Journal of Food Composition and Analysis 23 (2010) 525–532

Original Article

Proximate composition, phenolic acids, and flavonoids characterization ofcommercial and wild nopal (Opuntia spp.)

Teresita Guevara-Figueroa a,b, Hugo Jimenez-Islas b, Marıa L. Reyes-Escogido a,d,Anne G. Mortensen c, Bente B. Laursen c, Li-Wei Lin a, Antonio De Leon-Rodrıguez a,Inge S. Fomsgaard c, Ana P. Barba de la Rosa a,*a Instituto Potosino de Investigacion Cientıfica y Tecnologica A.C. San Luis Potosı, San Luis Potosı, Mexicob Instituto Tecnologico de Celaya, Celaya, Guanajuato, Mexicoc Department of Integrated Pest Management, Faculty of Agricultural Sciences, Aarhus University, Aarhus, Denmarkd CIATEJ-Merida Yucatan, Mexico

A R T I C L E I N F O

Article history:

Received 1 December 2008

Received in revised form 19 November 2009

Accepted 9 December 2009

Keywords:

Cactus pear

Flavonoids

High plateau

Nopal

Nopalitos

Prickly pear

Phenolic acids

Phytochemicals

Protein profiles

Wild Opuntias

Arid zone plant resources

Biodiversity

Food analysis

Food composition

A B S T R A C T

The proximate composition, phenolic acid, and flavonoid characterization of two commercial and eight

wild Opuntia spp., as well two processed products, were analyzed. Commercial varieties were obtained

from a local market; wild varieties were collected from the Central and High Plateau zones of Mexico,

and Opuntia tablets were purchased at a natural commodities store. Spines from fresh cladodes were

removed and clean cladodes were freeze-dried and milled to obtain nopal powder. Differences in

proximate composition were determined: wild nopal blanco collected from the High Plateau had the

highest protein content, while tapon-II had the highest fiber content. Nopal tablets had low protein and

carbohydrate content but had the highest ash content. The wild morado, tempranillo, blanco, and

cristalino varieties had the highest total phenolic acid content, while the commercial varieties had the

highest total flavonoid contents. Very low amounts of flavonoids were found in nopal tablets. LC-MS/MS

was used to identify six phenolic acids (gallic, coumaric, 3,4-dihydroxybenzoic, 4-hydroxybenzoic,

ferulic, and salicylic acid), and only tapon-II contained all six. The five flavonoids (iso-quercitrin,

isorhamnetin-3-O-glucoside, nicotiflorin, rutin, and narcissin) were found in all varieties; nicotiflorin

was predominant, followed by narcissin.

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1. Introduction

The cactus pear (Opuntia spp.) is native to Mexico and welladapted to arid and semi-arid zones (Reynolds et al., 2003; Stintzingand Carle, 2005). About 1500 species of cactus produce edible stemsknown as pads, vegetable, phylloclades or cladodes; synonyms arenopales or pencas. Due to agricultural problems linked to increasingarid zones and declining water resources, Opuntia spp. is gainingimportance as an effective food source (Stintzing et al., 2001).Because of the high genetic diversity of Opuntia spp., a great numberof varieties can be grown worldwide. Its ecological advantages canbe attributed to crassulacean acid metabolism (CAM) that allowsCO2 uptake at night, thus minimizing water loss during photosyn-thesis. In Mexico, Opuntia spp. is cultivated on approximately 3

* Corresponding author. Tel.: +52 444 8342000.

E-mail address: apbarba@ipicyt.edu.mx (A.P. Barba de la Rosa).

0889-1575/$ – see front matter � 2010 Elsevier Inc. All rights reserved.

doi:10.1016/j.jfca.2009.12.003

million hectares; most commercial and wild species are located inthe Central and High Plateau zones (Flores, 2001; Reyes-Aguero etal., 2005). The cladodes are covered with spines and multicellularhairs or trichomes, which form the areole characteristic of theCactaceae family. The wild nopal may have small or large spines, andthe cladodes usually present different hues of green, although someare reddish or purple (Reyes-Aguero et al., 2005). The maincommercial variety is Opuntia ficus-indica, which has fewer spines(Vigueras and Portillo, 2001). The young cladodes, also known asnopalitos, are consumed as vegetables (Stintzing et al., 2001;Stintzing and Carle, 2005).

There is a global trend toward the use of natural phytochem-icals present in fruits, vegetables, oilseeds and herbs as antiox-idants or functional foods. Opuntia appears to be a good source ofthose compounds (Lee et al., 2002). Opuntia spp. is an importantfood and medicinal source; the cladodes and fruits are used fortreating arteriosclerosis, diabetes, gastritis and hypercholesterol-emia (Trejo-Gonzalez et al., 1996; Galati et al., 2003a; Decorde et

Table 1Physical characteristics of Opuntia spp.

