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6/8/2015
1
FA1104 Training School
Qualitative, physicochemical and phytochemical indicators of cherry fruit quality
[2-4 June 2015]
Maria Isabel Gil
CEBAS – CSIC, Murcia, Spain
Determination of phytochemical components with
advanced analytical methods – Part I
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
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
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
They are the large plant secondary metabolites, widely distributed in the plant
kingdom.
Structure: presence of at least one aromatic hydrocarbon ring (phenol) or more
(polyphenols) attached with one or more hydroxyl groups.
Phenolic and polyphenolic compounds
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Phenolic and polyphenolic compounds
They are commonly found conjugated to sugars and organic acids.
They can be grouped as:
1. FLAVONOIDS: Flavonols,
Flavones,
Isoflavones,
Flavan-3-ols,
Flavanones,
Anthocyanidins
2. NON-FLAVONOIDS: phenolic acids and hydroxycinnammates
Jaganath and Crozier. 2008. Overview of health-promoting compounds in fruit and vegetables. In: Improving the health-promoting properties of fruit and
vegetable products. Tomás-Barberán, F.A., Gil, M.I., (Eds.), CRC Press. Woodhead Publishing, 3-37.
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
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:
Structures of the main flavonoid subgroups
Jaganath and Crozier. 2008. Overview of health-promoting compounds in fruit and vegetables. In: Improving the health-promoting properties of fruit and
vegetable products. Tomás-Barberán, F.A., Gil, M.I., (Eds.), CRC Press. Woodhead Publishing, 3-37.
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
They appear to be the most widespread in plant foods. The main flavonols are:
Flavonols
Structures of common flavonol aglycones
Jaganath and Crozier. 2008. Overview of health-promoting compounds in fruit and vegetables. In: Improving the health-promoting properties of fruit and
vegetable products. Tomás-Barberán, F.A., Gil, M.I., (Eds.), CRC Press. Woodhead Publishing, 3-37.
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FA1104 Training School
Determination of phytochemical components with advanced analytical methods
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.
Jaganath and Crozier. 2008. Overview of health-promoting compounds in fruit and vegetables. In: Improving the health-promoting properties of fruit and
vegetable products. Tomás-Barberán, F.A., Gil, M.I., (Eds.), CRC Press. Woodhead Publishing, 3-37.
Structures of flavan-3-ol monomers
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Anthocyanidins
Anthocyanidins 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.
Jaganath and Crozier. 2008. Overview of health-promoting compounds in fruit and vegetables. In: Improving the health-promoting properties of fruit and
vegetable products. Tomás-Barberán, F.A., Gil, M.I., (Eds.), CRC Press. Woodhead Publishing, 3-37.
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.
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Anthocyanidins
The common anthocyanidins are pelargonidin, cyanidin, peonidin, delphinidin, petunidin
and malvidin.
Jaganath and Crozier. 2008. Overview of health-promoting compounds in fruit and vegetables. In: Improving the health-promoting properties of fruit and
vegetable products. Tomás-Barberán, F.A., Gil, M.I., (Eds.), CRC Press. Woodhead Publishing, 3-37.
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.
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Phenolic and polyphenolic compounds
Phenolics are commonly found conjugated to sugars and organic acids and can
be grouped as:
1. FLAVONOIDS: Flavonols,
Flavones,
Isoflavones,
Flavan-3-ols,
Flavanones,
Anthocyanidins
NON-FLAVONOIDS: benzoic acid , hydroxycinnammic acid derivatives
Jaganath and Crozier. 2008. Overview of health-promoting compounds in fruit and vegetables. In: Improving the health-promoting properties of fruit and
vegetable products. Tomás-Barberán, F.A., Gil, M.I., (Eds.), CRC Press. Woodhead Publishing, 3-37.
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
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
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. Benzoic acid and hydroxycinnamic acid derivatives
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
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.
2. Benzoic acid and hydroxycinnamic acid derivatives
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FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Nutrients
Sugars and organic acids
Vitamin C
Major phytochemicals
Carotenoids: beta-carotene, lutein, alpha-carotene,
betacryptoxanthin, zeaxanthin and phytoene
Phenolic compounds: anthocyanidins, flavonols and
hydroxicinnamates
N-containing compounds: Melatonin y seratonin
Sweet Cherry (Prunus avium L.)
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Phenylpropanoids and the
indolamine biosynthetic
route
Gonzalez-Gomez, D., Lozano, M., Fernandez-Leon, M.F., Bernalte, M.J., Ayuso, M.C., Rodriguez, A.B. 2010. Sweet cherry phytochemicals: Identification and characterization by HPLC-DAD/ESI-MS in six sweet-cherry cultivars grown in Valle del Jerte (Spain). J. Food Composition Analysis 23, 533–539.
