Up-Cycling of Olea europaea L. Ancient Cultivars Side Products: Study of a Combined Cosmetic-Food Supplement Treatment Based on Leaves and Olive Mill Wastewater Extracts.

In recent years, a reversal of the global economic framework has been taking place: from the linear model, there has been a gradual transition to a circular model where by-products from the agri-food industry are taken and transformed into value products (upcycling) rather than being disposed of. Olive tree pruning represents an important biomass currently used for combustion; however, the leaf part of the olive tree is rich in phenolic substances, including hydroxytyrosol. Mill wastewater is also discarded, but it still contains high amounts of hydroxytyrosol. In this study, cosmetic and food supplement formulations were prepared using biophenols extracted from leaves and wastewater and were tested in a placebo-controlled study on healthy volunteers using a combined cosmetic and food supplement treatment. A significant improvement in skin health indicators (collagen density, elasticity, etc.) and a 17% improvement against Photo-induced Irritative Stimulus was observed.

Prediction models for assessing anthocyanins in grape berries by fluorescence sensors: Dependence on cultivar, site and growing season.

Fluorescence sensors are useful tools for the non-destructive assessment of grape berry anthocyanins. The Multiplex (Mx) sensor here studied provides two anthocyanin indices: ANTHR = log(1/Chl-fluorescence_R) and ANTHRG = log(Chl-fluorescence_R/Chl-fluorescence_G), based on the chlorophyll (Chl) fluorescence excited with red (R) and green (G) light. These indices were calibrated against wet chemistry. The dependence of anthocyanin prediction models on cultivar, season and site was studied on four cultivars in two Italian regions during three consecutive years. The 2010 global model (all cultivars at both growing sites) gave relative prediction errors on anthocyanin content less than 14.1% (ANTHR) and 19.0% (ANTHRG). The ANTHRG was independent of season, maintaining a relative error of about 20% in both 2011 and 2012. In field applications of the calibrated Mx, it showed its ability to detect inter-plot and inter-season differences on both growing sites.

Lipophilization of Hydroxytyrosol-Enriched Fractions from Olea europaea L. Byproducts and Evaluation of the in Vitro Effects on a Model of Colorectal Cancer Cells.

A hydroxytyrosol (HTyr)-enriched fraction containing HTyr 6% w/w, derived from Olea europaea L. byproducts and obtained using an environmentally and economically sustainable technology, was lipophilized under green chemistry conditions. The effects of three fractions containing hydroxytyrosyl butanoate, octanoate, and oleate, named, respectively, lipophilic fractions 5, 6, and 7, and unreacted HTyr on the human colon cancer cell line HCT8-ß8 engineered to overexpress estrogen receptor ß (ERß) were evaluated and compared to those of pure HTyr. The experimental data demonstrated that HTyr and all fractions showed an antiproliferative effect, as had been observed by the evaluation of the cellular doubling time under these different conditions (mean control, 32 ± 4 h; HTyr 1, 65 ± 9 h; fraction 5, 64 ± 11 h; fraction 6, 62 ± 14 h; fraction 7, 133 ± 30 h). As evidenced, fraction 7 containing hydroxytyrosyl oleate showed the highest activity. These results were related to the link with ER-ß, which was assessed through simultaneous treatment with an inhibitor of ERß.

Hydrolyzable Tannins from Sweet Chestnut Fractions Obtained by a Sustainable and Eco-friendly Industrial Process.

Sweet Chestnut (Castanea sativa Mill.) wood extracts, rich in Hydrolyzable Tannins (HTs), are traditionally used in the tanning and textile industries, but recent studies suggest additional uses. The aim of this work is the HPLC-DAD-ESI-MS characterization of Sweet Chestnut aqueous extracts and fractions obtained through a membrane separation technology system without using other solvents, and the evaluation of their antioxidant and antiradical activities. Total tannins range between 2.7 and 138.4 mM; gallic acid ranges between 6% and 100%; castalagin and vescalagin range between 0% and 40%. Gallic Acid Equivalents, measured with the Folin-Ciocalteu test, range between 0.067 and 56.99 g/100 g extract weight; ORAC test results for the marketed fractions are 450.4 and 3050 µmol/g Trolox Equivalents/extract weight. EC50 values, measured with the DPPH test, range between 0.444 and 2.399 µM. These results suggest a new ecofriendly and economically sustainable method for obtaining chestnut fractions with differentiated, stable and reproducible chemical compositions. Such fractions can be marketed for innovative uses in several sectors.

Characterisation of the polyphenol content in the kiwifruit (Actinidia deliciosa) exocarp for the calibration of a fruit-sorting optical sensor.

