6/8/2015 FOOD CONSTITUENTS. Determination of phytochemical components with advanced analytical methods Part I. Phenolic and polyphenolic compounds

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1 Qualitative, physicochemical and phytochemical indicators of cherry fruit quality [2-4 June 2015] Determination of phytochemical components with advanced Part I Maria Isabel Gil CEBAS CSIC, Murcia, Spain Nutrients macronutrients (lipids, carbohydrates, proteins) micronutrients (minerals and vitamins) e.g. vitamin C, vitamin E Non-nutrients other constituents with biological properties beyond nutrition Plant secondary metabolites Phytochemicals Phytonutrients Bioactive compounds Health-promoting compounds FOOD CONSTITUENTS Fruits and vegetables contain a variety of plant secondary metabolites that may potentially benefit human health They are the large plant secondary metabolites, widely distributed in the plant kingdom. Phenolic and polyphenolic compounds Structure: presence of at least one aromatic hydrocarbon ring (phenol) or more (polyphenols) attached with one or more hydroxyl groups. Phenolic and polyphenolic compounds They are commonly found conjugated to sugars and organic acids. They can be grouped as: 1. FLAVONOIDS:, Flavones, Isoflavones, Flavan-3-ols, Flavanones, 2. NON-FLAVONOIDS: phenolic acids and hydroxycinnammates 1. Flavonoids They are polycyclic structures consisting of 15 C atoms based on C6-C3-C6 skeleton. According to the cyclization and the degree of unsaturation and oxidation of the three-carbon-segment they can be classified into several groups, the main ones being: They appear to be the most widespread in plant foods. The main flavonols are: Structures of the main flavonoid subgroups Structures of common flavonol aglycones 1

2 Flavan-3-ols They are the most complex subclass of flavonoids ranging from the simple monomers (+)- catechin and its isomer ( )-epicatechin to the oligomeric and polymeric proanthocyanidins which are also known as condensed tannins. are mainly present in nature as their sugar-conjugated derivatives, the anthocyanins, and are particularly evident in fruit and flower tissue where they are responsible for red, blue and purple colours. Structures of flavan-3-ol monomers They are involved in the protection of plants against excessive light by shading leaf mesophyll cells and also have an important role to play in attracting pollinating insects. The common anthocyanidins are pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin. Phenolic and polyphenolic compounds Phenolics are commonly found conjugated to sugars and organic acids and can be grouped as: Structures of anthocyanidins Anthocyanins such as cyanidin-3-o-rutinoside, cyanidin-3-o-glucoside and peonidin-3- rutinoside have been reported in sweet cherries (Prunus avium) and sour cherries. 1. FLAVONOIDS:, Flavones, Isoflavones, Flavan-3-ols, Flavanones, NON-FLAVONOIDS: benzoic acid, hydroxycinnammic acid derivatives 2. Benzoic acid and hydroxycinnamic acid derivatives The C6-C1 benzoic acids and the C6-C3 hydroxycinammates. The simples of the class is C6-C1 phenolic acids such as gallic acid. Structure ellagic acid, gallic acid and sanguiin H6, an ellagitannin found in berries 2. Benzoic acid and hydroxycinnamic acid derivatives The most common hydroxycinnamates are p- coumaric, caffeic and ferulic acids which frequently accumulate as their respective tartrate esters, coutaric, caftaric and fertaric acids. Quinic acid conjugates of caffeic acid, namely 3-, 4- and 5-O-caffeoylquinic acid, are commonly found in fruits and vegetables. Ellagic acid has been reported to be present in berries, particularly raspberries (Rubus idaeus), strawberries (Fragaria x ananassa) and blackberries although it is more common the present of ellagitannins, such as sanguiin H-6. 2

