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Summary • 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.

Experiments were conducted on Phillyrea latifolia plants grown under a dense overstorey of Pinus pinea (shade plants) or on seashore dunes (sun plants) in a coastal area of Tuscany (42° 46′ N, 10° 53′ E). Total integrated photon flux densities averaged 1.67 and 61.4 m mol m−2 d−1 for shade and sun sites, respectively. A leaf morphological–structural analysis, a qualitative and quantitative analysis of phenylpropanoids of leaf tissue and leaf surface, and a histochemical localization of flavonoids were conducted. The area of sun leaves reached 57% of that of shade leaves, whereas leaf angle (β), sclerophylly index (ratio of leaf d. wt:leaf area), and trichome frequency (trichome number mm−2 ) were markedly greater in leaves exposed to full solar radiation than in leaves acclimated to shade. The total thickness of sun leaves was 78% higher than that of shade leaves, mostly owing to a greater development of both palisade parenchyma and spongy mesophyll. The concentration, but not the composition, of leaf tissue phenylpropanoids varied significantly between sun and shade leaves, with a marked increase in flavonoid glycosides in sun leaves. Flavonoids occurred almost exclusively in the upper epidermal cells of shade leaves. By contrast, flavonoids largely accumulated in the upper and lower epidermis, as well as in the mesophyll tissue of leaves that were acclimated to full sunlight. Flavonoid glycosides were found exclusively in the secretory products of glandular trichomes of P. latifolia leaves exposed to high levels of light; luteolin 7-O- glucoside and quercetin 3-O-rutinoside were the major constituents. By contrast, verbascoside and an unidentified caffeic acid derivative constituted 72% of total phenylpropanoids secreted by glandular trichomes of shade leaves, whereas they were not detected in glandular trichomes of sun leaves. These findings suggest that the light-induced synthesis of flavonoids in glandular trichomes of P. latifolia probably occurs in situ and concomitantly inactivates other branch pathways of the general phenylpropanoid metabolism. This is the first report of the key role of glandular trichomes and of flavonoid glycosides in the integrated mechanisms of acclimation of P. latifolia to excess light.

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-β8 engineered to overexpress estrogen receptor β (ERβ) 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-β, which was assessed through simultaneous treatment with an inhibitor of ERβ.

The introgressed cultivar of Parainema coffee was studied in two Honduras plantations that differed in their tree-shading system, namely intercropping as opposed to agroforestry. The cumulative sunlight effect on plants was determined by means of a non-destructive index of leaf flavonols, applied here for the first time to the coffee culture. The coffee seeds selectively harvested from shaded and full sunlight plants were analyzed for both their chemical composition and the derived cup quality. The high and constant shading of the agroforestry system determined both the heaviest coffee beans and an excellent rating of the beverage. Green beans under shade contained higher levels of caffeine and lower levels of trigonelline and mono-caffeoylquinic acids (other than chlorogenic acid) than those from sun-exposed plants. Banana-tree shading produced no differences in the green bean chemicals with respect to sunlight. The employed leaf flavonol optical index allowed to quantify the spatial variability of shading within the coffee plantations. Regardless of the light regime and site, Parainema coffee was classified as a “specialty”, an aspect that, together with its high seed weight and resistance to disease, can further favor its spread among coffee producers.

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(FRFR/FRFUV), where FRFR and FRFUV 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. Copyright © 2013 John Wiley & Sons, Ltd.

A sustainable extractive technology followed by membrane separation methods was applied to Olea europaea L. (leaves and pitted olive pulp) and Cynara scolymus L. by-products (leaves and stems), to obtain different standardized commercial extracts for application in the functional food industry, pharmaceutical and cosmetic fields. The entire treatment, PCT registered, consists of water extraction of the aforementioned vegetal material and the following steps of fractionation: (1) microfiltration (only for Olea); (2) ultrafiltration (only for Cynara); (3) nanofiltration (only for Olea); (4) reverse osmosis and final concentration by evaporation at low temperature or spray-dried technique. Thanks to these steps, different fractions enriched with phenolic compounds that have an economic value can be obtained. These fractions were characterized and quantified by HPLC/DAD-ESI/MS and then tested for their antiradical and antioxidant properties. The EC50 values by DPPH test were 6.76 × 10−3 mM for the Olea green leaves (GL) soft extract, 5.44 × 10−3 mM for the Olea dried leaves (DL) soft extract, and 1.22 × 10−3 mM in the case of the Olea OH-Tyr soft extract. The EC50 value of the Cynara GL soft extract was 3.25 × 10−3 mM. The ORAC results were 3632 ± 110.7 µmoL TE/g for OH-Tyr Olea soft extract, 1410 ± 62.1 µmoL TE/g for GL Olea soft extract, and finally 760 ± 12 µmoL TE/g for GL Cynara soft extract, confirming the highest antioxidant activity of hydroxytyrosol.

