Influence of cultivar and origin on the flavonol profile of fruits and cladodes from cactus Opuntia ficus-indica

    Influence of cultivar and origin on the flavonol profile of fruits and cladodes from cactus Opuntia ficus-indica Tamer E. Moussa-Ayoub, El-Sayed A. Abd El-Hady, Helmy T. Omran, Salah K. El-Samahy, Lothar W. Kroh, Sascha Rohn PII: DOI: Reference:

S0963-9969(14)00559-6 doi: 10.1016/j.foodres.2014.08.021 FRIN 5443

To appear in:

Food Research International

Received date: Accepted date:

13 June 2014 24 August 2014

Please cite this article as: Moussa-Ayoub, T.E., Abd El-Hady, E.-S.A., Omran, H.T., El-Samahy, S.K., Kroh, L.W. & Rohn, S., Influence of cultivar and origin on the flavonol profile of fruits and cladodes from cactus Opuntia ficus-indica, Food Research International (2014), doi: 10.1016/j.foodres.2014.08.021

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Influence of cultivar and origin on the flavonol profile of fruits and cladodes

T

from cactus Opuntia ficus-indica

IP

Tamer E. Moussa-Ayoub 1, 2, El-Sayed A. Abd El-Hady 2, Helmy T. Omran 2,

1

SC R

Salah K. El-Samahy 2, Lothar W. Kroh 1, Sascha Rohn 3

Technische Universität Berlin, Institute of Food Technology and Food Chemistry, Food Chemistry and Analysis

Department, TIB 4/3-1, Gustav-Meyer-Allee 25, D-13355 Berlin, Germany. 2Suez Canal University, Agriculture

NU

Faculty, Food Technology Department, 41522 Ismailia, Egypt. 3University of Hamburg, Hamburg School of Food Science, Institute of Food Chemistry, Grindelallee 117, D-20146 Hamburg, Germany.

MA

Abstract

Flavonols are hypothesized to be the most important polyphenolic antioxidants. This present study aimed at investigating flavonols in cactus O. ficus-indica fruits from two Egyptian

D

cultivars in comparison to three common Sicilian cultivars, and two further cultivars from South

TE

Africa. Besides, cactus O. ficus-indica cladodes from Egyptian cultivars have been investigated as well. HPLC-DAD analyses showed that cactus O. ficus-indica fruits and cladodes are mainly

CE P

characterized by isorhamnetin glycosides. These flavonols were found only in the fruit‘s peel and the cladodes, but not in the pulp. However, all analyzed cultivars exhibited the same flavonol profile which might therefore serve as a chemical fingerprint with regard to

AC

genuineness of cactus O. ficus-indica fruits and cladodes or even food products containing whole cactus fruits as ingredients. The findings obtained have been confirmed either by enzymatic hydrolysis of flavonol glycosides with following analysis of the aglycons and by HPLC-ESI-MSn. The concentration of total flavonols ranged from 2.2 to 4.1 and 6.3 to 7.6 mg/g (dw) in the fruit peels and the cladodes, respectively. Further, fruit peels and cladodes exhibited high total phenolic contents and antioxidant activities compared to those of the fruit pulps. These investigations valorize cactus fruit's peel and cladode in comparison to fruit's pulp, and further may provide additional data for using flavonols in chemotaxonomic studies of cactus Opuntia spp. and authenticity of cactus O. ficus-indica products.

Key words: Opuntia ficus-indica, flavonols, fruits, cladodes.

ACCEPTED MANUSCRIPT 1. Introduction The rising consumer's awareness in health issues leads to manifold studies which investigate the correlation between consumer's health and his daily diet. Many of these studies report a positive

T

correlation between consumption of plant foods rich in polyphenols (i.e. flavonoids) and a

IP

reduction of the risk to suffer from degenerative diseases such as cancer or cardiovascular

SC R

diseases (Hollman, Hertog, & Katan, 1996; Vinson, Su, Zubik, & Bose, 2001; Scalbert, Manach, Morand, Rémésy, & Jiménez, 2005). The most interesting flavonoid subclass, the flavonols and their glycosides, is ubiquitous and predominant in fruits and vegetables and they are mostly

NU

accumulated in the fruit's skin or outer tissues (Herrmann, 1976). Figure 1 shows the chemical structure of the most common flavonols quercetin, isorhamnetin, kaempferol, and myricetin. In the context of polyphenol-rich health-beneficial plants, the genus Opuntia seems to be very

MA

interesting. Opuntia, the most economic and impressive member of the Cactaceae, is approximately consisting of more than 250 species (Britton & Rose, 1963). The cactus Opuntia plants are very capable for cultivation in arid and semiarid regions (Russell & Felker, 1987). Up

D

to now, the most propagated and world-wide distributed cactus for the commercial (food)

TE

production is from Opuntia ficus-indica (Inglese, Basile, & Schirra, 2002; Griffith, 2004). Cactus O. ficus-indica produces both, edible fruits and fleshy flattened stems termed ―cladodes‖.

CE P

Regionally, cladodes are used as a green feed for animals. Besides, the young cladodes commercially termed ―nopalitos‖ are used as fresh or processed vegetables for human consumption as well as being used in traditional medicine, or as functional constituents for food

AC

and pharmaceutical products (Russell & Felker, 1987; Stintzing & Carle, 2005).

Cactus O. ficus-indica fruits and cladodes contain different phytochemicals exhibiting potent antioxidant activity and further functional properties (Stintzing, Schieber, & Carle, 2001; Piga, 2004; Stintzing & Carle, 2005; Livrea & Tesoriere, 2006). In most studies dealing with the phytochemical compounds of cactus O. ficus-indica, mainly betalains have been investigated. Only a few studies reported specific flavonoids in cactus Opuntia spp. fruits or even cladodes and flowers. However, information about presence of specific flavonoids in cactus O. ficusindica is not only rare but also kind of inconsistent: Some studies reported that isorhamnetin or its glycosides are the only flavonoids found in O. ficus-indica plant tissues, and others mentioned them as predominant derivatives. For instance, Arcoleo, Ruccia, & Cusmano (1961) found isorhamnetin in the extract of O. ficus-indica flowers. But recently, De Leo, Bruzual De

ACCEPTED MANUSCRIPT Abreu, Pawlowska, Cioni, & Braca (2010) detected different flavonols and identified them as isorhamnetin, quercetin and kaempferol glycosylated derivatives in the flowers of O. ficusindica with isorhamnetin-3-O-robinobioside as the predominant compound. In cactus O. ficus-

T

indica cladodes, different flavonols have been found but with kind of inconsistency with regard

IP

to the abundant flavonols (Ginestra et al., 2009; Guevara-Figueroa et al., 2010; Santos-Zea,

SC R

Gutierrez-Uribe, & Serna-Saldivar, 2011). For the fruits from O. ficus-indica, information about flavonoids is also very inconsistent. While Galati et al. (2003) reported that the main flavonoids identified in cactus juice, pressed from Sicilian O. ficus-indica fruits are isorhamnetin

NU

glycosides, Kuti (2004) reported that quercetin seems to be the predominant derivative followed by kaempferol and isorhamnetin present only small quantities. Recently, Moussa-Ayoub, ElSamahy, Kroh, & Rohn (2011a) found only isorhamnetin aglycon after hydrolysis of the

MA

flavonoids glycosides in an Egyptian cultivar from cactus O. ficus-indica.