No. Variety Speciesa Opuntia Size (cm) of cladode spines Locality of collection

1 Blanco C ficus-indica 16–17 0.2–0.5 Central

2 Manso C ficus-indica 16–17 0.2–0.5 Central

3 Amarillo W ficus-indica 12–14 0.2–0.5 H.P.

4 Blanco W ficus-indica 12 0.5 H.P.

5 Cristalino W ficus-indica 12–14 0.3 H.P.

6 Duraznillo W Leucotricha 12 0.5 H.P.

7 Morado W Robusta 11 0.5–1.0 H.P.

8 Tapon-I W Tapon 8–12 1–1.5 H.P.

9 Tapon-II W Tapon 9 1–1.5 H.P.

10 Tempranillo W – 12 0.5–1.0 H.P.

C = cultivated; W = wild;a Following description of Pimienta-Barrios et al. (2003). Central = Guanajuato State, Mexico, H.P. = Mexican High Plateau: San Luis Potosı and Zacatecas States.

T. Guevara-Figueroa et al. / Journal of Food Composition and Analysis 23 (2010) 525–532526

al., 2008). Most phytochemical studies of Opuntia are performedusing commercial raw materials and sometimes do not take intoconsideration the potential modification of the biological activityof nutrients by processing and agriculture (Hove et al., 2003;Decorde et al., 2008).

The purpose of the present study was to characterize theproximate composition of commercial and wild Opuntia spp. and todetermine the qualitative and quantitative phenolic acid andflavonoid profiles of fresh nopal in comparison to differentprocessed products.

2. Materials and methods

2.1. Plant material

The materials used for this study are listed in Table 1 and shownin Fig. 1A. Two different commercial (Blanco and Manso) nopalitoswere obtained from a local market in Guanajuato State (CentralMexico). Cladodes (nopales) from wild Opuntia spp. were collectedfrom Central Mexico (Guanajuato State) and High Plateau zones (SanLuis Potosi and Zacatecas States). Cladode collection was performedin the morning; and four to five cladodes were cut from differentplants. The spines of commercial and wild cladodes were removedmanually (Reyes-Aguero et al., 2005). The size of cladodes and spineswas measured from the base to the top. Clean cladodes were cut intosmall pieces and freeze-dried (Labconco Corp., MO, USA). Driedcladode samples were milled in a coffee grinder (Braun, Mexico) andthe powder was sieved through a size 80 mesh (Fig. 1B) and kept inplastic containers at 4 8C until use. Two commercial products wereincluded in this work: nopal tablets purchased at a naturalcommodities store and commercial nopal powder kindly donatedby the manufacturer (Valtierrilla, Guanajuato, Mexico). Bothproducts are obtained from commercial cladode varieties.

2.2. Proximate analysis

All determinations were done in a certified laboratory (ISO9000) following the AOAC (1980) official protocols. The Kjeldahlmethod was used for total nitrogen determination (AOAC method954.01) using a Kjeltec System. Protein was calculated from totalnitrogen using a factor of 6.25. The Soxhlet method (AOAC method920.39) was used for total fat determination. Total fat was obtainedfrom 6 h hexane extraction. Crude fiber was obtained after samplesdigestion with boiling diluted acid and alkali (AOAC method962.09). For protein, fat and crude fiber, the Tecator-Foss systemswere used (Foss, MD, USA). Moisture was determined from sampleweight loss after oven drying at 110 8C for 4 h (AOAC method925.09). Ash was calculated after heating the sample at 550 8C for2 h. Carbohydrates were determined by difference. All sampleswere analyzed in triplicate.

2.3. Protein extraction

Freeze-dried nopal powders were mixed (1:10) with a Tris–HCl0.1 M, pH 8.0 buffer containing 0.5 M NaCl, and the mixtures wereagitated for 2 h. After centrifugation for 4 8C at 10,000 � g for20 min, soluble proteins were precipitated with 4 volumes of cold-acetone containing 10% TCA, 1% PVPP and 2% 2-mercaptoethanol(2-ME). The suspension was sonicated (GE-505, UltrasonicProcessor, USA) for 15 min and centrifuged. The supernatantwas incubated at �20 8C for 2 h, and protein was pelleted bycentrifugation at 13,000 � g for 20 min at 4 8C (Huerta-Ocampo etal., 2009). Total protein was quantified using the BioAssay kit (Bio-Rad, Hercules CA, USA).

2.4. SDS-PAGE patterns

Protein pellets were resuspended in Laemmli buffer andproteins were separated in 15% SDS-PAGE at 20 mA/gel (Barbade la Rosa et al., 1992). The proteins were visualized with SyproRuby stain (Invitrogen, Carlsbad, CA) and photo-documented in aGel-Doc apparatus (Bio-Rad).

2.5. Alcohol extraction

Three grams of freeze-dried cladodes were extracted with47 mL of ethanol at room temperature for 48 h under mechanicalshaking. The mixture was filtered through a cheese-cloth andcentrifuged at 6500 � g for 10 min at 4 8C. The supernatant wasevaporated at 50 8C in a rotary vacuum evaporator. Residues wereresuspended in 1 mL of dimethyl sulfoxide (DMSO) (Tequida-Meneses et al., 2002).

2.6. Total phenol content

Total phenol content was quantified using Folin-Ciocalteureagent. Samples (20 mL) from the DMSO suspensions describedabove were mixed with 1 mL of Folin reagent (Sigma–Aldrich, MO,USA). After 8.5 min, 300 mL of 20% Na2CO3 were added, and thismixture was incubated at room temperature for 2 h. Theabsorbance was then measured at 765 nm with a spectrometer(Agilent 8453, Palo Alto, CA, USA). The results were expressed asmg of gallic acid equivalent (GAE)/g dry mass plant material(Luximon-Ramma et al., 2002).