INTRODUCTION: Kiwifruit contains high amounts of anti-oxidants beneficial to health. Its quality is influenced by ripening time, genotype, cultivation techniques, climate and storage conditions after harvest. OBJECTIVE: The aim of the present study was to characterise the phenolic content by HPLC methods and to evaluate the performance of a portable optical sensor (Multiplex 3), for in vivo non-destructive phenolic compound assessment in kiwifruits. METHODS: Kiwifruits peel extracts were characterised by reverse-phase (RP) HPLC with diode-array detector (DAD) and electrospray ionisation (ESI) with MS using the Zorbax SB-Aq. column from Agilent. The fluorimetric sensor method is based on the screening of fruit chlorophyll fluorescence excitation and allows the UV absorbance of intact fruit skin to be measured. The flavonol index, FLAV, was calculated as log(FRF(R)/FRF(UV)), where FRF(R) and FRF(UV) are the chlorophyll fluorescence excited with red and UV light. RESULTS: Hydroxycinnamic acids, procyanidins, and quercetin glycosides were the main polyphenol classes detected by HPLC-DAD-ESI/MS in the kiwifruit skin. A good linear regression (R² = 0.88) was found between the fluorimetric sensor FLAV index and flavonol chromatographic analysis of the fruits. The FLAV index was able to detect the higher content of flavonols in sun-exposed fruits with respect to mid-shaded and shaded ones in accordance with the destructive analysis. CONCLUSION: The fluorimetric sensor represents a rapid and non-invasive tool to: (i) monitor the flavonol accumulation in kiwifruit and to assess its quality concerning the healthy anti-oxidant properties; (ii) evaluate the effect of environmental and agronomical factors related to the fruit quality; and (iii) select fruits with the largest flavonol content, and consequently less susceptible to pathogen attack, in order to improve their storage durability.

Simultaneous determination of anthocyanins, coumarins and phenolic acids in fruits, kernels and liqueur of Prunus mahaleb L.

In the fresh tissues of Prunus mahaleb L., three classes of phenolics were characterised: phenolic acid derivatives (main compound being o-coumaric acid glucoside), quercetin glycosides, and anthocyanins (cyanidin 3,5-diglucoside, cyanidin 3-sambubioside, cyanidin 3-xylosyl-rutinoside and cyanidin 3-rutinoside). Coumarin was also identified. The kernels showed a high content of coumarin (0.87 mgg(-1)) which is the main class of metabolites in this sample, but present in pitted berries as well (0.63 mgg(-1)). Flavonoids are mainly concentrated in the skin and pulp (0.55 mgg(-1)). In 'Mirinello di Torremaggiore' liqueur, produced from P. mahaleb L. in accordance with traditional procedures, anthocyanins make up 16.5%, phenolic acids 43.3%, coumarin 36.2% and flavonoids 4% of total compounds. Anthocyanins are the main class in solid residues from liqueur production (70%). These findings point out that solid residues of P. mahaleb can be considered an interesting and innovative source of appreciable amounts of cyanidin glycosides (3.3 mgg(-1)).

Antimicrobial and antiviral activity of hydrolysable tannins.

Hydrolysable tannins (HTs), secondary metabolites widely distributed in the plant kingdom, are generally multiple esters of gallic acid with glucose. HTs have been shown to be effective antagonists against viruses, bacteria and eukaryotic microorganisms. The present review examines the antimicrobial and antiviral activity of HTs, the mechanism(s) of action, and some structure-activity relationships.

Extraction and HPLC analysis of phenolic compounds in leaves, stalks, and textile fibers of Urtica dioica L.

In the present study the phenolic composition of leaves, stalks, and textile fiber extracts from Urtica dioica L. is described. Taking into account the increasing demand for textile products made from natural fibers and the necessity to create sustainable "local" processing chains, an Italian project was funded to evaluate the cultivation of nettle fibers in the region of Tuscany. The leaves of two nettle samples, cultivated and wild (C and W), contain large amounts of chlorogenic and 2- O-caffeoylmalic acid, which represent 71.5 and 76.5% of total phenolics, respectively. Flavonoids are the main class in the stalks: 54.4% of total phenolics in C and 31.2% in W samples. Anthocyanins are second in quantitative importance and are present only in nettle stalks: 28.6% of total phenolics in C and 24.4% in W extracts. Characterization of phenolic compounds in nettle extracts is an important result with regard to the biological properties (antioxidant and antiradical) of these metabolites for their possible applications in various industrial activities, such as food/feed, cosmetics, phytomedicine, and textiles.

Optically-assessed preformed flavonoids and susceptibility of grapevine to Plasmopara viticola under different light regimes.