Nowadays, there are many different cultivars of cactus O. ficus-indica fruits produced in

D

different countries, and intensively exported to European fresh fruit markets. Furthermore,

TE

different products from cactus Opuntia spp. fruits and cladodes have become more available in different markets. Therefore, an evaluation of the flavonol profiles seems to be necessary in

CE P

order to clarify, if this profile might bear the possibility to authentify the cactus O. ficus-indica fruits and cladodes or even products originating from them. Although advertised for containing Opuntia ficus-indica fruit ingredients, there are already some products in the market that do not

AC

contain it and the product is only with cheaper plant material and colors. With regard to future product quality control, analysis has to be fast to be applicable in e.g. the labs of a customs office or in other authorities that have to decide quickly if the products are authentic. Moreover, information about antioxidant active flavonols is almost absent in cactus O. ficus-indica cultivated in Egypt, where up to now cactus fruit is only a hardly exploited food crop and the fruit peels and the cladodes are considered as by-products or even waste.

Therefore, the present study aimed at comparing flavonol profiles and contents in fruit's pulp and peel from two different cultivars of red and yellow-orange colored cactus O. ficus-indica fruits cultivated in Egypt (locally named cv. Farawla and cv. Shamia), in comparison to three most common Sicilian cultivars of red ‗Rosa‘, yellow-orange ‗Gialla‘, and greenish-white ‗Bianca‘, and two further cultivars of red and yellow-orange colored fruits cultivated in South

ACCEPTED MANUSCRIPT Africa (specific cultivars unknown). Besides fruits, flavonol profiles and contents were also investigated in the corresponding cladodes from both Egyptian cultivars. Moreover, total phenolic contents and the antioxidant activity have been evaluated in the pulp, peel and cladode

T

of the different origins in order to evaluate the potential of each plant part for future (food)

SC R

IP

applications.

2. Materials and Methods

NU

2.1. Plant sample preparation.

Fresh samples from seven cultivars of cactus O. ficus-indica fruits were collected from three origins: Red and yellow-orange fruit cultivars were collected from an Egyptian orchard (3 times

MA

5 kg randomly collected in the orchard). Fruits from the common cultivars of red ‗Rose‘, yellow-orange ‗Gialla‘ and greenish-white ‗Bianca‘ were collected from a Sicilian orchard (3 times 5 kg randomly collected in the orchard). Further, red and yellow-orange fruit cultivars

D

from South Africa were bought at a German local market (5 kg each). The fruits' pulps and peels

TE

were coded as ‗Pu 1 to 7‘, and ‗Pe 1 to 7‘, respectively (Table 1). The glochides and the two distal parts (top and bottom parts, approximately 1 cm) have been removed. Then fruits were

CE P

peeled longitudinally, pulps and peels were separated and after that, seeds were removed from the pulps. Pulps and peels were lyophilized separately and stored in closed bottles at -18ºC until analysis. Besides, cactus O. ficus-indica cladodes were harvested from the same plants from

spines.

AC

Egypt. These were coded as ‗Cl 1‘ and ‗Cl 2‘, respectively, and lyophilized after removing the

2.2. Chemicals and reagents RAPIDASE C80MAX as commercial pectinase preparation (from Aspergillus niger) was provided by DSM (Heerlen, Netherlands). Cellulase (0.033U/mg, E.C.3.2.1.4, from Trichoderma viride) was purchased from Fluka (Fluka Chemie AG, Switzerland). As commercial

standards

isorhamnetin-3-O-rutinoside,

isorhamnetin-4′-O-glucoside,

and

isorhamnetin were purchased from Extrasynthese (Genay, France). HPLC solvents and further chemicals were purchased from Carl Roth GmbH (Karlsruhe, Germany).

2.3 Extraction for analysis.

ACCEPTED MANUSCRIPT Analyses were carried out with extracts of freeze-dried pulps and peels, respectively. 100 mg of dried cactus sample per 1mL aqueous methanol (70%) were sonicated for 15 minutes and vortexed. Prior to the analyses, all extracts were again vortexed and centrifuged at 10.000 x g for

T

10 minutes (Hermle Z233MK, Hermle Labortechnik GmbH, Wehingen, Germany). All extracts

IP

were sonicated for 5 min, filtrated through syringe filters (nylon, 0.45 µm, Carl Roth GmbH,

2.4. Identification and quantification of flavonols.

SC R

Karlsruhe, Germany) prior to HPLC-DAD analysis.

NU

Flavonol glycosides were quantified using HPLC-DAD analysis according to method described by Moussa-Ayoub et al. (2011a). Analytical separation of the flavonols was carried out on a 4.6 x 250 mm i.d., 5 µm, Fluofix IEW425 column (Wako Pure Chemical Industries, Osaka, Japan),

MA

with a gradient mixture consisting of eluent A (water/acetic acid/acetonitrile; 94.5/0.5/5 v/v/v) and eluent B (acetonitrile). The gradient used for all extracts was as follows: 0–8 min, 0% B; 9– 29 min, 4% B; 30–44 min, 8% B; 45–49 min, 22% B; 50–64 min, 28% B; 65 min, 45% B; 66–

TE

wavelengths 365, 325 and 280 nm.

D

108 min, 0% B. The flow rate was 1 ml per min. The detection was performed at the three Besides, HPLC-DAD/ESI-MSn measurements were carried out according to Schmidt et al.

CE P

(2010), in order to identify the flavonol aglycon. The extracts were separated on a Phenomenex Prodigy column (3.0 x 150 mm, 3 µm, 100 Å) with a security guard C18 (4 x 3.0mm, 3 µm, 100 Å) at a temperature of 30 °C using a gradient mixture consisting of eluent A (water/acetic acid;

AC

99.5/0.5 v/v) and eluent B (acetonitrile). The gradient used was as follows: 0-12 min, 5-7% B; 12-25 min, 7-9% B; 25-45 min, 9-12% B; 45-100 min, 12-15% B; 100-150 min, 15% isocratic; 150-155 min, 15-50% B; 155-165 min, 50% isocratic; 165-170 min, 50-5% B; 170-175 min, 5% isocratic. The flow rate used was 0.4 mL per min, and the measured detector wavelengths were set at 280, 325, and 365 nm. The flavonols were identified as deprotonated molecular ions and characteristic mass fragment ions by HPLC-DAD/ESI-MSn using an Agilent series 1100 ion trap mass spectrometer in negative ionization mode. Nitrogen was used as the drying gas (12 L per min, 350 °C) in addition to nebulizer gas (40 psi) with a capillary voltage of ~3500 V. Helium was used as the collision gas in the ion trap. The mass optimization for the ion optics of the mass spectrometer was performed for quercetin m/z 301.

2.5. Enzymatic hydrolysis for determining flavonol aglycons

ACCEPTED MANUSCRIPT As described by Moussa-Ayoub et al. (2011a), 100 mg dried cactus pulps or peels were treated with 1 mL of an enzyme mixture (RAPIDASE C80MAX diluted with water 1:10 and 40 mg cellulases added). The samples were vortexed for 1 min, and then incubated at 50 ˚C for 16

T

hours. Successively, all samples were vortexed every 15 min for first 2 h, and every 30 min for

IP

the next 6 h. After that, samples were subjected to 100 ˚C for 5 min to stop the enzymatic action,

SC R

and then diluted with pure methanol to a final concentration of 80%. The diluted samples were vortexed five times for 1 min, sonicated and then filtrated prior to HPLC-DAD analysis.

NU

2.6. Determination of the total phenolic content.

According to Moussa-Ayoub, El-Samahy, Rohn & Kroh (2011b), total phenolic content was measured directly at a wavelength of 280 nm against a calibration curve of gallic acid using a

MA

UV/Vis spectrophotometer (Shimadzu, Japan). Determination was carried out in aqueous methanolic (70%) extracts of the samples. The results were expressed as milligrams gallic acid

D

equivalents (GAE) per 100 mg of dry sample.