Shikimate serves as the precursor of
tryptophan and phenylalanine
through different metabolic routes.
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Identification and quantification of phenolic compounds in sweet cherries (P. avium)
Flavonols: 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.
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, 2010.
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
HPLC separation of
polyphenols in sweet-
cherry at different
wavelengths
Gonzalez-Gomez, D., Lozano, M., Fernandez-Leon, M.F., Bernalte, M.J., Ayuso, M.C., Rodriguez, A.B. 2010. Sweet cherry phytochemicals: Identification and characterization by HPLC-DAD/ESI-MS in six sweet-cherry cultivars grown in Valle del Jerte (Spain). J. Food Composition Analysis 23, 533–539.
Anthocyanins
Flavonols
Phenolic acids
Flavan-3-ols
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
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).
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)
Gonzalez-Gomez, D., Lozano, M., Fernandez-Leon, M.F., Bernalte, M.J., Ayuso, M.C., Rodriguez, A.B. 2010. Sweet cherry phytochemicals: Identification and characterization by HPLC-DAD/ESI-MS in six sweet-cherry cultivars grown in Valle del Jerte (Spain). J. Food Composition Analysis 23, 533–539.
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Gonzalez-Gomez, D., Lozano, M., Fernandez-Leon, M.F., Bernalte, M.J., Ayuso, M.C., Rodriguez, A.B. 2010. Sweet cherry phytochemicals: Identification and characterization by HPLC-DAD/ESI-MS in six sweet-cherry cultivars grown in Valle del Jerte (Spain). J. Food Composition Analysis 23, 533–539.
Six different sweet-cherry cultivars (Prunus avium L.) grown in
Valle del Jerte area (Spain)
HPLC and ESI-MS separation of anthocyanin standard pigments
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FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Gonzalez-Gomez, D., Lozano, M., Fernandez-Leon, M.F., Bernalte, M.J., Ayuso, M.C., Rodriguez, A.B. 2010. Sweet cherry phytochemicals: Identification and characterization by HPLC-DAD/ESI-MS in six sweet-cherry cultivars grown in Valle del Jerte (Spain). J. Food Composition Analysis 23, 533–539.
Sweet-cherry cultivar (Prunus avium L.) PICO NEGRO Valle del
Jerte area (Spain)
Chromatographic separation of anthocyanin pigments in Pico Negro cherry
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Cinnamic acid derivatives:
chlorogenic acid, its isomer neochlorogenic acid and cryptochlorogenic acid
p-coumaric acid and ferulic acid
Phenolic acid content in different cultivars
Kaulmann, A., Jonville, M-C., Schneider, Y-J., Hoffmann, L., Bohn, T. 2014. Carotenoids, polyphenols and micronutrient profiles of Brassica oleraceae and plum varieties and their contribution to measures of total antioxidant capacity. Food Chemistry 155, 240–250.
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Phytochemical characterization of Prunus avium L.
Bastos, C., Barros, L., Dueñas, M., Calhelha, R.C., Queiroz, M.J.R.C., Santos-Buelga, C., Ferreira, I.C.F.R. 2015. Chemical characterisation and bioactive properties
of Prunus avium L.: The widely studied fruits and the unexplored stems. Food Chemistry, 173, 1045-1053.
3- phenolic acid derivatives, 3-anthocyanins and 6 flavonoids
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
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. 2015.
Chemical characterisation and bioactive properties of Prunus avium L.: The widely studied fruits and the
unexplored stems. Food Chemistry, 173, 1045-1053.
Phytochemical characterization of Prunus avium L. 3- phenolic acid derivatives, 3-anthocyanins and 6 flavonoids
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Wojdyło, A., Nowicka, P., Laskowski, P. Oszmianski, J. 2014. Evaluation of sour sherry (Prunus cerasus L.) fruits for their polyphenol content, antioxidant properties, and nutritional components. J. Agric. Food Chem., 62, 12332−12345.
Sour Cherry (Prunus cerasus L.)
Phenolic compounds : 41
Flavonols: 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).
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Wojdyło, A., Nowicka, P., Laskowski, P. Oszmianski, J. 2014. Evaluation of sour sherry (Prunus cerasus L.) fruits for their polyphenol content, antioxidant properties, and nutritional components. J. Agric. Food Chem., 62, 12332−12345.
Sour Cherry (Prunus cerasus L.)
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FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Wani, A.A., Singh, P., Gul, K., Wani, M.H., Langowski, H.C. 2014. Sweet cherry (Prunus avium): Critical factors affecting the composition and shelf life. Food Packaging and Shelf-life, 1, 86-99.