The role of flavonoids in the response of plants to Plasmopara viticola, the phytopathogen agent of downy mildew, was studied in the Vitis vinifera L. cultivar Sangiovese. Grapevines in the vineyard were exposed to two light regimes, 100% and 35% of full sunlight in order to induce differences in total leaf polyphenolic content. Epidermal leaf phenolic compounds were assessed optically, using the Dualex chlorophyll fluorescence-based portable leaf-clip. Dualex data were calibrated by means of HPLC analysis of extracts from the same measured leaves. Good correlations were obtained with total flavonoid contents, which consist mainly of quercetin 3-O-glucuronide. From the Dualex non-destructive measurements, we showed that full-sun exposed leaves contained 75% more flavonoids than shaded leaves. Inoculation of leaves with P. viticola sporangia resulted in a significantly lower infected leaf area in sun-lit leaves compared with shaded ones, as seen from subsequent analysis of the downy mildew severity. These results indicated an inverse relationship between preformed flavonoids and the susceptibility of grapevines to downy mildew. The rapid optical method for the non-destructive assessment of flavonoids presented here could be useful for large scale screening and predicting V. vinifera susceptibility to P. viticola.

Morpho-anatomical, physiological and biochemical adjustments in response to root zone salinity stress and high solar radiation in two Mediterranean evergreen shrubs, Myrtus communis and Pistacia lentiscus.

Salt- and light-induced changes in morpho-anatomical, physiological and biochemical traits were analysed in Myrtus communis and Pistacia lentiscus with a view to explaining their ecological distribution in the Mediterranean basin. In plants exposed to 20 or 100% solar radiation and supplied with 0 or 200 mm NaCl, measurements were conducted for ionic and water relations and photosynthetic performance, leaf morpho-anatomical and optical properties and tissue-specific accumulation of tannins and flavonoids. Net carbon gain and photosystem II (PSII) efficiency decreased less in P. lentiscus than in M. communis when exposed to salinity stress, the former having a superior ability to use Na(+) and Cl(-) for osmotic adjustment. Morpho-anatomical traits also allowed P. lentiscus to protect sensitive targets in the leaf from the combined action of salinity stress and high solar radiation to a greater degree than M. communis. Salt and light-induced increases in carbon allocated to polyphenols, particularly to flavonoids, were greater in M. communis than in P. lentiscus, and appeared to be related to leaf oxidative damage. Our data may conclusively explain the negligible distribution of M. communis in open Mediterranean areas suffering from salinity stress, and suggest a key antioxidant function of flavonoids in response to different stressful conditions.

On the role of flavonoids in the integrated mechanisms of response of Ligustrum vulgare and Phillyrea latifolia to high solar radiation.

The role of flavonoids in mechanisms of acclimation to high solar radiation was analysed in Ligustrum vulgare and Phillyrea latifolia, two Mediterranean shrubs that have the same flavonoid composition but differ strikingly in their leaf morpho-anatomical traits. In plants exposed to 12 or 100% solar radiation, measurements were made for surface morphology and leaf anatomy; optical properties, photosynthetic pigments, and photosystem II efficiency; antioxidant enzymes, lipid peroxidation and phenylalanine ammonia lyase; synthesis of hydroxycinnamates and flavonoids; and the tissue-specific distribution of flavonoid aglycones and ortho-dihydroxylated B-ring flavonoid glycosides. A denser indumentum of glandular trichomes, coupled with both a thicker cuticle and a larger amount of cuticular flavonoids, allowed P. latifolia to prevent highly damaging solar wavelengths from reaching sensitive targets to a greater degree than L. vulgare. Antioxidant enzymes in P. latifolia were also more effective in countering light-induced oxidative load than those in L. vulgare. Consistently, light-induced accumulation of flavonoids in L. vulgare, particularly ortho-dihydroxylated flavonoids in the leaf mesophyll, greatly exceeded that in P. latifolia. We conclude that the accumulation of flavonoid glycosides associated with high solar radiation-induced oxidative stress and, hence, biosynthesis of flavonoids appear to be unrelated to 'tolerance' to high solar radiation in the species examined.

Nondestructive evaluation of anthocyanins in olive (Olea europaea) fruits by in situ chlorophyll fluorescence spectroscopy.

Anthocyanins (Anths) in olive (Olea europaea L.) fruits at different degrees of pigmentation were assessed nondestructively by measuring chlorophyll fluorescence (ChlF). The method is based on the comparison of the ChlF excitation spectra from olives with different pigmentation from green to green-red, reddish-purple, and purple. The logarithm of the ratio between the fluorescence excitation spectra (logFER) from two different colored zones gave the difference in the absorption spectrum between them. The absorbance spectrum derived from the logFER between a red olive and the same olive devoid of the skin showed the typical Anth green band (at 550 nm). It matched that recorded by microspectrophotometry on a single pulp cell and the in vitro absorbance spectrum of the olive skin extract. As expected, the in vivo Anths absorption maximum increased in intensity going from less to more mature olives and was higher in the sun-exposed olive side with respect to the sun-shaded side. Absolute quantitative nondestructive determination of Anths for each olive sample was obtained by the logFER calculated for two excitation wavelengths, 550 and 625 nm, of ChlF at 740 nm. Going from green to purple skin colors, the Log[ChlF(625)/ChlF(550)] was fairly well-correlated to the extract Anths concentration. Finally, the relationship between the Anths and the other main phenolics present in the olives analyzed by high-performance liquid chromatography was evaluated. The main result was a net increase of verbascoside with increasing Anths content. On the basis of our results, the development of a new rapid and noninvasive method for the monitoring of olive development and ripening can be envisaged.