TE

2.7. Determination of the antioxidant activity 2.7.1. Trolox equivalent antioxidant capacity (TEAC) assay

CE P

Antioxidant activity was monitored using the trolox equivalent antioxidant capacity (TEAC) assay (method modified by Rohn, Rawel, & Kroll, 2004). According to the total phenolic content, extracts are adequately diluted in duplicate with PBS buffer. A 500 µL aliquot of the

AC

[2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] diammonium salt (ABTS) is added to 100 µL of diluted extract, or rather the blank value of PBS buffer, in disposable plastic cuvettes. To initiate the reaction, 200 µL of potassium persulphate are added to the samples in a regular interval of 15 s. The mixture developed a dark green color and after 6 min, the reading of each cuvette was done again in the same regular time intervals. The procedure was also conducted for the Trolox calibration curve: 10 different concentrations of Trolox (0.05-0.5 mM /L-1) were used. Results were expressed as µM Trolox equivalents (TE) per 100 mg DM.

2.7.2 Electron paramagnetic resonance (EPR) spectrometry The degradation of the stable synthetic radical Fremy‘s salt (potassium nitrosodisulfonate) in presence of antioxidants in the cactus extracts (50 mg/ 1mL aqueous methanol) was monitored as applied by Rösch, Bergmann, Knorr, and Kroh (2003). Appropriate extract dilutions (1:15)

ACCEPTED MANUSCRIPT were prepared and 100 µL aliquots were allowed to react for 30 min with an equal volume of a solution of Fremy‘s salt (1 mM in phosphate buffer, pH 7.4). EPR spectra of Fremy‘s radical were obtained with a Miniscope MS100 spectrometer (Magnettech GmbH, Berlin, Germany).

T

The antioxidant activity expressed as mM Fremy‘s salt reduced by 100 µL diluted extract, was

IP

calculated by comparison with a control reaction with 100 µl Fremy‘s salt 1 mM and 100 µL

SC R

phosphate buffer.

2.7.3. Statistical Analysis

NU

The analysis of variance (ANOVA) was carried out to test the possibility of significance of treatment effect. LSD, as described by Ott (1984), was used to perform all possible pair comparisons between means of different treatments. Means having the same letter are not

D

3. Results and Discussion

MA

significantly different at p ≤ 0.05.

TE

3.1. Flavonol profile and content in O. ficus-indica fruits and cladodes. As already mentioned above, inconsistent information about the presence of flavonols in cactus

CE P

O. ficus-indica fruits is very obvious. While there are some reports on quercetin being the most dominating flavonol in O. ficus-indica, some think of isorhamnetin. Moreover, information about these antioxidant compounds is almost absent for some production regions such as Egypt.

AC

Therefore, a comparison between different cultivars grown in Egypt and other locations was carried out to clear any data inconsistencies. In order to characterize the flavonols in the aqueous methanolic extracts of fruit's pulp and fruit's peel, HPLC-DAD analyses were carried out prior to and after enzymatic hydrolysis for evaluating the profile of flavonol glycosides and the corresponding aglycons, followed by HPLC-ESI-MSn measurements to support results obtained. Additionally, similar analyses were performed in order to characterize the flavonols in the aqueous methanolic extracts of cladodes from the Egyptian cultivars.

3.1.1. Flavonol glycosides and aglycons in O. ficus-indica fruits. 3.1.1.1. Influence of cultivar. In the present study, three cultivars of red colored fruits from Egypt, Sicily, and South Africa, three cultivars of yellow-orange colored fruits from Egypt, Sicily, and South Africa, and one

ACCEPTED MANUSCRIPT cultivar of greenish-white colored fruits from Sicily have been investigated. HPLC-DAD analyses (prior to enzymatic hydrolysis) showed presence of five prominent flavonol derivatives in all O. ficus-indica fruit peels (Figure 2A-E), while all pulps did not exhibit any flavonol

T

(profile), at all. All analyzed fruit peels exhibited the same flavonol profile, but the amounts of

IP

the single compounds differed between cultivars. According to retention times and spectra of the

SC R

flavonol glycosides detected, isorhamnetin-3-O-rutinoside was identified as predominant flavonol derivative (Figure 2).

NU

In order to identify the flavonol aglycons, enzymatic hydrolysis was carried out as described by Moussa-Ayoub et al. (2011a). With this method, a gentle enzymatic hydrolysis of the flavonol glycosides using commercial preparation of pectolytic enzymes and cellulases is performed, as

MA

this does not produce any further degradation products such as protocatechuic acid which are known to be formed thermally-induced under drastic acidic conditions, e.g. during acidic hydrolysis. Flavonol glycosides are quantitatively deglycosylated under hydrolysis conditions

D

using hydrochloric acid (pH 1-2). Unfortunately, the resulting flavonol aglycon can be further

TE

degraded oxidatively: Oxidation of phenolic compounds can be either very fast under alkaline conditions (pH > 7), or under very acidic, proton-catalyzed conditions such as during the acidic

CE P

hydrolysis (Buchner, Krumbein, Rohn, & Kroh, 2006; Moussa-Ayoub et al. 2011a). Preferentially, the more gentle enzymatic hydrolysis using commercial pectolytic enzymes might even increase level of flavonols detected, since cactus fruits and cladodes exhibit viscous

AC

properties and cell-wall components may retain part of the flavonols. As expected for the present study, results showed that enzymatic hydrolysis resulted in disappearing of all flavonol glycosides and produced only isorhamnetin aglycon in the fruits' peels from all cultivars investigated. However, Galati et al. (2003) investigated flavonols in juice pressed from whole fruits (pulps and peels) with a combination of red (95%) and yellow-orange (5%) cactus O. ficus-indica cultivars. They reported that isorhamnetin derivatives are the main flavonols found in O. ficus-indica fruits' juice and very small amounts of rutin and kaempferol-3-O-rutinoside. Later, Moussa-Ayoub et al. (2011a, b) found only isorhamnetin derivatives in both O. ficusindica and O. dillenii fruits. In contrast, Kuti (2004) mentioned quercetin as the predominant flavonol aglycon in cactus fruits from O. ficus-indica and three other Opuntia species subjected to acidic hydrolysis, but unlikely that study did not give any information about flavonol glycosides before hydrolysis. He reported concentrations of total flavonol aglycons produced by

ACCEPTED MANUSCRIPT acidic hydrolysis being 0.069, 0.093, 0.054, and 0.010 mg/g (fw) in fruits from O. ficus-indica, O. lindheimeri, O. streptacantha and O. stricta, respectively, which is significantly lower than values found in the present study with regard to O. ficus-indica fruits. As mentioned above, this

T

might be due to further degradation of the aglycons under acidic hydrolysis conditions.

IP

However, in the present study the amount of flavonol glycosides prior enzymatic hydrolysis and

SC R

aglycons after enzymatic hydrolysis ranged between 2.2 - 4.1 and 0.88 - 1.96 mg/g (DM), respectively (Figure 3A and B).

NU

To underline the presence of isorhamnetin as the only aglycon, HPLC-ESI-MSn measurements were carried out. Results showed the presence of different isorhamnetin derivatives with mainly isorhamnetin-3-O-rutinoside (m/z 624), isorhamnetin diglycosides (m/z 610), isorhamnetin

MA

triglycosides (m/z 786), and further amounts of other isorhamnetin glycosides in O. ficus-indica fruit's peel. Exemplarily, isorhamnetin-3-O-rutinoside, the dominating compound, as obvious from the chromatogram (Figure 2), provided its typical fragments at m/z 623.9, m/z 431.1 m/z

D

315.1 and 300.4 (Figure 4). Fragmentations patterns for isorhamnetin glycosides were similar to

TE

those of the literature, exemplarily given also by Abad-García et al. (2012) and Yeddes et al. (2013).

