.

[Essential fatty acids for ectodermic organs]. / Gli acidi grassi "essenziali" per organi "ectodermici".

Damage of exocrine pancreas liver and lungs was induced by CCl4, probably because an unbalancement of essential fatty acids (EFA). The present research is made on the rats, injected with CCl4 subcutaneously and kept with diets at variable composition in linoleic acid, proceeding to the extraction of these organs for the gaschromatografic determination of fatty acids. CCl4 reduces the arachidonic acid in liver, pancreas and lungs of the injected rats and the dietary linoleic acid does not modify this picture in the organs.

[Alpha-linolenic acid and its metabolic derivatives in the tissues of rats treated with CCl4]. / L'acido alfa-linolenico e derivati di conversione nei tessuti di ratti trattati con CCl4.

The poly-unsaturated fatty acids of w-3 series were studied in the tissues of rats injected with CCl4 and also the effect of the alpha-linolenic acid added to the diet. Gascromathographic technique of fatty acids was applied on samples of liver, muscle, lungs, pancreas and adipose tissue. CCl4 was seen to increase in the liver and in the muscle the docosahexaenoic acid; the dietetic alpha-linolenic acid inhibits, in muscle, lungs and pancreas, the conversion of linoleic in arachidonic acid.

[Alpha-linolenic acid in the trophism of striated muscle]. / L'acido alfa-linolenico nel trofismo del muscolo striato.

Erucic and alpha-linolenic acids are probably related to a damage of muscle. The research was defined to the question if CCl4 that causes lesions in the tissue of the rat) damages also the muscle and if the dietary unsaturated fatty acids are effective in the prevention. The alpha-linolenic acid causes a strong reduction of the docosaesaenoic acid in the muscle, while the reduction of arachidonic acid is quite similar in all studied organs.

Deterioration of protein fraction by Maillard reaction in dietetic milks.

The development of the Maillard reaction in pasteurized, UHT and in-bottle sterilized dietetic milks was studied. In these products damage caused by heat treatments could increase as a result either of the addition of various ingredients or of manufacturing processes that alter their content of reducing carbohydrates. Protein damage was evaluated by measuring furosine by reversed-phase ion-paired HPLC. The levels of furosine detected made it possible to assess the amounts of biologically unavailable lysine. In all milks analysed blocked lysine values were < 340-350 mg/g total lysine, the level at which lysine becomes the limiting amino acid in milk. Pasteurized dietetic milks had levels of blocked lysine similar to that in ordinary pasteurized cows' milk. In some UHT and in-bottle sterilized dietetic milks their different composition resulted in an increase in the blocked lysine content. In some in-bottle sterilized milks, protein damage greatly reduces the beneficial effects of milk as a dietary supplement. Lactose-free milks, which are more susceptible to protein deterioration because of their higher content of reducing carbohydrates, were also analysed after storage at 20 degrees C and at < or = 4 degrees C. At the end of their recommended storage times, they contained limited amounts of blocked lysine only if they had been stored at < or = 4 degrees C.