3 Nutrients Sugars and organic acids Vitamin C Major phytochemicals Sweet Cherry (Prunus avium L.) Carotenoids: beta-carotene, lutein, alpha-carotene, betacryptoxanthin, zeaxanthin and phytoene Phenolic compounds: anthocyanidins, flavonols and hydroxicinnamates N-containing compounds: Melatonin y seratonin Phenylpropanoids and the indolamine biosynthetic route Shikimate serves as the precursor of tryptophan and phenylalanine through different metabolic routes. Gonzalez-Gomez, D., Lozano, M., Fernandez-Leon, M.F., Bernalte, M.J., Ayuso, M.C., Rodriguez, A.B Sweet cherry phytochemicals: Identification and characterization by HPLC-DAD/ESI-MS in six sweetcherry cultivars grown in Valle del Jerte (Spain). J. Food Composition Analysis 23, Identification and quantification of phenolic compounds in sweet cherries (P. avium) : rutin and quercetin-3-oglucoside Flavan-3-ols: some procyanidin derivatives such as catechin and epicatechin. Phenolic acids: chlorogenic, neochlorogenic and p-coumaroylquinic acids. Anthocyanins: cyanidin-3-o-glucoside, cyanidin-3-o-rutinoside, peonidin-3-o-glucoside, peonidin-3-o-rutinoside and pelargonidin-3-o-rutinoside. HPLC separation of polyphenols in sweetcherry at different wavelengths Anthocyanins Phenolic acids Flavan-3-ols Ballistreri et al., 2013; Fazzari et al., 2008; Gao & Mazza, 1995; Gonzalez-Gomez et al., 2010; Goncalves et al., 2004; Liu et al., 2011; Pacifico et al., 2014; Serra et al., 2010; Serra et al., 2011; Serradilla et al., 2011; Usenik et al., 2008, Individual anthocyanins and phenolic compounds by HPLC/DAD-MS in six different sweet-cherry cultivars (Prunus avium L.) grown in Valle del Jerte area (Spain) Six different sweet-cherry cultivars (Prunus avium L.) grown in Valle del Jerte area (Spain) Protected Designation of Origin (PDO) The most abundant anthocyanin pigment was cyanidin-3-rutinoside (105 mg/100 g fw in Pico Negro sweet-cherry cultivar). The most abundant phenolic compound was p-coumaroylquinic acid (130 mg/100 g fw in Pico Negro sweet-cherry cultivar). HPLC and ESI-MS separation of anthocyanin standard pigments 3

4 Sweet-cherry cultivar (Prunus avium L.) PICO NEGRO Valle del Jerte area (Spain) Phenolic acid content in different cultivars Cinnamic acid derivatives: chlorogenic acid, its isomer neochlorogenic acid and cryptochlorogenic acid p-coumaric acid and ferulic acid Chromatographic separation of anthocyanin pigments in Pico Negro cherry Kaulmann, A., Jonville, M-C., Schneider, Y-J., Hoffmann, L., Bohn, T Carotenoids, polyphenols and micronutrient profiles of Brassica oleraceae and plum varieties and their contribution to measures of total antioxidant capacity. Food Chemistry 155, Phytochemical characterization of Prunus avium L. 3- phenolic acid derivatives, 3-anthocyanins and 6 flavonoids Phytochemical characterization of Prunus avium L. 3- phenolic acid derivatives, 3-anthocyanins and 6 flavonoids P. avium fruits extracts: Phenolic profile at 370 nm (B) and anthocyanins at 520 nm (C) Bastos, C., Barros, L., Dueñas, M., Calhelha, R.C., Queiroz, M.J.R.C., Santos-Buelga, C., Ferreira, I.C.F.R Chemical characterisation and bioactive properties of Prunus avium L.: The widely studied fruits and the unexplored stems. Food Chemistry, 173, Bastos, C., Barros, L., Dueñas, M., Calhelha, R.C., Queiroz, M.J.R.C., Santos-Buelga, C., Ferreira, I.C.F.R Chemical characterisation and bioactive properties of Prunus avium L.: The widely studied fruits and the unexplored stems. Food Chemistry, 173, Sour Cherry (Prunus cerasus L.) Phenolic compounds : 41 Sour Cherry (Prunus cerasus L.) : rutin and quercetin-3-oglucoside, kaempferol, isorhamnetin, apigenin Flavan-3-ols: (+)catechin and (-) epicatechin, procyanidin B1, dimer, trimer and tetramers Phenolic acids: neochlorogenic, chlorogenic and p-coumaroylquinic acids, caffeoylquinic and dicaffeoylquinic acids. Anthocyanins: 11 (cyanidin-3-o-glucoside and diglucoside, peonidin-3-o-glucoside, peonidin- 3-O-rutinoside and pelargonidin-3-o-rutinoside). Wojdyło, A., Nowicka, P., Laskowski, P. Oszmian ski, J Evaluation of sour sherry (Prunus cerasus L.) fruits for their polyphenol content, antioxidant properties, and nutritional components. J. Agric. Food Chem., 62, Wojdyło, A., Nowicka, P., Laskowski, P. Oszmian ski, J Evaluation of sour sherry (Prunus cerasus L.) fruits for their polyphenol content, antioxidant properties, and nutritional components. J. Agric. Food Chem., 62,