CE P

But being the most important result for the present study, the complete fragmentation pattern of derivatives detected, produced only isorhamnetin (m/z 315) as the only aglycon in the fruit's peel. Therefore, results obtained herein underline to a large extent results obtained by Galati et

AC

al. (2003) and Moussa-Ayoub et al. (2011a, b), of isorhamnetin being the main flavonol to be found in cactus O. ficus-indica fruits. Further, HPLC-ESI-MSn analyses also confirmed that cactus O. ficus-indica fruit's pulp does not contain any flavonols at all. This is supported by Tesoriere, Fazzari, Allegra, & Livrea (2005) who showed that edible pulp from O. ficus-indica fruit did not contain significant amounts of flavonols. They investigated edible pulps of red, yellow and white Sicilian cultivars from O. ficus-indica, and found only a very low amount of kaempferol (2.7 µg/ 100g fw) in the yellow cultivar. This is very low and might not be appropriate for using it as an analytical marker substance for foods that contain cactus as an ingredient (beverages etc.).

Similarly to O. ficus-indica fruits, isorhamnetin glycosides also characterize some common fruits such as pears (Fernandez de Simon, Perez-Ilzarbe, Hernandez, Gomez-Cordoves, &

ACCEPTED MANUSCRIPT Estrella, 1992; Schieber, Keller, & Carle, 2001), or sea buckthorn (Rösch et al., 2003). Isorhamnetin-3-O-rutinoside represents 50% of total flavonol glycosides in sea buckthorn juice (Rösch et al., 2003), while reaching a maximum 24% of total isorhamnetin glycosides in cactus

T

fruit peels as investigated in the present study. The flavonol isorhamnetin is the primary

IP

metabolite during the in vivo metabolism of quercetin (Crespy et al., 1999; Graefe et al., 2001;

SC R

Lan et al., 2008). Pharmacological properties and bioactivity of isorhamnetin or its glycoside derivatives were investigated in different studies. For instance, an activity of isorhamnetin against skin cancer (Kim et al., 2011) and cell injuries (Bao & Lou, 2006; Zhang et al., 2011)

NU

has been reported. Its glycoside isorhamnetin-3-O-rutinoside exhibited potential antimicrobial effects (Agnese, Pérez, & Cabrera, 2001), and positive effect against human myelogenous erythroleukaemia cells (Boubaker et al., 2011). Furthermore, Ito et al. (1999) concluded that

MA

anti-tumor activity of isorhamnetin-3-O-rutinoside and isorhamnetin-3-O-glucoside might be comparable to or even stronger than that of green tea polyphenol epigallochatechin gallate. Recently, Ku, Kim, Lee, Kim, & Bae (2013) attributed the high anticoagulant and

D

profibrinolytic effects of isorhamnetin-3-O-galactoside ‗compared to those of quercetin-3-O-

TE

galactoside‘ to the positive regulation of anticoagulant function by the methoxy group of

CE P

isorhamnetin-3-O-galactoside.

Results obtained herein showed that flavonol profiles in all cultivars have not been influenced by further characteristics of the fruits such as their color. Although the seven cultivars

AC

investigated in the present study exhibited different fruit colors, all fruits' peels produced the same flavonol profile and composition. The different fruit colors are due to differences in betalain contents in fruits investigated. Betalains, being the responsible of the attractive colors of plant parts from some limited higher plants restricted to Caryophyllales (e.g., cactus Opuntia spp.), contribute to a large extent to a characterization/classification of cactus fruit cultivars. Betalains, the derivatives of betalamic acid, are classified depending on their chemical structures. ―Red to purple‖ colors are represented by betacyanins and ―yellow to orange‖ colors by betaxanthins. Conjugation of betalamic acid with cyclo-dihydroxyphenylalanine is producing the betacyanins: betanidin or isobetanidin, and the glycoslation and/or acylation of these aglycons result in further betacyanins. Betaxanthins are resulting from conjugation of betalamic acid with different amines and protein/or non-protein amino acids (Delgado-Vargas et al., 2000; Strack, Vogt, & Schliemann, 2003; Tanaka, Sasaki, & Ohmiya, 2008). In the present study, the

ACCEPTED MANUSCRIPT red cactus fruits contained mainly betacyanins, and yellow-orange cactus fruits contained almost only betaxanthins, while both betalain derivatives have not been found in greenish-white fruits at all (data not shown). As mentioned above, these different cultivars from the same cactus

T

Opuntia species ―Opuntia ficus-indica‖ produce similar flavonol profiles even in greenish-white

IP

and yellow-orange fruits (Figure 2D and E). It was only observed that red fruit cultivars

SC R

contained highest contents of flavonol glycosides slightly compared to yellow-orange cultivars, and greenish-white cultivar from same origin (Figure 3A). The specific profiles of flavonoid occurrence arise by expression of combination of regulatory genes (Holton & Cornish, 1995;

NU

Mol, Grotewold, & Koes, 1998). Therefore, each cactus species might have a specific flavonol profile being determined genetically. For instance, Moussa-Ayoub et al. (2011b) reported that flavonol profile of cactus O. dilenii fruits is significantly different from those of cactus O. ficus-

MA

indica fruits. In line with results obtained for the different cultivars of cactus O. ficus-indica, Vinha et al. (2005) reported that 18 different cultivars of olive fruits, although collected from

TE

3.1.1.2. Influence of location.

D

different locations, exhibited the same phenolic profile.

Different factors such as maturity and ripeness, climate, or exposure to UV-B radiation, etc.

CE P

affect content of flavonols. As mentioned above, the specific flavonol profile might be determined by the genotype. Results show that the geographically different locations of production did not influence the flavonol composition (Figure 2A-C). All fruit peels from all

AC

cultivars investigated, contained the same five isorhamnetin derivatives. Among cultivars collected from the three locations, fruits from South Africa showed the highest flavonol content compared to fruits from Egypt and Sicily. As it was not the aim of this study to evaluate the influence of ecophysiological factors in a kind of agricultural study taking replicates in the field, several cultivation periods etc. into account, these results have to be regarded as trends, showing the similarity of the flavonol profile and values in a similar range of a power of ten, at least. Studies reported that flavonols formation might be accelerated by increased sun light exposure. For instance, Price, Breen, Valladao, & Watson (1995) investigated the impact of sun exposure level on the concentration of flavonols in wine and reported that quercetin glycosides had 4.5, 14.8, and 33.7 mg/L in wines produced from shaded, moderate, and highly light exposed grapes, respectively. Further, Stewart et al. (2000) investigated the influence of location on the flavonols in twenty different varieties of tomatoes. They reported that highest concentrations of flavonols

ACCEPTED MANUSCRIPT found in tomatoes originating from warm sunny climates in Spain and Israel. Schirrmacher, Schnitzler, & Graßmann (2004) mentioned also that field-grown plants have much higher flavonoid contents than those in greenhouse-grown plants. This might explain the increase in

T

flavonol levels in cactus fruits from South Africa compared to fruits from Egypt and Sicily. As

IP

already mentioned, information about specific cultivation practices was not available, but it was

SC R

tried to get/collect the full colored fruits at an approximate similar maturity and ripening stage.

Flavonol levels in cactus fruits are higher than those in edible parts of more famous fruits such

NU

as papaya, cherry, banana, or watermelon (Lako et al., 2007). This valorizes cactus O. ficusindica fruits as a promising source of flavonols especially in arid and semiarid regions, where malnutrition and lack in water resources cause severe problems. However, results obtained

MA

herein showed that isorhamnetin is exclusively occurring in the fruits' peels. This fact of being flavonols accumulated in the fruit's peel was observed also in common fruits such as apple and kiwi (Wijngaard, Röβle, Brunton, 2009). Stewart et al. (2000) reported that flavonols in 20

D

varieties of tomatoes had an amount between 1.3 to 22.2 μg/g (fw), and 98% of them to be

TE

found in the fruit skin. With regard to fruits‘ by-products, flavonol levels (2.2-4.1 mg/g dw) (Figure 3) found in cactus fruits' peels might be even higher than those found in some other

CE P

common fruits. For example, Schieber et al. (2001) found that flavonols ranged (0.97-1.85 mg/g dw) and (10.3-43 mg/L) in pomace and juice from three different apple varieties, respectively.