2.7. Total flavonoid content

To determine the total flavonoid content, 0.75 mL samples ofthe DMSO suspension were mixed with an equal volume of 2%AlCl3�6H2O. The mixture was shaken and allowed to rest for10 min, and the absorbance was then measured at 365.7 nm. The

[(Fig._1)TD$FIG]

Fig. 1. (A) Cladodes of different Opuntia spp. varieties studied in this work: commercial varieties included 1 = blanco; 2 = manso, and wild varieties included 3 = blanco,

4 = amarillo, 5 = blanco, 6 = cristalino, 7 = duraznillo, 8 = morado, 9 = tapon-I, 10 = tapon-II, 11 = tempranillo. (B) Powders obtained from cladodes after freeze-drying.

T. Guevara-Figueroa et al. / Journal of Food Composition and Analysis 23 (2010) 525–532 527

total flavonoid content was expressed as mg of quercetinequivalent (QE)/g dry mass plant material (Tequida-Meneseset al., 2002).

2.8. Phenolic acids and flavonoids analysis

The extraction of phenolic acids and flavonoids wasperformed using the Accelerated Solvent Extraction 200 (ASE)system (DIONEX, Denmark) following the manufacturer’sprotocol. Flavonoids were extracted with 70% methanol (HPLCgrade, Rathburn Chemicals Ltd., Scotland), and phenolic acidswere extracted using 80% methanol containing 1% acetic acid(99–100% for HPLC, Baker, Holland). After a 30–40 min extrac-tion, extracts were transferred to amber flasks and stored at�20 8C until use. Samples were diluted with one volume ofwater for flavonoids and with chromatographic A-eluent for

phenolic acids. An Applied Biosystems MDS SCIEX API 3200liquid chromatography-ion trap quadropole mass spectrometer(LC-MS/MS) with turbo electrospray ionization in negativemultiple reaction monitoring (MRM) mode was used forchemical analysis of flavonoids. The chromatographic separationof the flavonoids was done using a Synergi 4 mm Polar column(2.0 mm � 250 mm). For the analysis of phenolic acids, an HP-1100 series liquid chromatography-mass spectrometer (LC/MS)with an atmospheric pressure ionization-electrospray ionizationchamber was used in negative single ion monitoring mode.Chromatographic separation was done using a Synergi 4 mmFusion-RP 80 A column. Flavonoid standards included rutin, iso-quercetin, nicotiflorin, narcissin and isorhamnetin-3-O-gluco-side, which were purchased from Extrasynthese (Lyon, France).Phenolic acid standards included 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, gallic acid, syringic acid and caffeic acid,

Table 2Phenolic acids analyzed and detected in commercial and wild Opuntia spp. Detected compounds are shown with an asterisk (*).

Short name Systematic name Structural formula CAS # Molar mass Detection

in LC/MS

Vanillic acid 4-Hydroxy-3-methoxybenzoic acid 121-34-6 168.15 Mass: 167;

Rt: 9.9 min

Ferulic acid* 3-(4-Hydroxy-3-methoxyphenyl)-2-propenoic acid 1135-24-6 194.19 Mass: 193;

Rt: 17.1 min

p-Coumaric acid* (E)-3(4-Hydroxyphenyl)-2-propenoic acid 501-98-4 164.16 Mass: 163;

Rt: 15.1 min

4-Hydroxybenzoic acid* 4-Hydroxybenzoic acid 99-96-7 138.12 Mass: 137;

Rt: 9.0 min

Syringic acid 4-Hydroxy-3,5-dimethoxybenzoic acid 530-57-4 198.175 Mass: 197;

Rt: 10.4 min

Protocatechuic acid 3,4-Dihydroxybenzoic acid 99-50-3 154.122 Mass: 153;

Rt: 6.7 min

Caffeic acid* 3,4-Dihydroxy-cinnamic acid 331-39-5 180.1598 Mass: 179;

Rt: 10.8 min

Salicylic acid* 2-Hydroxybenzoic acid 69-72-7 138.1226 Mass: 137;

Rt: 5.2 min

Gallic acid* 3,4,5-Trihydroxybenzoic acid 149-91-7 170.1214 Mass: 169;

Rt: 5.2 min

Synapic acid 3,5-Dimethoxy-4-hydroxycinnamic acid 530-59-6 224.2128 Mass: 223;

Rt: 21.9 min

T. Guevara-Figueroa et al. / Journal of Food Composition and Analysis 23 (2010) 525–532528

Table 3Flavonoids analyzed and detected in commercial and wild Opuntia spp.

Short name Systematic name Structural formula CAS # Molar mass Detection in LC-MS/MS

Rutin Quercetin-3-O-rutinoside 153-18-4 610.51 611.0/302.9; Rt: 7.3 min

Iso-quercitrin Quercetin-3-O-b-glucopyranoside 482-35-9 464.38 465.0/302.8; Rt: 10.4 min

Nicotiflorin Kaempferol-3-O-rutinoside 17650-84-9 594.53 595.1/286.9; Rt: 10.7 min

Narcissin Isorhamnetin-3-O-rutinoside 604-80-8 624.56 625.2/316.7; Rt: 11.1 min

Isorhamnetin-3-O-glucoside 5041-82-7 478.41 479.8/217.6; Rt: 16.0 min

T. Guevara-Figueroa et al. / Journal of Food Composition and Analysis 23 (2010) 525–532 529

which were purchased from Sigma–Aldrich and Fluka (Den-mark). The flavonoid and phenolic acid standards used are listedin Tables 2 and 3, respectively. More details regarding theanalytical method can be found elsewhere (Barba de la Rosa etal., 2009).