This paper describes a circular economy process based on environmentally and economically sustainable procedures which was applied to the sector of olive oil processing on an industrial scale. Olea europaea L. tissues and by-products represent a renewable and low-cost source of polyphenols, in particular hydroxytyrosol (HTyr), a naturally occurring compound well known for its biological properties. Specifically, green leaves (GL), dried leaves (DL), and pitted olive pulp were treated with water in a pneumatic extractor to obtain the corresponding polyphenolic extracts. Three standardized fractions, named Soft Extract Olea GL, Soft Extract Olea DL, and Soft Extract Olea HTyr resulted after the following two steps: a separation process carried out by membrane technology, and a concentration step performed under reduced pressure and low temperature. The polyphenolic fractions showed antiradical activity and have potential industrial applications in the food, nutraceutical, pharmaceutical, feed, and agronomic fields. Novel functionalized extracts containing hydroxytyrosol methyl carbonate (HTyr-MC) were obtained from Soft Extract Olea HTyr through an innovative approach based on green chemistry procedures, which appear to be a promising tool to increase the applications of the polyphenolic extracts.

A rapid and inexpensive HPLC method has been developed for simultaneous separation of the three main classes of polyphenol in the leaves of Cistus salvifolius L. Time devoted to extraction of polyphenols, which was performed using small volume of solvent, did not exceed 120 min. We identified three ellagitannins (punicalagin and related compounds), a total of ten glycosyl derivatives of quercetin and myricetin, and two coumaroyl glucosyl kaempferols by use of both diode-array detection (DAD) and mass spectrometry. The polyphenol composition of C. salvifolius leaves, which may contribute to the metabolic plasticity of the species, may explain its distribution in infertile soils of the Mediterranean area, and may also indicate this shrub is an important source of metabolites of potential use in human health care.

Fruit quality compounds, such as antioxidant phenolics and chlorophyll, were assessed in situ by using a fluorescence method applied by a portable sensor. Indices of anthocyanins (ANTH) and flavonols (FLAV) localized on the fruit surface were obtained based on their screening of chlorophyll fluorescence excitation. The chlorophyll content was estimated by the far red to red chlorophyll fluorescence ratio (CHL index), due to the partial reabsorption of red fluorescence by chlorophyll itself. In kiwifruits, the CHL index was found to be well linearly correlated to the chlorophyll content determined by wet chemistry on the same fruit samples. Full sunlight exposed kiwifruits possessed a higher content of chlorophyll than shaded kiwifruits. This is an important parameter to know for assessing fruit quality and storability. Based on the estimation of the red-pigmented anthocyanins, we defined a new rapid method to determine the maturity level of olives after harvest, giving the proportion of red and green olives, important for the quality of the olive oil produced. In plums, ANTH and FLAV were found to be linearly correlated to the actual content of compounds measured by HPLC analysis of skin extracts. These indices can be, therefore, used to predict the phenolic antioxidant potential of plums and to define their maturity stage.

Lactuca sativa L. plants (cv. Audran) developed in greenhouse or in open air, were analysed for their polyphenol compounds (caffeic acid derivatives, quercetin and kaempferol glycosides) to verify whether these two different growing environments affected both the qualitative and quantitative phenol patterns. The lettuce extracts from greenhouse and open-air samples were compared and directly analysed by HPLC/DAD, HPLC/MS and HPTLC. All open-air samples had higher flavonol contents than the greenhouse ones. The applied rapid and sensitive HPTLC method could be routinely employed to determine the leaf flavonol content of a large number of lettuce samples.

Ultraviolet radiation (UVR) blocking properties of textiles depend on fibre type, fabric construction and nature of finishing chemicals. Natural dyes can provide vegetable fibres with strong colours if mordants are used. In this study UV protection properties of dyeing extracts from Mediterranean flora ( Helichrysum italicum Roth, Rubia peregrina L., Daphne gnidium L., Lavandula stoechas L., Cynara scolymus L.) were tested in combination with fabrics made of vegetable fibres (cotton and flax) and different types of mordants (potassium alum and chestnuts tannins). Pre- and post-dyeing solutions were analyzed quali-quantitatively by HPLC/DAD/ESI-MS in order to calculate the dye uptake on fabrics during the dyeing process. UVR transmittance of fabrics was measured using a spectrophotometer equipped with an integrating sphere. After the dyeing process only flax fabrics mordanted with alum and dyed with Lavandula and Rubia, and flax fabrics mordanted with tannin and dyed with Rubia and Helychrysum reached the minimum protection level. A very good protection level was reached by flax mordanted with alum and dyed with Helychrysum. Chestnut tannins provided a slight support in phenols uptake for Lavandula and Helichrysum plant dyes with respect to potassium alum, but without an improvement of the fabrics UV protection properties; flax samples dyed with Helichrysum extract showed an improvement in UV protection properties when mordanted with potassium alum instead of tannins. Colour fastness and the persistence of UV protection characteristics were also tested after light/sun exposure and after several washes.

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.

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.