AC

3.1.3. Flavonol glycosides and aglycons in O. ficus-indica cladodes Cactus cladodes are widely investigated for their nutritional value, medicinal properties as well as their use a potential vegetable food product, but only few investigations have been reported on the flavonol content of O. ficus-indica cladodes, so far. Information about cactus O. ficusindica cladodes from cultivars produced in Egypt is more or less absent. Although there is a high potential of such biomass resources in Egypt, the scientific research approaches are only marginal in this field. Furthermore, use of cactus cladodes, commonly consumed as traditional vegetables in Mexico and Latin America, are up to now comparatively negligible in Egypt. Therefore, the present study aimed at giving preliminary information about flavonols in cladodes from cactus O. ficus-indica grown in Egypt. Cladode samples from a cultivar producing red fruits as well as other samples from cultivar producing yellow-orange fruits were collected from Egypt and subjected to the same analysis carried out as with the corresponding

ACCEPTED MANUSCRIPT fruits as described above. Results of the HPLC-DAD analysis (prior to enzymatic hydrolysis) showed that O. ficus-indica cladodes from both cultivars produced flavonols profiles similar to those obtained from cactus O. ficus-indica fruits' peels. In accordance, cladodes contained the

T

same five prominent flavonol derivatives found in fruit peels from Egyptian, Sicilian, as well as

IP

South African cultivars (Figure 2F). Results presented in Figures 3A show that cactus cladodes

SC R

contain valuable amounts of flavonols compared to their corresponding fruits. Similarly to results of the fruits, the enzymatic hydrolysis produced isorhamntin as the only aglycon. HPLCESI-MSn measurements showed presence of the same flavonol derivatives to be found also in fruit

peels;

isorhamnetin-3-O-rutinoside,

isorhamnetin

diglycosides,

isorhamnetin

NU

the

triglycosides and other isorhamnetin glycosides were found in small amounts. The complete fragmentation pattern of all derivatives revealed only isorhamnetin aglycon (m/z 315). The

MA

results obtained herein underline to some extent those obtained by Ginestra at al. (2009) and Santos-Zea et al. (2011) from cactus O. ficus-indica cladodes collected from Italy and Mexico, respectively. They also identified five prominent flavonols as isorhamnetin derivatives. While

D

Santos-Zea et al. (2011) detected them in cladodes from two different cultivars from O. ficus-

TE

indica separately; Ginestra at al. (2009) detected them in a whole mix of three different cultivars from O. ficus-indica cladodes. Both studies illustrated that cactus O. ficus-indica cladodes are

CE P

characterized with mainly isorhamnetin derivatives. In contrast, Guevara-Figueroa et al. (2010) detected isoquercitrin, isorhamnetin-3-O-glucoside, kaempferol-3-O-rutinoside, rutin and isorhamnetin-3-O-rutinoside in two commercial and three wild varieties of O. ficus-indica

AC

cladodes cultivated in Mexico. Further, they reported that the most abundant flavonols were kaempferol-3-O-rutinoside and isorhamnetin-3-O-rutinoside. Results obtained in the present study, showed that kaempferol was not found at all. The concentrations of flavonols in the present cladodes are comparatively higher (6.3-7.3 mg/g DW) than those found in cladodes from Italy (4.8 mg/g) (Ginestra at al., 2009), as well as the cladodes from Mexico (1.1-3.5 mg/g) (Guevara-Figueroa et al., 2010). Further, the level of aglycon reported herein by enzymatic hydrolysis is significantly higher than that reported by Santos-Zea et al. (2011).

3.2. Total phenolic content in O. ficus-indica fruits and cladodes Besides flavonols, cactus plants contain quite a lot more of phenolic compounds, being also able to contribute to the antioxidant activity. To give a rough estimation, photometric determination

ACCEPTED MANUSCRIPT of the total phenolic content was carried out. The concentrations of total phenolic contents in pulps found herein (Figure 3C) are comparable to those reported in the literature (0.242-0335 mg/mL) by Stintzing et al. (2005) and (0.45-0.454 mg/g pulp) by Díaz Medina, Rodírguez-

T

Rodríguez, & Díaz Romero (2007), but lower than those (approx. 5.5 mg/g pulp dw) found by

IP

Piga, Del Caro, Pinna, & Agabbio (2003) and higher than those (0.172 mg/g juice) found by

SC R

Chavez -Santoscoy, Gutierrez-Uribe, & Serna-Saldívar (2009). Galati et al. (2003) reported 0.746 mg/mL as a mean value of total phenolic compounds found in juice produced from the whole fruit. With regard to cladodes, total phenolic contents obtained herein were lower than

NU

those obtained by Jaramillo-Flores at al. (2003), but were in the same range as reported by Guevara-Figueroa et al. (2010). However, results of the present study showed that cactus O. ficus-indica fruit's peel and cladodes contain quite high amounts of phenolic compounds in

MA

comparison to fruit's pulp in all investigated cultivars (Figure 3C). Similar to the flavonol content, red fruits exhibited the highest phenolic content compared to others from each location, while the greenish-white fruits exhibited the lowest content, at all. With regard to influence of

D

location, the South African cultivars had higher phenolic contents than those from Sicilian and

TE

Egyptian cultivars. This might be due to the expected difference in ecophysiological conditions between the different locations. But as mentioned above, it was not the aim of this study to

CE P

evaluate the influence of ecophysiological factors in a kind of agricultural study taking replicates in the field, several cultivation periods etc. into account, these results have to be

AC

regarded as trends.

3.3. Antioxidant activity of O. ficus-indica fruits and cladodes 3.3.1. Trolox equivalent antioxidant capacity (TEAC). TEAC assay was carried out for all cactus fruit pulps and peels samples, as well as for the cladodes. Cactus O. ficus-indica fruit's peel and cladodes exhibited high antioxidant capacities in comparison to cactus fruit's pulp (Figure 5A). But antioxidant capacities of fruits' pulps obtained herein were higher than those (2.24-2.60 mmol/kg pulp) reported by Stintzing et al. (2005) and comparable to those (4.20-5.31 μmol/g pulp) reported by Butera et al. (2002). With regard to antioxidant capacity of the whole fruit, Fernández-López, Almela, Obón, & Castellar (2010) reported a value of 6.7 μmol Trolox/g fresh fruit. Obviously, antioxidant capacities of all analyzed cactus O. ficus-indica tissues were almost in line with their corresponding total phenolic content underlining that the phenolic compounds are the main antioxidants (Figures

ACCEPTED MANUSCRIPT 3C & 5A). Castrejón, Eichholz, Rohn, Kroh, & Huyskens-Keil (2008) reported a high correlation of the antioxidant activity measured with TEAC with the total phenolic content for all maturity stages of different four cultivars from highbush blueberry (Vaccinium corymbosum

SC R

3.3.2. Electron paramagnetic resonance (EPR) spectrometry.

IP

T

L.).

Information about antioxidant activity of cactus fruits and cladodes, using the electron paramagnetic resonance (EPR) spectrometry assay is not available, so far. This technique was

NU

used in present study to support results obtained by the traditionally used TEAC assay. This might give additional information about the antioxidant capacity of the plant tissues investigated. Further, as suggested by Verhagen et al. (2003), more than one assay should be

MA

performed when investigating the antioxidant activity of a natural compound. As expetced, EPR results also showed that cactus O. ficus-indica fruit peels and cladodes degraded more amounts of the free radical Fremy's salt than fruit pulps (Figure 5B). Moreover, Fremy‘s salt degradation

D

curves showed that radical scavenging is quite different depending on the plant tissue (Figure

TE

5C). Antioxidant activity of all analyzed cactus O. ficus-indica tissues evaluated by TEAC assay and EPR assay were also in line with their corresponding contents of total phenolics even in the

4. Conclusion

CE P

cladodes and greenish-white fruits.