2.9. Statistical analysis

Assays were done in triplicate. Variance analysis and Tukey’stest with a confidence interval of 95% performed using GraphPadPrism 5 software (GraphPad Software, Inc.).

3. Results and discussion

The comparative morphological characteristics of commer-cial and wild Opuntia varieties analyzed in this work are shownin Table 1 and Fig. 1. In Mexico, different varieties of O. ficus-

indica have been traditionally known as amarillo, blanco andmorado, among other names based on their morphologicalcharacteristics (Pimienta-Barrios et al., 2003; Reyes-Aguero etal., 2005).

3.1. Proximate analysis

The proximate composition of commercial and wild nopales aswell as that of nopal products is shown in Table 4. Wild nopalblanco had the highest protein content (19.0% of dry weight). Thetablets purchased in the natural commodities market had thelowest protein content (4.2%). Among the varieties analyzed,tapon-II and cristalino showed the highest fat concentrations (1.8and 1.5%, respectively). No fat was detected in nopal tablets, butthey had the highest fiber and ash content at 51.6 and 37.6%,respectively. Among cladodes, differences were found in carbohy-drate content, where the highest value was found in morado(80.9%) ant the lowest value in tapon-II (42.4%). The tablets onlycontained 6.7% carbohydrates; this suggests that thermal proces-sing may have resulted in the observed high fiber content (Martin-Cabrejas et al., 2006). Differences in proximate composition amongcultivated and wild varieties might have been due to differences insoils and climatic environment (Rodrıguez-Felix, 2002; Stintzingand Carle, 2005; Rodrıguez-Garcıa et al., 2007). However, nodefinitive conclusions can be reached since our intent was not tocompare varieties, and future work should include information for

Table 4Proximate composition of different commercial and wild Opuntia spp.a.

No. Variety Component (%)

Protein Lipids Fiber Ash Carbohydrates

1 Blanco C 6.7j 0.1d 15.0c 17.3b 61.4c

2 Manso C 16.0c 0.1d 10.8d 18.8b 55.1g

3 Amarillo W 15.1e 0.6c 6.2h 14.9c 63.2c

4 Blanco W 19.0a ND ND 18.3b 62.7c,d

5 Cristalino W 9.4h 1.5a,b 7.7g 14.8c 66.5b

6 Duraznillo W 13.5g 1.1a 7.1f 19.7b 69.8f

7 Morado W 13.9f ND ND 5.2d 80.9a

8 Tapon-I W 15.1d ND ND 18.9b 66.0b

9 Tapon-II W 17.4b 1.8a 20.4b 19.5b 42.4h

10 Tempranillo W 13.4g ND 5.5i 19.3b 61.9d.e

11 Tablets P 4.2k 0 51.6a 37.6a 6.7i

a As dry weight; C = commercial varieties; W = wild varieties; P = commercial product purchased at the store; ND = not determined. Means of triplicates in the same column

with different letters are significantly different at P<0.05.

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certified reference and/or quality control materials (Phillips et al.,2007).

3.2. Protein profiles

Protein extraction is more difficult from cladode samples, butwe have developed a methodology to allow us to obtain cleanprotein samples (Huerta-Ocampo et al., 2009). The electrophoreticpatterns of soluble proteins obtained from wild Opuntias areshown in Fig. 2. The tempranillo variety showed the mostdifferences among all wild varieties, with a main band atapproximately 18 kDa (Fig. 2, lane 9). The band observed around50 kDa in all varieties may correspond to the Rubisco large subunitreported for other plants (Saravanan and Rose, 2004). Opuntia spp.plants could be a source of novel proteins; enzymes asarylamidases, lipases, proteinases and glucosidases have beenfound in plant extracts of the Cacteaceae family. A new plantprotease extracted from O. ficus-indica fruit has been reported tohave milk-clotting activity (Teixeira et al., 2008).

3.3. Total phenolic and flavonoid content

The total phenolic acid and flavonoid contents in the cladodes ofdifferent Opuntia varieties are shown in Fig. 3. A higherconcentration of total phenols (black bars) was found in the wildmorado variety collected from the Mexican High Plateau(19.9 mgGAE/g), followed by tempranillo (19.4 mgGAE/g), blancoand cristalino (17.8 mgGAE/g). Differences in total phenol content[(Fig._2)TD$FIG]

Fig. 2. SDS-PAGE patterns of different wild Opuntias spp. collected from the Mexican

Central and High Plateau zones: 1 = molecular weight standard, 2 = amarillo,

3 = blanco, 4 = cristalino, 5 = duraznillo, 6 = morado, 7 = tapon-I, 8 = tapon-II,

9 = tempranillo.

among the varieties analyzed could arise from different climaticconditions (Reyes-Aguero et al., 2005). The tapon varieties andnopal powders had the lowest phenolic content, with values lessthan 2 mgGAE/g (Fig. 3). The blanco and manso commercialvarieties had total phenol contents of 5.25 and 11.7 mgGAE/g, butthe commercial powders only contained 1.7 mg; it is well knownthat inappropriate air temperature during cladode drying results inthe loss of total phenolic acids (Hove et al., 2003), thus attentionmust be taken on drying procedure. Total phenol values have beenreported as 8–9 mg/100 g fresh weight and 180.3 mg/g (Lee et al.,2003), these differences might have arisen from differentmethodologies and Opuntia samples used in different studies.

