Presentación de PowerPoint - Inra - Centre Bordeaux … physicochemical and phytochemical indicators of cherry fruit quality [2-4 June 2015] CEBAS

6/8/2015

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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


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



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




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.



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





They appear to be the most widespread in plant foods. The main flavonols are:

Flavonols

Structures of common flavonol aglycones



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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.



Structures of flavan-3-ol monomers



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.



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.



Anthocyanidins

The common anthocyanidins are pelargonidin, cyanidin, peonidin, delphinidin, petunidin

and malvidin.



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.




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





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



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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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.)



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.



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.



HPLC separation of

polyphenols in sweet-

cherry at different

wavelengths


Anthocyanins

Flavonols

Phenolic acids

Flavan-3-ols



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)





Six different sweet-cherry cultivars (Prunus avium L.) grown in

Valle del Jerte area (Spain)

HPLC and ESI-MS separation of anthocyanin standard pigments

http://www.google.es/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRw&url=http://ehealthforum.com/blogs/neckpainrelief/study-shows-that-tart-cherry-juice-will-help-you-sleep-b28181.html&ei=jYRoVbbCC4O5UcH1gagI&bvm=bv.94455598,d.d24&psig=AFQjCNGibeZNKAqjlh84MRHtPedQOgT2iw&ust=1432999432665022

http://www.google.es/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRw&url=http://www.adventuresofaglutenfreemom.com/2011/06/the-best-mayo-free-chicken-salad/&ei=jYRoVbbCC4O5UcH1gagI&bvm=bv.94455598,d.d24&psig=AFQjCNGibeZNKAqjlh84MRHtPedQOgT2iw&ust=1432999432665022

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Sweet-cherry cultivar (Prunus avium L.) PICO NEGRO Valle del

Jerte area (Spain)

Chromatographic separation of anthocyanin pigments in Pico Negro cherry



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.



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



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



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).



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.)

http://www.google.es/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRw&url=http://blog.farmmade.com/growing-delicious-cherries/&ei=doVoVdqdHYL4UIm4gNAF&bvm=bv.94455598,d.d24&psig=AFQjCNGibeZNKAqjlh84MRHtPedQOgT2iw&ust=1432999432665022

http://www.google.es/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0CAcQjRw&url=http://www.traversebayfarms.com/tart-cherry-vs-black-cherry.htm&ei=jYRoVbbCC4O5UcH1gagI&bvm=bv.94455598,d.d24&psig=AFQjCNGibeZNKAqjlh84MRHtPedQOgT2iw&ust=1432999432665022

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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



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



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



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.



Importance of Plant Material

Fresh

Critical Aspects

Phytochemical analysis

Freeze-dried Frozen



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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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)



Extraction and quantification



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



Phenolic compounds: Quantification

Chlorogenic acid (330nm) Phenolic Acids

Flavonoids Rutin (quercetin 3-rhamnosyl-(1-6)-glucoside) (360nm)

Anthocyanins

Cyanidin-3-rutinoside (520 nm)



mA

U

Time (min)

Chlorogenic acid

caffeoyltartaric acid

caffeoylmalic acid

quercetin-3-O-(6”-O-malonyl)-glucoside

chicoric acid

Phenolic compounds: Quantification



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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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.




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

Documents

Presentación de PowerPoint - Inra - Centre Bordeaux … physicochemical and phytochemical indicators of cherry fruit quality [2-4 June 2015] CEBAS