Cynara cardunculus is a diploid (2n = 2x = 34) species, native to the Mediterranean basin, which belongs to the family of Asteraceae. It includes globe artichoke, cultivated cardoon, as well as their progenitor wild cardoon. The species is a source of biophenols and its leaf extracts have been widely used in herbal medicine as hepatoprotectors and choleretics since ancient times. Globe artichoke leaves have been found to be rich in compounds originating from the metabolism of phenylpropanoids however, to our knowledge, the leaf polyphenolic composition of the two other forms within the species, cultivated and wild cardoon, have not yet been properly investigated. Two main classes of polyphenols have been detected by HPLC/DAD and HPLC/MS analyses: caffeoyl esters and flavonoids. The compounds which are the result of esterification of caffeoylquinic acid moiety with succinic acid, previously detected in other members of the Asteraceae family, were detected in cardoon leaves for the first time.

• 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 tissuespecific 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 djustment. 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.

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.

Separation, identification and quantification of polyphenols was carried out on leaves of Pistacia lentiscus L., an evergreen member of the family Anacardiaceae, using semi-preparative HPLC, HPLC-photodiode array detection and HPLC-MS analysis, together with 1H- and 13C NMR. Three major classes of secondary metabolites were detected: (i) gallic acid and galloyl derivatives of both glucose and quinic acid; (ii) flavonol glycosides, i.e. myricetin and quercetin glycosides; and (iii) anthocyanins, namely delphinidin 3-O-glucoside and cyanidin 3-O-glucoside. Low amounts of catechin were also detected. The concentration of galloyl derivatives was extremely high, representing 5.3% of the leaf dry weight, and appreciable amounts of myricetin derivatives were also detected (1.5% on a dry weight basis). These findings may be useful in establishing a relationship between the chemical composition of the leaf extract and the previously reported biological activity of P. lentiscus, and may also assign a new potential role of P. lentiscus tissue extracts in human health care. Copyright © 2002 John Wiley & Sons, Ltd.

The production of olive oil yields a considerable amount of waste water, which is a powerful pollutant and is currently discarded. Polyphenols and other natural antioxidants, extracted from olives during oil extraction process, partially end up in the waste waters. Experimental and commercial olive oil waste waters from four Mediterranean countries were analyzed for a possible recovering of these biologically interesting constituents. Identification and quantitation of the main polyphenols were carried out by applying HPLC-DAD and HPLC-MS methods. Representative samples of ripe olives were also analyzed at the same time to correlate, if possible, their polyphenolic profiles with those of the corresponding olive oil waste waters. The results demonstrate that Italian commercial olive oil waste waters were the richest in total polyphenolic compounds with amounts between 150 and 400 mg/100 mL of waste waters. These raw, as yet unused, matrices could represent an interesting and alternative source of biologically active polyphenols.

Identification and quantification of flavonol glycosides and secoiridoids was carried out on leaves of Ligustrum vulgare L. (Oleaceae) by means of HPLC-DAD and HPLC-MS analysis. In addition to previously reported secoiridoids (oleuropein, ligustaloside A, ligustaloside B, and ligstroside) four kaempferol glycosides (kaempferol 3-O-glucoside 7-O-rhamnoside, kaempferol 3, 7-O-dirhamnoside, kaempferol 3-O-rhamnoside, and kaempferol 3-O-glucoside) and two quercetin glycosides (quercetin 3-O-glucoside 7-O-rhamnoside and quercetin 3,7-O-dirhamnoside) were present in leaves of L. vulgare L. Although secoiridoids accounted for nearly the 76% of the total leaf polyphenols content (with ligustaloside A as the main component), kaempferol glycosides were also accumulated in the leaves of L. vulgare L. to a relatively high extent (23%). Contribution of quercetin derivatives was minor under our experimental conditions. Our findings suggest that flavonol glycosides may have a central role in both the ecology and the biology of L. vulgare L.

In this study polyphenolic compounds extracted from olive fruits of five registered cultivars were analyzed. A solid-liquid extraction (LSE) procedure with Extrelut cartridge (diatomaceous earth) using different eluents was developed to obtain polyphenolic compounds. HPLC-DAD and HPLC-MS methods were applied for the quali-quantitative analysis of each fraction obtained from LSE. The results of this work show that the LSE procedure with diatomaceous earth cartridge supplies a rapid and reproducible fractioning method able to obtain a quantitative recovery of all compounds and to collect fractions directly analyzed by HPLC. A comparison among different cultivars shows significant quantitative differences in some polyphenols, such as verbascoside, anthocyanic compounds, and oleuropein derivatives.

The aim of this investigation was to obtain qualitative and quantitative profiles of the flavonoid and biflavonoid composition of six cypress species—Cupressus funebris L.,Cupressus sempervirens L.,Cupressus glabra L.,Cupressus arizonica L.,Cupressus goveniana L., andCupressus lusitanica L. HPLC-diode-array detection (DAD), HPLC-MS, and HPTLC were used to identify the individual compounds. A chromatographic method was optimized for identification and quantification of the main flavonoid glycosides and biflavonoids. The flavonoids identified and calibrated were: rutin, quercetin glucoside, quercetin rhamnoside, and kaempferol 3-O-rhamnoside. The biflavonoids identified and calibrated were: cupressuflavone, amentoflavone, robustaflavone, hinokiflavone, methylrobustaflavone, methylamentoflavone, and dimethylcupressuflavone.