Flavonoids were found in smaller amounts than phenolic acids(Fig. 3, grey bars). In contrast to the pattern observed for totalphenolic acids, the blanco and manso commercial varieties had thehighest flavonoid content at 9.8 and 5.9 mgQE/g, respectively.Nopal tablets had very low flavonoid content (0.5 mgQE/g). Whencompared to fresh cladodes, nopal powders had lower flavonoidcontent (3.1 mgQE/g); it has also been reported that temperaturetends to increase the degradation of flavonoids (Runha et al., 2001).

3.4. Characterization of phenolic acids and flavonoids

LC-MS/MS was used to identify six phenolic acids (Table 5);ferulic acid and salicylic acid (SA) were found in all varieties.Tapon-I and tapon-II had the highest levels of ferulic acid at 347.7and 226.2 mg/g dry weights, respectively. Ferulic acid has beenassociated with anti-inflammatory, anti-diabetic and neuropro-tective properties, and has been reported to be a potent antioxidantthat prevents cellular damage against UV (Srinivasan et al., 2007).The blanco, amarillo and cristalino varieties had the highest levels[(Fig._3)TD$FIG]

Fig. 3. Total phenolic acid (mgGAE/g) and flavonoid (mgQE/g) content of samples

from Fig. 1. Commercial varieties 1 = blanco; 2 = manso; and wild varieties

3 = amarillo, 4 = blanco, 5 = cristalino, 6 = duraznillo, 7 = morado, 8 = tapon-I,

9 = tapon-II, 10 = tempranillo, 11 = commercial nopal powder, 12 = commercial

tablets. C = commercial; W = wild, P = nopal products.

Table 5Quantification of phenolic acids (mg/g).a.

No. Variety Gallic Coumaric 3,4-Dihydroxy-benzoic 4-Hydroxy benzoic Ferulic Salicylic

1 Blanco C ND ND ND 5.0e 5.6e 6.0d

2 Manso C ND ND 2.7e 19.6c 15.3e 7.8d

3 Amarillo W ND ND 0.6e 21.4b,c 43.2c 29.6a,b

4 Blanco W ND ND 3.6e 31.9b 27.4d 35.4a

6 Cristalino W 6.4c ND 25.1b 18.1c 27.3d 27.9b

6 Duraznillo W 12.6b ND 6.3d,e ND 7.7e 5.8d

7 Morado W ND ND 18.9c 14.6c,d 28.9d 16.3c

8 Tapon-I W ND 161.8a 14.7c,d 47.2a 347.7a 11.9c,d

9 Tapon-II W 23.7a 140.8b 24.2b,c 16.1c,e 226.2b 15.3c

10 Tempranillo W ND ND 10.0d 8.6c,e 23.2d,e 14.6c

11 Powder P 6.9c ND 50.2a 23.1b,c 31.6d 5.9d

a As dry weight; C = commercial varieties; W = wild varieties; P = commercial nopal powder; ND = not detected. Means of triplicates in the same column with different

letters are significantly different at P<0.05.

Table 6Quantification of flavonoids (mg/g).a.

No. Variety Iso-quercitrin Isorharmnetin, 3-O-glucoside Nicotiflorin Rutin Narcissin

1 Blanco C 22.9f,g 99.3b 1465.0a 53.0e 762.2b,c

2 Manso C 71.7f,e 112.4b 463.7d 36.2e,f 384.1e

3 Amarillo W 396.7a 322.1a 1173.0b 261.7a 1371.0a

4 Blanco W 267.8c 127.6b 317.7e 168.9c 567.1d

5 Cristalino W 343.6b 268.8a 1108.0b 147.0d 571.4d

6 Duraznillo W 50.0f 45.9b 28.9f 23.6f 220.1f

7 Morado W 106.0e 99.3b 910.7c 140.1d 555.9d

8 Tapon-I W 107.7e 96.6b 97.1f 55.8e 146.9f

9 Tapon-II W 158.7d,e 71.9b 84.9f 58.5e 197.1f

10 Tempranillo W 139.2e 56.7b 821.5c 193.9b 699.1c

11 Powder P 33.7f,g 98.3b 300.3e 129.0d 860.2b,c

a As dry weight; C = commercial varieties; W = wild varieties; P = commercial nopal powder. Means of triplicates in the same column with different letters are significantly

different at P<0.05.