AC

This present study provided information about flavonol profiles in both cactus O. ficus-indica fruits and cladodes from cultivars grown in Egypt, cultivars from Sicily, as well as cultivars from South Africa. Results showed that regardless betalain content, cactus O. ficus-indica can be characterized clearly by the presence of flavonols, in particular isorhamnetin glycosides. The flavonols accumulate only in the fruit's peel and the cladodes, while they are not present in the pulp. Interestingly, cactus O. ficus-indica fruit peels and cladodes from all cultivars investigated, exhibited same flavonol profile composed of the same five prominent isorhamnetin derivatives. Based on present results and literature, it is suggested that this profile of isorhamnetin glycosides might serve as a probable biochemical fingerprint for both cactus O. ficus-indica peels and cladodes. These findings might help effectively for the evaluation of authenticity of O. ficus-indica fruits or cladodes-based products. Although advertised for containing Opuntia ficusindica fruits ingredients, there are already some products in the market that do not contain it and

ACCEPTED MANUSCRIPT the product is only with cheaper plant material and colors. With regard to future product quality control, analysis has to be fast and to be applicable in e.g. the labs of a customs office or in other authorities that have to decide quickly if the products are authentic. The hypothesis of being

T

phenolics or flavonoids might serve as biochemical indicators of genuineness of fruit products

IP

are reported and taken into account in different studies (Fernandez de Simon, et al., 1992;

SC R

Tomas-Lorente, Garcia-Viguera, Ferreres, & Tomas-Barberan, 1992; Silva et al., 2000). The present study valorizes potency of cactus O. ficus-indica cladodes and fruit's peel which have not been taken into account until now in some cultivation regions such as Egypt. Therefore, based on their contents of flavonols, total phenolics, as well as their antioxidant activity, O.

MA

NU

ficus-indica fruit peels and cladodes should be exploited strongly for food or further products.

Acknowledgment

This work was partially financed and supported by the Egyptian Ministry of Higher Education

D

& Scientific Research. The authors gratefully thank Prof. Paolo Inglese (Università degli Studi

TE

di Palermo, Italy) for kind cooperation and providing the Sicilian cactus O. ficus-indica fruit

References

CE P

cultivars investigated in this present study.

AC

Abad-García, B., Garmón-Lobato, S., Berrueta, L. A., Gallo, B., & Vicente, F. (2012). On line characterization of 58 phenolic compounds in citrus fruit juices from Spanish cultivars by highperformance liquid chromatography with photodiode-array detection coupled to electrospray ionization triple quadrupole mass spectrometry. Talanta, 99, 213–224. Arcoleo, A., Ruccia, M., & Cusmano, S. (1961). Flavonoid pigments from Opuntia. I. Isorhamnetin from flowers Opuntia ficus-indica Miller. Annal Chimica (Rome), 51, 751–781. Agnese, A. M., Pérez, C., & Cabrera, J. L. (2001). Adesmia aegiceras: antimicrobial activity and chemical study. Phytomidicine, 8, 389–394. Bao, M., & Lou, Y. (2006). Isorhamnetin prevent endothelial cell injuries from oxidized LDL via activation of p38 MAPK. European Journal of Pharmacology, 547, 22–30. Boubaker, J., Bhouri, W., Ben Sghaier, M., Ghedira, K., Dijoux Franka, M. G., & GhekirGhedira, L. (2011). Ethyl acetate extract and its major constituent, isorhamnetin 3-O-rutinoside,

ACCEPTED MANUSCRIPT from Nitraria retusa leaves, promote apoptosis of human myelogenous erythroleukaemia cells. Cell Proliferation, 44, 453–461.

cactus family. Carnegie Institution of Washington, No. 248, Vo. I.

T

Britton, N. L., & Rose, J. N. (1919). The Cactaceae: description and illustrations of plants of the

IP

Buchner, N., Krumbein, A., Rhon, S., & Kroh, L. W. (2006). Effect of thermal processing on

SC R

the flavonols rutin and quercetin. Rapid Communications in Mass Spectrometry, 20, 3229–3235. Butera, D., Tesoriere, L., Di Gaudio, F., Bongiorno, A., Allegra, M., Pintaudi, A., et al. (2002). Antioxidant activities of Sicilian prickly pear (Opuntia ficus indica) fruits extracts and reducing

NU

properties of its betalains: betanin and indicaxanthins. Journal of Agricultural and Food Chemistry, 50, 6895–6901.

Castrejón, Eichholz, Rohn, Kroh, & Huyskens-Keil (2008). Phenolic profile and antioxidant

MA

activity of highbush blueberry (Vaccinium corymbosum L.) during fruit maturation and ripening. Food Chemistry, 109, 564–572.

Chavez-Santoscoy, R. A., Gutierrez-Uribe, J. A., & Serna-Saldívar, S. O. (2009). Phenolic

D

composition, antioxidant capacity and in vitro cancer cell cytotoxicity of nine prickly pear

TE

(Opuntia spp.) juices. Plant Foods for Human Nutrition, 64, 146–152. Clark, W. D., & Parfitt, B. D. (1980). Flower flavonoids of Opuntia series Opuntiae.

CE P

Phytochemistry, 19, 1856–1857.

Clark, W. D., Brown, G. K., & Mays, R. L. (1980). Flowers flavonoids of Opuntia subgenus cylindropuntia. Phytochemistry, 19, 2042–2043.

AC

Crespy, V., Morand, C., Manach, C., Besson, C., Demigne, C., & Remesy, C. (1999). Part of quercetin absorbed in the small intestine is conjugated and further secreted in the intestinal lumen. American Journal of Physiology, 277, G120–G126. Delgado-Vargas, F., Jiménez, A. R., & Paredes-López, O. (2000). Natural Pigments: Carotenoids, Anthocyanins, and Betalains — Characteristics, Biosynthesis, Processing, and Stability. Critical Reviews in Food Science and Nutrition, 40, 173–289. Díaz Medina, E. M., Rodríguez-Rodríguez, E. M., & Díaz Romero, C. (2007). Chemical characterization of Opuntia dillenii and Opuntia ficus indica fruits. Food Chemistry, 103, 38– 45. Fernandez de Simon, B., Perez-Ilzarbe, J., Hernandez, T., Gomez-Cordoves, C., & Estrella, I. (1992). Importance of phenolic compounds for the characterization of fruit juices. Journal of Agricultural and Food Chemistry, 40, 1531–1535.

ACCEPTED MANUSCRIPT Fernández-López, J. A., Almela, L., Obón, J. M., & Castellar, R. (2006). Determination of Antioxidant Constituents in Cactus Pear Fruits. Plant Foods for Human Nutrition, 65, 253–259. Inglese, P., Basile, F., & Schirra, M. (2002). Cactus pear fruit production. In P. S. Nobel (Eds.),

T

Cacti: biology and uses (pp. 163–182). University of California Press.Galati, E., Mondello, M.,

IP

Giuffrida, D., Dugo, G., Miceli, N., & Pergolizzi, S. (2003). Chemical characterization and

SC R

biological effects of Sicilian Opuntia ficus-indica (L.) mill. fruit juice: Antioxidant and antiulcerogenic activity. Journal of Agricultural and Food Chemistry, 51, 4903–4908. Ginestra, G., Parker, M. L., Bennett, R. N., Robertson, J., Mandalari, G., Narbad, A., et al.