Factors influencing phytochemicals of sweet cherry (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),
Serrano et al., 2005, 2009; Díaz-Mula et al., 2009; Serradilla et al., 2011, 2012
(Goncalves et al., 2004; Schmitz-Eiberger and Blanke, 2012)
(Gonc¸alves et al., 2004, 2007; Serrano et al., 2009; Faniadis et al., 2010)
The composition of phytochemicals affects visual and sensory properties, taste and flavour of sweet cherry fruit
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Total phenolics and vitamin C (mg/serving), carotenoids (g/serving). Serving = 100 g of fruit (80% flesh + 15% peel)
Phytochemicals in PLUM cultivars
Gil, M.I., Tomás-Barberán, F.A., Hess-Pierce, B., Kader, A.A. 2002. Antioxidant capacities, phenolic compounds, carotenoids and vitamin
C of nectarine, peach, and plum cultivars from California. J. Agric. Food Chem., 50, 4976-4982.
83 2.5 54.8 Santa Rosa
113 4.2 82.4 Angeleno
87 9.0 42.0 Wickson
87 10.2 57.4 Red Beaut
231 2.9 109.2 Black Beaut
Carotenoids Vitamin C Total Phenolics Cultivars
1. Genotype
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Phytonutrients in PEEL and FLESH of apricot cultivars
Ruiz, D., Egea, J., Tomás-Barberán, F.A, Gil, M.I. 2005. Characterization and quantitation of phenolic compounds in new apricot (Prunus
armeniaca L.) varieties. J. Agric. Food Chem., 53, 9544-9552.
(mg/100 g FW)
101.7 -- 18.5 Flesh Z 207/4
Mauricio
Currot
Cultivars
76.9 43.0 92.7 Peel
136.8 93.1 333.1 Peel
-- 29.2 10.4 Flesh
-- 7.5 15.0 Flesh
119.5 87.9 101.1 Peel
Flavonols Hydroxycinnamic Procyanidins Fruit part
1. Genotype
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Total content
Phytochemical analysis
Spectrophotometric assay: Low specificity, interference of other
substances and different reactivity of the molecules present.
HPLC: More accurate and provide information on individual compounds.
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Importance of Plant Material
Fresh
Critical Aspects
Phytochemical analysis
Freeze-dried Frozen
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Estimation of the Phytochemical Content
Critical Aspects
FRESH WEIGHT: Errors when the fruit is expanding rapidly
DRY WEIGHT: Eliminate variations in water between two samples
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FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Critical Aspects
External tissues have higher content than internal tissues
Flavonols Hydroxycinnamics Procyanidins Apricot
101.7 -- 18.5 Flesh
136.8 93.1 333.1 Skin
The concentration of phytochemicals in the edible portion
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)
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Extraction and quantification
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Freeze-dried sample (0.5 g)
Phenolic compounds: Hydroalcoholic extract
10 mL of Acetone/Water/Acetic Acid (70:29:1)
Centrifugation for 10 min at 3000 rpm
Concentration and filtration (0.45 µm polyethersulphone)
Vortexfor 1 min on ice
HPLC mobile phase: Water + 1% formic acid (A) and methanol (B) (flow rate 1 ml min-1)
Evaporation and filtration throughout C-18 Sep-pack cartridge)
HPLC/UV-DAD
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Phenolic compounds: Quantification
Chlorogenic acid (330nm) Phenolic Acids
Flavonoids Rutin (quercetin 3-rhamnosyl-(1-6)-glucoside) (360nm)
Anthocyanins
Cyanidin-3-rutinoside (520 nm)
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
mA
U
Time (min)
Chlorogenic acid
caffeoyltartaric acid
caffeoylmalic acid
quercetin-3-O-(6”-O-malonyl)-glucoside
chicoric acid
Phenolic compounds: Quantification
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
50 mg freeze-dried
Proanthocyanidins: Phloroglucinolysis
800 ŋL of extraction medium (phloroglucinol and 20 mg ascorbate)
Stop reaction in an ice bath + 40mM sodium acetate
Incubation for 20 min at 50ºC
Vortex for 1 min
HPLC mobile phase: water + 2.5% acetic acid (A) and acetonitrile (B) flow rate 1 ml min-1
HPLC/UV-DAD/ESI-MSn
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FA1104 Training School
Determination of phytochemical components with advanced analytical methods
Proanthocyanidins: Quantification
Catechin (280 nm)
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.
FA1104 Training School
Determination of phytochemical components with advanced analytical methods
FA1104 Training School
Qualitative, physicochemical and phytochemical indicators of cherry fruit quality
[2-4 June 2015]
Francisco Tomás Barberán
CEBAS – CSIC, Murcia, Spain
Determination of phytochemical components with
advanced analytical methods – Part II