5 Factors influencing phytochemicals of sweet cherry (Gonc alves et al., 2004, 2007; Serrano et al., 2009; Faniadis et al., 2010) (Usenik et al., 2008; Díaz-Mula et al., 2009; Serrano et al., 2009; González-Gómez et al., 2010; Serra et al., 2011; Serradilla et al., 2012; Ballistreri et al., 2013), 1. Genotype Phytochemicals in PLUM cultivars Cultivars Total Phenolics Vitamin C Carotenoids Serrano et al., 2005, 2009; Díaz-Mula et al., 2009; Serradilla et al., 2011, 2012 Wickson Black Beaut (Goncalves et al., 2004; Schmitz-Eiberger and Blanke, 2012) Red Beaut Santa Rosa Angeleno The composition of phytochemicals affects visual and sensory properties, taste and flavour of sweet cherry fruit Wani, A.A., Singh, P., Gul, K., Wani, M.H., Langowski, H.C Sweet cherry (Prunus avium): Critical factors affecting the composition and shelf life. Food Packaging and Shelf-life, 1, Total phenolics and vitamin C (mg/serving), carotenoids ( g/serving). Serving = 100 g of fruit (80% flesh + 15% peel) Gil, M.I., Tomás-Barberán, F.A., Hess-Pierce, B., Kader, A.A Antioxidant capacities, phenolic compounds, carotenoids and vitamin C of nectarine, peach, and plum cultivars from California. J. Agric. Food Chem., 50, Genotype Phytonutrients in PEEL and FLESH of apricot cultivars Phytochemical analysis Total content Cultivars Fruit part Procyanidins Hydroxycinnamic Spectrophotometric assay: Low specificity, interference of other Currot Peel substances and different reactivity of the molecules present. HPLC: More accurate and provide information on individual compounds. Mauricio Peel Z 207/ Peel (mg/100 g FW) Ruiz, D., Egea, J., Tomás-Barberán, F.A, Gil, M.I Characterization and quantitation of phenolic compounds in new apricot (Prunus armeniaca L.) varieties. J. Agric. Food Chem., 53, Critical Aspects Phytochemical analysis Critical Aspects Estimation of the Phytochemical Content Fresh Frozen Freeze-dried Importance of Plant Material FRESH WEIGHT: Errors when the fruit is expanding rapidly DRY WEIGHT: Eliminate variations in water between two samples 5

6 mau 6/8/2015 Critical Aspects The concentration of phytochemicals in the edible portion External tissues have higher content than internal tissues Extraction and quantification Apricot Skin Procyanidins Hydroxycinnamics Sweet cherry: Edible portion of the fruit (skin plus flesh) > were slightly greater than in the flesh by itself because the skin is relatively small proportion of the fruit. (Chaovanalikit and Wrolstad, 2004) Phenolic compounds: Hydroalcoholic extract Phenolic compounds: Quantification Freeze-dried sample (0.5 g) Vortexfor 1 min on ice 10 ml of Acetone/Water/Acetic Acid (70:29:1) Phenolic Acids Chlorogenic acid (330nm) Centrifugation for 10 min at 3000 rpm Evaporation and filtration throughout C- 18 Sep-pack cartridge) HPLC/UV-DAD Flavonoids Rutin (quercetin 3-rhamnosyl-(1-6)- glucoside) (360nm) Concentration and filtration (0.45 µm polyethersulphone) Anthocyanins HPLC mobile phase: Water + 1% formic acid (A) and methanol (B) (flow rate 1 ml min -1 ) Cyanidin-3-rutinoside (520 nm) Phenolic compounds: Quantification Proanthocyanidins: Phloroglucinolysis chicoric acid Chlorogenic acid 50 mg freeze-dried 800 ŋl of extraction medium (phloroglucinol and 20 mg ascorbate) caffeoyltartaric acid quercetin-3-o-(6 -O-malonyl)-glucoside Vortex for 1 min HPLC/UV-DAD/ESI-MSn caffeoylmalic acid Incubation for 20 min at 50ºC Stop reaction in an ice bath + 40mM sodium acetate Time (min) HPLC mobile phase: water + 2.5% acetic acid (A) and acetonitrile (B) flow rate 1 ml min -1 6

7 Proanthocyanidins: Quantification Catechin (280 nm) Qualitative, physicochemical and phytochemical indicators of cherry fruit quality [2-4 June 2015] Determination of phytochemical components with advanced Part II The average degree of polymerization is calculated as the molar ratio of all the flavan- 3-ol units (phloroglucinol adducts + terminal units) to ( )-epicatechin and (+)-catechin, which correspond to terminal units. Francisco Tomás Barberán CEBAS CSIC, Murcia, Spain 7