NU

(2009). Anatomical, chemical and biochemical characterization of cladodes from Prickly pear (Opuntia ficus-indica (L.) Mill). Journal of Agriculture and Food Chemistry, 57, 10323–10330. Graefe, E. U., Wittig, J., Mueller, S., Riethling, A. K., Uehleke, B., Drewelow, B., et al. (2001).

MA

Pharmacokinetics and bioavailability of quercetin glycosides in humans. Journal of Clinical Pharmacology, 41, 492–499.

Griffith, M. P. (2004). The Origins of an important cactus crop, Opuntia ficus-indica

D

(Cactaceae): new molecular evidence. American Journal of Botany, 91, 1915–1921.

TE

Guevara-Figueroa, T., Jimenez-Islas, H., Reyes-Escogido, M. L., Mortensen, A. G., Laursen, B. B., Lin, L., et al. (2010). Proximate composition, phenolic acids, and flavonoids characterization

525–532.

CE P

of commercial and wild nopal (Opuntia spp.). Journal of Food Composition and Analysis, 23,

Herrmann, K. (1976). Flavonols and flavones in food plants: a review. International Journal of

AC

Food Science and Technology, 11, 433–448. Hollman, P. C. H., Hertog, M. G. L., Katan, M. B. (1996). Analysis and health effects of flavonoids. Food Chemistry, 57, 43–46. Holton, T. A, & Cornish, E. C. (1995). Genetics and Biochemistry of Ant hocyanin Biosynthesis. The Plant Cell, 7, 1071–1083. Ito, H., Miyake, M., Nishitani, E., Mori, K., Hatano, T., Okuda, T., et al. (1999). Anti-tumor promoting activity of polyphenols from Cowania mexicana and Coleogyne ramosissima. Cancer Letters, 143, 5–13. Kim, J. H., Lee, H., Park, Y., Ra, K. S., Shin, K., Yu, K., et al. (2013). Mucilage removal from cactus cladodes (Opuntia humifusa Raf.) by enzymatic treatment to improve extraction efficiency and radical scavenging activity. LW T - Food Science and Technology, 51, 337–342.

ACCEPTED MANUSCRIPT Ku, S., Kim, T. H., Lee, S., Kim, S. M., & Bae, J. (2013). Antithrombotic and profibrinolytic activities of isorhamnetin-3-O-galactoside and hyperoside. Food and Chemical Toxicology, 53, 197–204.

T

Kuti, J. O. (2004). Antioxidant compounds from four Opuntia cactus pear fruit varieties. Food

IP

Chemistry, 85, 527–533.

SC R

Lako, J., Trenerry, V. C., Wahlqvist, M., Wattanapenpaiboon, N., Sotheeswaran, S., & Premier, R. (2007). Phytochemical flavonols, carotenoids and the antioxidant properties of a wide selection of Fijian fruit, vegetables and other readily available foods. Food Chemistry, 101,

NU

1727–1741.

Lan, K., He, J., Tian, Y., Tan, F., Jiang, X., Wang, L., et al. (2008). Intra-herb pharmacokinetics interaction between quercetin and isorhamnetin. Acta Pharmacologica Sinica, 29, 1376–1382.

MA

De Leo, M., Bruzual De Abreu, M., Pawlowska, A. M., Cioni, P. L., & Braca, A. (2010). Profiling the chemical content of Opuntia ficus-indica flowers by HPLC–P DA-ESI-MS and GC/EIMS analyses. Phytochemistry Letters, 3, 48–52.

D

Livrea, M. A., & Tesoriere, L. (2006). Health benefits and bioactive components of the fruits

Development, 8, 73–90.

TE

from Opuntia ficus-indica [L.] Mill. Journal of the Professional Association for Cactus

CE P

Mol, J., Grotewold, E., & Koes, R. (1998). How genes paint flowers and seeds. Trends in Plant Science, 3, 212–217.

Moussa-Ayoub, T. E., El-Samahy, S. K., Kroh, L. W., & Rohn, S. (2011a). Identification and

AC

quantification of flavonol aglycons in cactus pear (Opuntia ficus-indica) fruit using a commercial pectinase and cellulase preparation. Food Chemistry, 124, 1177–1184. Moussa-Ayoub, T. E., El-Samahy, S. K., Rohn, S., & Kroh, L. W. (2011b). Flavonols, betacyanins content and antioxidant activity of cactus Opuntia macrorhiza fruits. Food Research International, 44, 2169–2174. Ott, L. (1984). An introduction to statistical methods and data analysis. 2nd edition, PWS publisher, Boston, M. A., USA. Piga, A. (2004). Cactus pear: A fruits of nutaceutical and functional importance. Journal of the Professional Association for Cactus Development, 6, 9–22. Piga, A., Del Caro, A., Pinna, I., & Agabbio, M. (2003). Changes in ascorbic acid, polyphenol content and antioxidant activity in minimally processed cactus pear fruits. LW T - Food Science and Technology, 36, 257–262.

ACCEPTED MANUSCRIPT Price, S. F., Breen, P. J., Valladao, M., & Watson, B. T. (1995). Cluster sun exposure and quercetin in Pinot noir grapes and wine. American Journal of Enology Viticulture, 46, 187–194. Rohn, S., Rawel, H. M., & Kroll, J. (2004). Antioxidant Activity of Protein-Bound Quercetin.

T

Journal of Agricultural and Food Chemistry, 52, 4725–4729.

IP

Rösch, D., Bergmann, M., Knorr, D., & Kroh, L. W. (2003). Structure–antioxidant efficiency

SC R

relationships of phenolic compounds and their contribution to the antioxidant activity of sea buckthorn juice. Journal of Agricultural and Food Chemistry, 51, 4233–4239. Russell, C. E., & Felker, P. (1987). The prickly-pears (Opuntia spp. Cactaceae): A source of

NU

human and animal food in semiarid regions. Economic Botany, 41, 433–445. Ku, S., Kim, T. H., Lee, S., Kim, S. M., & Bae, J. (2013). Antithrombotic and profibrinolytic activities of isorhamnetin-3-O-galactoside and hyperoside. Food and Chemical Toxicology, 53,

MA

197–204.

Santos-Zea, L., Gutierrez-Uribe, J. A., & Serna-Saldivar, S. O. (2011). Comparative analyses of total phenols, antioxidant activity, and flavonol glycoside profile of cladode flours from

D

different varieties of Opuntia spp. Journal of Agriculture and Food Chemistry, 59, 7054–7061.

TE

Scalbert, A., Manach, C., Morand, C., Rémésy, C., & Jiménez, L. (2005). Dietary polyphenols and the prevention of diseases. Critical Reviews in Food Science and Nutrition, 45, 287–306.

CE P

Schmidt, S., Zietz., M., Schreiner, M., Rohn, R., Kroh, L. W., & Krumbein, A. (2010). Identification of complex, naturally occurring flavonoid glycosides in kale (Brassica oleracea var. sabellica) by high-performance liquid chromatography diode-array detection/electrospray

2009–2022.

AC

ionization multi-stage mass spectrometry. Rapid Communication in Mass Spectrometry, 24,

Schirrmacher, G., Schnitzler, W. H., & Graßmann, J. (2004). Determination of secondary plant metabolites and antioxidative capacity as new parameter for quality evaluation – indicated by the new Asia salad Gynura bicolor. Journal of Applied Botany and Food Quality, 78, 133–134. Schieber, A., Keller, P., & Carle, R. (2001). Determination of phenolic acids and flavonoids of apple and pear by high-performance liquid chromatography. Journal of Chromatography A, 910, 265–273. Silva, B. M., Andrade, P. M., Mendes, G. C., Valentão, P., Seabra, R. M., & Ferreira, M. A. (2000). Analysis of phenolic compounds in the evaluation of commercial quince jam authenticity. Journal of Agricultural and Food Chemistry, 48, 2853–2857.