T. Guevara-Figueroa et al. / Journal of Food Composition and Analysis 23 (2010) 525–532 531

of salicylic acid (35.4, 29.6 and 27.9 mg/g, respectively). There isconsiderable interest in salicylic acid and its precursors withregard to their functions in plants, particularly in the use of 4-HBAas a polyester precursor and 3,4-DHBA (3,4-dihydroxybenzoicacid) as an antioxidant (Dong et al., 2001). 3,4-DHBA was found inall varieties with the exception of commercial blanco; nopalpowder had the highest value at 50.2 mg/g. 4-Hydroxybenzoic acidwas also found in all varieties with the exception of duraznillo.Gallic acid was found only in three wild varieties (cristalino,duraznillo and tapon-II), but was found in the commercialpowders. Coumaric acid only was found in purple varieties suchas tapon-I and tapon-II at 161.8 and 140.8 mg/g, respectively.

Gallic acid at concentrations of 0–50 mM (0–12 h) inhibited thegrowth of 3T3-L1 pre-adipocytes and the induced apoptosis inthese cells, suggesting that phenolic compounds could form thebasis for antiobesity drugs (Ross et al., 2000; Hsu and Yen, 2006,2008; Hsu et al., 2006).

In contrast to the pattern observed for phenolic acids,flavonoids were found in all Opuntia varieties analyzed (Table6). All flavonoids detected were found at higher concentrations inthe amarillo variety, with the exception of nicotiflorin, which wasfound at 1465 mg/g in commercial blanco followed by 1173 mg/g inamarillo. Most report of flavonoids in Opuntia provide qualitativebut not quantitative analysis (Qiu et al., 2002; Lee et al., 2003;Stintzing and Carle, 2005; Muhammad et al., 2006). Galati et al.(2003a) determined in pear prickly the ascorbic acid, totalpolyphenols and flavonoids content, and isorhamnetin derivatives.Kutti (2003) found flavonoids, ascorbic acid and carotenoids inprickly pear while Tesoriere et al. (2005) studied biothiols,flavonols, tocopherols and carotenoids.

Flavonoids of Opuntia ficus-indica var. saboten were found mainlyin the glycosidic form particularly as isorhamnetin glycosides(54.6 mg/mL) (Galati et al., 2003b). Small amounts of isorhamnetin-3-glucoside, rutin and kaempferol-3-rutinoside were also detected;

these flavonoids exhibit neuroprotective actions, and quercitrin 3-methylether appears to be the most potent (Dok-Go et al., 2003).Berger et al. (2007) reported that green pads contain 16.6 mg/gquercitrin and purple pads contain 4.2 mg/g; these values werelower than those found in this work (Table 6). A concentration of3.3 mg/g isorhamnetin-3-O-glucoside has been reported for bothgreen and purple pads; in this work we found concentrationsranging from 45.9 to 322.1 mg/g. The flavonoids quercitrin andkaempferol have been shown to suppress stress in cardiomyocytes(Psotova et al., 2004). It has been reported that Amaranthus spinosus

Linn. leaves showed anti-diabetic, antinflammatory and anticarci-nogenic activity, effects attributed to rutin (Suryavanshi et al., 2007).

4. Conclusion

Differences in the proximate composition, phenolic acids andflavonoids composition of different Opuntia spp. were found.Protein, total phenolic acids and flavonoids were found at verylow amounts in nopal products; this could be the result ofmanipulations occurring during processing. Phenolic acids andflavonoids in Opuntia cladodes have been only partially identifiedand have not often been quantitatively determined. For the firsttime, we have qualitatively and quantitatively analyzed the phenolicacids and flavonoids of different varieties of commercial and wildOpuntia spp. These results could help the future selection of varietiesfor improvement of nutritional and nutraceutical nopal character-istics. There is a clear need for certified reference materials forflavonoids and phenolic acids in natural plant foods, in order to allowvalidation of methods and comparison of results between studies.

Acknowledgments

This work was supported by FOMIX-GTO, Contract No. GTO-2006-C01-31809. T. Guevara thanks the MsSc 204171 and

T. Guevara-Figueroa et al. / Journal of Food Composition and Analysis 23 (2010) 525–532532

mix-204171 fellowships from CONACyT-Mexico and Lin LW thankthe IPICyT fellowship. We also gratefully thank Stine Krogh-Steffensen, Carsten Christophersen and all the workers at theFlakkebjerg Research Center, and Marıa del Pilar Barba forproximate analysis.

References

AOAC, 1980. Association of Official Analytical Chemists Official Methods of Analysis.Chapter 45, pp. 4–5.

Barba de la Rosa, A.P., Fomsgaard, I.S., Laursen, B., Mortensen, A.G., Olvera-Martınez,L., Silva-Sanchez, C., Mendoza-Herrera, A., De Leon-Rodrıguez, A., GonzalezCastaneda, J., 2009. Amaranth (Amaranthus hypochondriacus) as an alternativecrop for sustainable food production: phenolic acids and flavonoids withpotential impact on its nutraceutical quality. Journal of Cereal Science 42,117–121.

Barba de la Rosa, A.P., Gueguen, J., Paredes-Lopez, O., Viroben, G., 1992. Fraction-ation procedures, electrophoretic characterization and amino acid compositionof amaranth seed proteins. Journal of Agricultural and Food Chemistry 40, 931–936.

Berger, J.M, Itagaki, Y., Nakanishi, K., 2007. The effect of ultraviolet-depleted light onthe flavonol contents of the cactus species Opuntia wilcoxii and Opuntia violacea.Chemistry and Biodiversity 4, 1525–1532.

