Francesco Pagano

Nutraceutical properties and polyphenolic profile of berry skin and wine of Vitis vinifera L. (cv. Aglianico)

Red grapes are rich in phenolics, flavonoids, anthocyanins and resveratrol, all substances which have been suggested as having nutraceutical and health benefits. The berry skin and wine of grape cultivar Vitis vinifera L. (cv. Aglianico), grown in Basilicata (Southern Italy) were examined to determinate the presence of the above mentioned compounds as well as to establish the inorganic cation profile. HPLC analysis coupled with LC-ESI/MS/MS detected high contents of total flavonols and anthocyanins in berry skin and wine. The wine made with the same grape used for berry skin assays showed a notable presence of quercetin-3-O-glucoside (39.4% of total flavonols), and malvidin and petunidin derivatives (63.9% and 10.8% of total anthocyanins, respectively). The strong antioxidant ROS-scavenging activity, determined by both DPPH and FRAP assays, and the high resveratrol content confer high sensory characteristics resulted to be associated with positive nutraceutical properties of these grapes and wine. The level of cis-resveratrol was lower than trans-resveratrol in both berry skin and wine reaching 44.1mg/kg and 0.3mg/l, respectively. The cation profile presents low levels of Ca, Cu, K, Fe, Zn and Cd compared to numerous, important red wines, such as Monastrell and Tempranillo.

Ultra high performance liquid chromatography with ion‐trap TOF‐MS for the fast characterization of flavonoids in Citrus bergamia juice

We have developed a fast ultra HPLC with ion-trap TOF-MS method for the analysis of flavonoids in Citrus bergamia juice. With respect to the typical methods for the analysis of these matrices based on conventional HPLC techniques, a tenfold faster separation was attained. The use of a core-shell particle column ensured high resolution within the fast analysis time of only 5 min. Unambiguous determination of flavonoid identity was obtained by the employment of a hybrid ion-trap TOF mass spectrometer with high mass accuracy (average error 1.69 ppm). The system showed good retention time and peak area repeatability, with maximum RSD% values of 0.36 and 3.86, respectively, as well as good linearity (R(2) ≥ 0.99). Our results show that ultra HPLC can be a useful tool for ultra fast qualitative/quantitative analysis of flavonoid compounds in citrus fruit juices.

Detailed polyphenolic profiling of Annurca apple (M. pumila Miller cv Annurca) by a combination of RP-UHPLC and HILIC, both hyphenated to IT-TOF mass spectrometry

Annurca apple, a Southern Italian cultivar, possesses not only a particular taste and flavor, different from other types of apple, but also several healthy properties. With the aim to thoroughly elucidate the polyphenolic profile of this variety, listed as Protected Geographical Indication product, an extensive qualitative profiling of Annurca apple polyphenolic peel extract was carried out, by employing a combination of ultra high performance reversed phase (RP-UHPLC) and hydrophilic liquid chromatography (HILIC) coupled to ion trap-time of flight (IT-TOF) mass spectrometry. A total of 63 compounds were tentatively identified, 25 of which not reported in Annurca apple extract so far. Furthermore, thanks to the different selectivity obtained with the HILIC, in combination with accurate mass measurements, an improved separation and detection of procyanidins, was obtained. Moreover, the obtained profiles were compared with those of a conventional variety, such as Red Delicious (RD), highlighting their differences. This work contributes to increase the knowledge about the polyphenolic fingerprint of this typical apple variety.

Evaluation of two sub-2μm stationary phases, core-shell and totally porous monodisperse, in the second dimension of on-line comprehensive two dimensional liquid chromatography, a case study: Separation of milk peptides after expiration date

Milk is a rich source of bioactive peptides of great interest for their healthy properties. These peptides are usually encrypted in the sequences of proteins and are released after time dependent proteolysis as very complex hydrolysates. In order to separate and identify the bioactive sequences, we developed an on-line comprehensive two dimensional liquid chromatography approach using the high performance combined with the ultra high performance conditions. A microbore reversed phase (C18 silica, 5 μm) column was employed in first dimension, while, in second dimension, two different UHPLC columns, packed with C18 silica, were tested: a new column based on monodisperse sub-2 μm fully porous particles with high surface area (50 mm × 3.0 mm, 1.9 μm d.p., from Supelco), and a column based on sub-2 μm core-shell particles (50 mm × 3.0 mm, 1.7μm d.p, from Phenomenex(®)). Both set-ups were compared, showing high peak capacity values with respect to a high efficiency monodimensional method, maintaining the same analysis time. Satisfactory selectivity was obtained through the use of different pH between the two dimension, while a very fast continuous shifted gradient in second dimension ensured a good employment of the 2D separation space.

Bioavailable Citrus sinensis Extract: Polyphenolic Composition and Biological Activity

Citrus plants contain large amounts of flavonoids with beneficial effects on human health. In the present study, the antioxidant and anti-inflammatory potential of bioavailable polyphenols from Citrus sinensis was evaluated in vitro and ex vivo, using the murine macrophages cell line J774A.1 and primary peritoneal macrophages. Following simulated gastro-intestinal digestion, the in vitro bioavailability of Citrus sinensis polyphenolic extract was assessed using the human cell line Caco-2 grown as monolayers on a transwell membrane. Data demonstrated a relative permeation of its compounds (8.3%). Thus, the antioxidant and anti-inflammatory effect of polyphenolic Citrus sinensis fraction (Cs) was compared to the bioavailable one (CsB). Results revealed that Citrus extract were able to reduce macrophages pro-inflammatory mediators, including nitric oxide, iNOS, COX-2 and different cytokines. Moreover, the effect of Citrus sinensis polyphenols was associated with antioxidant effects, such as a reduction of reactive oxygen species (ROS) and heme-oxygenase-1 (HO-1) increased expression. Our results provide evidence that the bioavailable polyphenolic constituents of the Citrus sinensis extract accumulate prevalently at intestinal level and could reach systemic circulation exerting their effect. The bioavailable fraction showed a higher anti-inflammatory and antioxidant potential compared to the initial extract, thus highlighting its potential nutraceutical value.