ACCEPTED MANUSCRIPT Stewart, A. J., Bozonnet, S., Mullen, W., Jenkins, G. I., Lean, M. E., & Grozier, A. (2000). Occurrence of flavonols in tomatoes and tomato-based products. Journal of Agricultural and Food Chemistry, 48, 2663–2669.

T

Stintzing, F. C., Schieber, A., & Carle, R. (2001). Phytochemical and nutritional significance of

IP

cactus pear. European Food Research and Technology, 212, 396–407.

SC R

Stintzing, F. C., & Carle, R. (2005). Cactus stems (Opuntia spp.): a review on their chemistry, technology, and uses. Molecular Nutrition and Food Research, 49, 175–194. Stintzing, F. C., Herbach, K. M., Mosshammer, M. R., Carle, R., Yi, W., Sellappan, S., et al.

NU

(2005). Color, betalain pattern, and antioxidant properties of cactus pear (Opuntia spp.) clones. Journal of Agricultural and Food Chemistry, 53, 442–451. Strack, D., Vogt, T., & Schliemann, W. (2003). Recent advances in betalain research.

MA

Phytochemistry, 62, 247–269.

Tanaka, Y., Sasaki, N., & Ohmiya, A. (2008). Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids. The Plant Journal, 54, 733–749.

D

Tesoriere, L., Fazzari, M., Allegra, M., & Livrea, M. A. (2005). Biothiols, Taurine, and Lipid-

TE

Soluble Antioxidants in the Edible Pulp of Sicilian Cactus Pear (Opuntia ficus-indica) Fruits and Changes of Bioactive Juice Components upon Industrial Processing. Journal of Agricultural

CE P

and Food Chemistry, 53, 7851–7855.

Tomas-Lorente, F., Garcia-Viguera, C., Ferreres, F., & Tomas-Barberan, F. A. (1992). Phenolic compounds analysis in the determination of fruit jam genuineness. Journal of Agricultural and

AC

Food Chemistry, 40, 1800–1804.

Valente, L. M., Paixon, D., Nascimento, A. C., Santos, P. F., Scheinvar, L. A., Moura, M. R., Tinoco, A. C., Gomes, L. F., & Silva, J. F. (2010). Antiradical activity, nutritional potential and flavonoids of the cladodes of Opuntia monacantha (Cactaceae). Food Chemistry, 123, 1127– 1131. Verhagen, H., Aruoma, O. I., van Delft, J. H., Dragsted, L. O., Ferguson, L. R., Knasmüller, S., et al. (2003). The 10 basic requirements for a scientific paper reporting antioxidant, antimutagenic or anticarcinogenic potential of test substances in in vitro experiments and animal studies in vivo. Food and Chemical Toxicology, 5, 603–610. Vinha, A. F., Ferreres, F., Silva, B. M., Valentão, P., Goncalves, A., Pereira, J. A., et al. (2005). Phenolic profiles of Portuguese olive fruits (Olea europaea L.): Influences of cultivar and geographical origin. Food Chemistry, 89, 561–568.

ACCEPTED MANUSCRIPT Vinson, J. A., Su, X., Zubik, L., & Bose, P. (2001). Phenol antioxidant quantity and quality in foods: Fruits. Journal of Agricultural and Food Chemistry, 49, 5315–5321. Wijngaard, H. H., Röβle, C., & Brunton, N. (2009). A survey of Irish fruit and vegetable waste

T

and by-products as a source of polyphenolic antioxidants. Food Chemistry, 116, 202–207.

IP

Yeddes, N., Chérif, J. K., Guyot, S., Sotin, H., & Ayadi, M. T. (2013). Comparative study of

tunisian Opuntia forms. Antioxidants, 2, 37–51.

SC R

antioxidant power, polyphenols, flavonoids and betacyanins of the peel and pulp of three

Zhang, N., Pei, F., Wei, H., Zhang, T., Yang, C., Ma, G., et al. (2011). Isorhamnetin protects rat

NU

ventricular myocytes from ischemia and reperfusion injury. Experimental and Toxicologic

AC

CE P

TE

D

MA

Pathology, 63, 33–38.

ACCEPTED MANUSCRIPT

Plant part

Color of the cultivar

Origin

Code

1

pulp

red

Egypt

2

pulp

yelloworange

Egypt

3

pulp

red

Sicily

Pu-3

4

pulp

yelloworange

Sicily

Pu-4

5

pulp

greenishwhite

Sicily

Pu-5

6

pulp

red

South-Africa

Pu-6

7

pulp

yelloworange

South-Africa

Pu-7

8

peel

red

Egypt

Pe-1

9

peel

yelloworange

Egypt

Pe-2

10

peel

red

Sicily

Pe-3

11

peel

yelloworange

Sicily

Pe-4

T

Sample No.

IP

Table 1. Description and code of the cactus samples used in this study

SC R

NU

MA

D TE

Pu-2

12

AC

CE P

Pu-1

peel

greenishwhite

Sicily

Pe-5

13

peel

red

South-Africa

Pe-6

14

peel

yelloworange

South-Africa

Pe-7

15

cladode

red

Egypt

Cl-1

16

cladode

yelloworange

Egypt

Cl-2

ACCEPTED MANUSCRIPT Figure Legends

Figure 1. Generic structure of the common flavonols: quercetin (R1= OH; R2= H), isorhamnetin

IP

T

(R1= OCH3; R2= H), kaempferol (R1= H; R2= H), and myricetin (R1= OH; R2= OH).

SC R

Figure 2. A-F. Flavonol profiles of cactus Opuntia ficus-indica depending on plant part, location, and variety (HPLC-DAD, 370 nm). (A) peel from a red Egyptian cultivar – Pe 1; (B) peel from a red Sicilian cultivar – Pe 3; (C) peel from a red South African cultivar – Pe 6; (D) peel from a greenish-white Sicilian cultivar – Pe 5; (E) peel from a yellow-orange South African

NU

cultivar – Pe 7; (F) cladode from a red Egyptian cultivar – Cl 1; (1) isorhamnetin-3-O-

MA

rutinoside; (2) further isorhamnetin glycosides.

Figure 3 A-C. Contents of flavonol glycosides, flavonol aglycons and total phenolic compounds of cactus Opuntia ficus-indica depending on plant part, location, and variety (A) Flavonol

D

glycosides prior to hydrolysis (expressed as µg isorhamnetin-3-O-rutinoside/ 100 mg dw); (B)

TE

Flavonol aglycons after hydrolysis (expressed as µg isorhamnetin/ 100 mg dw); (C) Total

CE P

phenolic compounds (expressed as mg gallic acid/ 100 mg dw).

Figure 4. Deprotonated mass spectra and fragmentation patterns of isorhamnetin-3-O-

AC

rutinoside.

Figure 5. A-C. Antioxidant activity of cactus Opuntia ficus-indica depending on plant part, location, and variety. (A) TEAC assay (expressed as µmol Trolox /100 mg dw); (B) Degradation of the free radical Fremy's salt after reacting with cactus extracts for 15 minutes; (C) Degradation kinetics of the free radical Fremy's salt by a cactus pulp, a peel, and a cladode.

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Figure 1

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Figure 2

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Figure 3

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Figure 4

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Figure 5

ACCEPTED MANUSCRIPT Highlights: Flavonols in cactus Opuntia ficus-indica fruits and cladodes have been investigated.



Isorhamnetin derivatives have been found only in the fruits' peel and the cladodes.



Cactus fruits and cladodes from different cultivars and origins had similar flavonol

IP

T



profiles.

Flavonols in cactus might serve as a chemical fingerprint with regard to genuineness of

SC R



products.

Antioxidant activities of cactus fruits' peels and cladodes are high compared to fruits'

CE P

TE

D

MA

NU

pulps.

AC