Decorde, K., Teissedre, P.L., Auger, C., Cristol, J.P., Rouanet, J.M., 2008. Phenolics frompurple grape, apple, purple grape juice and apple juice prevent early athero-sclerosis induced by an aterogenic diet in hamsters. Molecular Nutrition andFood Research 52, 400–407.

Dok-Go, H., Lee, K.H., Kim, H.J., Lee, E.H., Lee, J., Song, Y.S., Lee, Y.-H., Jin, Ch., Lee, Y.S.,Cho, J., 2003. Neuroprotective effects of antioxidative flavonoids, quercetin, (+)-dihydroquercetin and quercetin 3-methyl ether, isolated from Opuntia ficus-indica var. saboten. Brain Research 965, 130–136.

Dong, D., Jiang, Sh., Ni, Y., Jiang, B., 2001. Synthesis and properties of thermotropiccopolyesters of p-hydroxybenzoic acid. European Polymers Journal 37, 611–617.

Flores, C., 2001. Produccion, industrializacion y comercializacion de nopalitos. In:Centro de Investigaciones economicas, sociales y tecnologicas de la Agroindus-tria y la Agricultura Mundial, Universidad Autonoma de Chapingo, Chapingo,Mexico, p. 27.

Galati, E.M., Tripodo, M.M., Trovato, A., d’Aquino, A., Monforte, M.T., 2003a. Biologi-cal activity of Opuntia ficus-indica cladodes II: effect on experimental hyper-cholesterolemia in rats. Pharmaceutical Biology 41, 175–179.

Galati, E.M., Mondello, M.R., Giuffrida, D., Dugo, G., Miceli, N., Pergolizzi, S., Taviano,M.F., 2003b. Chemical characterization and biological effects of Sicilian Opuntiaficus indica (L.) Mill. fruit juice: antioxidant and antiulcerogenic activity. Journalof Agricultural and Food Chemistry 51, 4903–4908.

Hove, L., Ndlovu, L.R., Sibanda, S., 2003. The effects of drying temperature onchemical composition and nutritive value of some tropical fodder shrubs.Agroforestry Systems 59, 231–241.

Hsu, C.L., Huang, S.L., Yen, G.C., 2006. Inhibitory effect of phenolic acids onproliferation of 3T3-L1 pre-adipocytes in relation to their antioxidant activity.Journal of Agricultural and Food Chemistry 54, 4191–4197.

Hsu, C.L., Yen, G.C., 2006. Induction of cell apoptosis in 3T3-L1 pre-adipocytes byflavonoids is associated with their antioxidant activity. Molecular Nutrition andFood Research 50, 1072–1079.

Hsu, C., Yen, G.C., 2008. Phenolic compounds: evidence for inhibitory effects againstobesity and their underlying molecular signaling mechanisms. Molecular Nu-trition and Food Research 53, 53–61.

Huerta-Ocampo, J.A., Briones-Cerecero, E.K., Mendoza-Hernandez, G., De Leon-Rodrıguez, A., Barba de la Rosa, A.P., 2009. Proteomic analysis of amaranth(Amaranthus hypochondriacus L.) leaves under drought stress. InternationalJournal of Plant Science 170, 990–998.

Kutti, J.O., 2003. Antioxidant compounds from four Opuntia cactus pear fruitvarieties. Food Chemistry 85, 527–533.

Lee, J.C., Kim, H.R., Kim, J., Jang, Y.S., 2002. Antioxidant property of an ethanol extractof the stem of Opuntia ficus-indica var. saboten. Journal of Agricultural and FoodChemistry 50, 6490–6496.

Lee, E.H., Hyoung, J.K., Song, Y.S., Changbae, J., Kyung-Tae, L., Cho, J., Lee, Y.S., 2003.Constituents of the stems and fruits of Opuntia ficus-indica var. saboten.Archives Pharmaceutical Research 26, 1018–1023.

Luximon-Ramma, A., Bahorun, T., Soobrattee, M.A., Aruoma, O.I., 2002. Antioxidantactivities of phenolic, proanthocyanidin, and flavonoid components in extractsof Cassia fistula. Journal of Agricultural and Food Chemistry 50, 5042–5047.

Martin-Cabrejas, M.A, Aguilera, Y., Benitez, V., Molla, E., Lopez-Andreu, F.J., Esteban,R.M., 2006. Effect of industrial dehydration on the soluble carbohydrates anddietary fiber fractions in legumes. Journal of Agricultural and Food Chemistry54, 7652–7657.

Muhammad, S., Hyoung, J.K., Chang, K.H., Changbae, J., Yong, S.L., 2006. Secondarymetabolites from Opuntia ficus-indica var. saboten. Phytochemistry 67, 1390–1394.

Pimienta-Barrios, E., Loza-Cornejo, S., Gonzalez del Castillo-Aranda, M.E., 2003.Caracterizacion anatomica y conductividad hidraulica teorica de los elementosde vaso de variedades silvestres y cultivadas de nopal tunero (Opuntia). ActaBotanica Mexicana 62, 21–30.

Phillips, K.M., Wolf, W.R., Patterson, K.Y., Sharpless, K.E., Holden, J.M., 2007. Refer-ence materials to evaluate measurement systems for the nutrient compositionof foods: results from USDA’s National Food and Nutrient Analysis Program(NFNAP). Analytical and Bioanalytical Chemistry 389, 219–229.