Evaluation of antioxidant effect of different extracts of Myrtus communis L.

Oxidative stress is involved in the pathogenesis of numerous diseases. Nevertheless, no optimal natural antioxidant has been found for therapeutics, therefore polyphenol antioxidants have been looked for in myrtle leaves, a plant that in folk medicine has been used as anti-inflammatory drug. Antioxidant-rich fractions were prepared from myrtle (Myrtus communis L.) leaves liquid-liquid extraction (LLE) with different solvents. All myrtle extracts were very rich in polyphenols. In particular, hydroalcoholic extracts contain galloyl-glucosides, ellagitannins, galloyl-quinic acids and flavonol glycosides; ethylacetate extract and aqueous residues after LLE are enriched in flavonol glycosides and hydrolysable tannins (galloyl-glucosides, ellagitannins, galloyl-quinic acids), respectively. Qualitative and quantitative analysis for the single unidentified compound was also performed. Human LDL exposed to copper ions was used to evaluate the antioxidant activity of the myrtle extracts. Addition of these extracts did not affect the basal oxidation of LDL but dose-dependently decreased the oxidation induced by copper ions. Moreover, the myrtle extracts reduce the formation of conjugated dienes. The antioxidant effect of three myrtle extracts decreased in the following order: hydroalcoholic extracts, ethylacetate and aqueous residues after LLE. The extracts had the following IC50: 0.36, 2.27 and 2.88 microM, when the sum of total phenolic compounds was considered after the correction of molecular weight based on pure compounds. Statistical analysis showed a significant difference among hydroalcoholic extracts vs. the ethylacetate and aqueous residues after LLE. These results suggest that the myrtle extracts have a potent antioxidant activity mainly due to the presence of galloyl derivatives.

Differential accumulation of flavonoids and hydroxycinnamates in leaves of Ligustrum vulgare under excess light and drought stress.

• The differential accumulation of various polyphenols, particularly of flavonoids and hydroxycinnamates, was studied in leaves of Ligustrum vulgare exposed to increasing sunlight under well watered or drought-stress conditions. • Light- and drought-induced changes in leaf polyphenol concentrations were normalized to the CO2 assimilation rate. The functional roles of flavonoids and hydroxycinnamates were analysed through tissue localization using multispectral fluorescence microimaging, and through efficiencies to scavenge superoxide radicals (O2 - ) and to screen UV wavelengths. • Clear effects of light and water treatments on leaf polyphenol concentrations were not observed, as the CO2 assimilation rate varied according to sunlight and water availability. However, biosynthesis of quercetin 3-O-rutinoside, luteolin 7-O-glucoside and echinacoside, which were efficient O2 - scavengers, increased sharply in response to solar radiation. By contrast, carbon for the synthesis of p-coumaric acid and monohydroxyflavones, efficient UV screeners but poor O2 - scavengers, did not vary depending on light treatments. Flavonoids accumulated in both the adaxial epidermis and the palisade tissue because of sunlight irradiance, whereas echinacoside occurred largely in abaxial tissues. • We hypothesize that flavonoids may serve antioxidant functions in response to excess light and drought stress, and that a coordinate control system between hydroxycinnamate and flavonoid pathways operated in L. vulgare exposed to excess light.

Antioxidant activity of galloyl quinic derivatives isolated from P. lentiscus leaves.

The antioxidant properties of galloyl quinic derivatives isolated from Pistacia lentiscus L. leaves have been investigated by means of Electron Paramagnetic Resonance spectroscopy (EPR) and UV-Vis spectrophotometry. Antioxidant properties have been also estimated using the biologically relevant LDL test. The scavenger activities of gallic acid, 5-O-galloyl, 3,5-O-digalloyl, 3,4,5-O-trigalloyl quinic acid derivatives, have been estimated against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, superoxide (O2) radical, and hydroxyl (OH) radical. On the whole, the scavenger activity raised as the number of galloyl groups on the quinic acid skeleton increased. The half-inhibition concentrations (IC50) of di- and tri-galloyl derivatives did not exceed 30 microM for all the tested free radicals. All the tested metabolites strongly reduced the oxidation of low-density lipoproteins (LDL), following a trend similar to that observed for the scavenger ability against OH radical.