Psotova, J., Chlopcikova, S., Miketova, P., Hrac, J., Simanek, V., 2004. Chemoprotec-tive effect of plant phenolics against anthracycline-induced toxicity on ratcardiomyocytes. Part II. Apigenin, baicalelin, kaempherol, luteolin and querce-tin. Phytotherapy Research 18, 516–521.

Qiu, Y., Chen, Y., Pei, Y., Mastsuda, H., Yoshikawa, M., 2002. Constituents with radicalscavenging effect from Opuntia dillenii: Structures of new a-Pyrones andflavonol glycoside. Chemistry and Pharmacology Bulletin 50, 1507–1510.

Reyes-Aguero, J.A., Aguirre-Rivera, J.R., Hernandez, H.M., 2005. Systematic notesand a detailed description of Opuntia ficus-indica (L.) Mill. (CACTACEAE). Agro-ciencia 39, 395–408.

Reynolds, S.G., Arias-Jimenez, E., Mondragon-Jacobo, C., Perez-Gonzalez, S., 2003. Elnopal (Opuntia spp.) como forraje, Estudio FAO Produccion y Proteccion Vegetal169. Rome, pp. 344–345 (ISSN 1014-1227, ISBN 92-5r-r304705-4).

Rodrıguez-Felix, A., 2002. Postharvest physiology and technology of cactus pearfruits and cactus leaves. Acta Horticultura 581, 191–199.

Rodrıguez-Garcıa, M.E., de Lira, C., Hernandez-Becerra, E., Cornejo-Villegas, M.A.,Palacios-Fonseca, A.J., Rojas-Molina, I., Reynoso, R., Quintero, L.C., Del-Real, A.,Zepeda, T.A., Munoz-Torres, C., 2007. Physicochemical characterization of nopalpads (Opuntia ficus indica) and dry vacuum nopal powders as a function of thematuration. Plant Foods for Human Nutrition 62, 107–112.

Ross, S.E., Hemati, N., Longo, K.A., Bennett, C.N., Lucas, P.C., Erickson, R.L., MacDou-gald, O.A., 2000. Inhibition of adipogenesis by Wnt signaling. Science 289, 950–953.

Runha, F.P., Cordeiro, D.S., Pereira, C.A.M., Vilegas, J., Oliveria, W.P., 2001. Productionof dry extracts of medicinal Brazilian plants by spouted bed process: develop-ment of the process and evaluation of thermal degradation during the dryingoperation. Food Bioproducts and Processing 79, 160–168.

Saravanan, R.S., Rose, J.K.C., 2004. A critical evaluation of simple extraction tech-niques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics4, 2522–2532.

Srinivasan, M., Sudheer, A.R., Menon, V.P., 2007. Ferulic acid: therapeutic potentialthrough its antioxidant property. Journal of Clinical and Biochemical Nutrition40, 92–100.

Stintzing, F.C., Carle, R., 2005. Cactus stems (Opuntia spp.): a review on thechemistry, technology, and uses. Molecular Nutrition and Food Research 49,175–194.

Stintzing, F.C., Schieber, A., Carle, R., 2001. Phytochemical and nutritional signifi-cance of cactus pear. European Food Research and Technology 212, 396–407.

Suryavanshi, V.L., Sathe, P.A., Baing, M.M., Singh, G.R., Lakshmi, S.N., 2007. Deter-mination of rutin in Amaranthus spinosus Linn. whole plant powder by HPLC.Chromatographia 65, 767–769.

Teixeira, G., Santa, A.R., Pais, M.S., Clemente, A., 2008. Enzymes of Opuntia ficus-indica (L.) Miller with potential industrial applications I. Applied Biochemistryand Biotechnology 8, 299–312.

Tequida-Meneses, M., Cortez-Rocha, M., Rosas-Burgos, E.C., Lopez Sandoval, S.,Corrales-Maldonado, C., 2002. Efecto de extractos alcoholicos de plantas sil-vestres sobre la inhibicion de crecimiento de Aspergillus flavus, Aspergillus nıger,Penicillium chrysogenum, Penicillum expansum, Fusarium moniliforme, y Fussarionpoae. Revista Iberoamericana de Micologıa 19, 84–88.

Tesoriere, L., Fazzari, M., Allegra, M., Livrea, M.A., 2005. Biothiols, taurine, and lipid-soluble antioxidants in the edible pulp of Sicilian cactus pear (Opuntia ficusindica) fruits and changes of bioactive juice components upon industrialprocessing. Journal of Agricultural and Food Chemistry 53, 7851–7855.

Trejo-Gonzalez, A, Ortiz, G., Puebla-Perez, A.M., Mejıa-Arreguın, S., Calva, E., 1996. Apurified extract from prickly pear cactus (Opuntia fuliginosa) controls experi-mentally induced diabetes in rats. Journal of Ethnopharmacology 55, 27–33.

Vigueras, A.L., Portillo, L., 2001. Uses of Opuntia species and the potential impact ofCactoblastis cactorum (Lepidoptera: Pyralidae) in Mexico. Florida Enthomolo-gists 84, 493–498.