Plant Foods Hum Nutr (2009) 64:146152 DOI 10.

1007/s11130-009-0117-0

ORIGINAL PAPER

Phenolic Composition, Antioxidant Capacity and In Vitro Cancer Cell Cytotoxicity of Nine Prickly Pear (Opuntia spp.) Juices
R. A. Chavez-Santoscoy & J. A. Gutierrez-Uribe & S. O. Serna-Saldvar

Published online: 26 May 2009 # Springer Science + Business Media, LLC 2009

Abstract Juices of nine prickly pears (Opuntia spp.) were characterized in terms of color, acidity, sugar content, phenolics, flavonoids, betalains and antioxidant activity and tested in vitro against four cancer cell lines. The juices had pHs, acidities and sugar ranging from 4.27 to 5.46, 0.03 to 0.27% and 8 to 14.7Brix, respectively. Juices also varied in color from white to purple and contained total phenolics, flavonoids, betaxanthins, betacyanins and antioxidant capacity ranging from 22 to 226g gallic acid eq/g, 95 to 374g quercetin eq/g, 3 to 189g/g, 1.6 to 300g/g and 17 to 25 micromoles Trolox eq./mL, respectively. Among the cancer lines tested, viability of prostate and colon cells were the most affected. Moradillo contained the highest flavonoids and diminished both prostate and colon cancer cell viability without affecting mammary or hepatic cancer cells. Rastrero reduced the growth of the four cancer cell lines without affecting normal fibroblast viability. The research shows intervarietal differences among prickly pears in terms of juice properties and phytochemicals that could prevent oxidative stress and cancer. Keywords Prickly pear . Flavonoids . Betalains . Antioxidant activity . Cancer cells

Introduction Mexico possesses approximately 90% of the world distribution of prickly pears and has been the most important
R. A. Chavez-Santoscoy : J. A. Gutierrez-Uribe : S. O. Serna-Saldvar (*) Departamento de Biotecnologa e Ingeniera de Alimentos, Tecnolgico de Monterrey, Av. Eugenio Garza Sada 2501 Sur, CP 64849 Monterrey, NL, Mxico e-mail: sserna@itesm.mx

producer with 79.4% of the total globe production (SAGARPA 2001 [1]). The Aztecs and other Mesoamerican civilizations used the cactus pads as a vegetable and the prickly pear as a seasonal fruit. The interest for these cactus derived foods has increased nationally and internationally due to their potential nutraceutical effects [26]. The cactus plant provides pads rich in soluble and insoluble dietary fibers and prickly pears rich in phenolics including betalains. These compounds are known to combat oxidative stress and chronic diseases [7, 8]. As a result, the economic and social impact of the cultivation of cactus, especially in arid and marginal areas has increased in Mexico [9]. According to Gurrieri et al. [3], the juice of Sicilian cultivars of prickly pear had a slightly acidic pH (5.1), an acidity of approximately 0.02% and a sugar composition of 6% glucose and 5 to 6% fructose. Tesoriere et al. [6, 10] found that the pulp of prickly pears contained phenolics and other antioxidants such as biothiols and concluded that they had a positive effect in the Redox balance of humans mainly due to reduced LDL hydroperoxides levels. The nutraceutical benefits have been attributed to the synergistic effects of betalains and flavonoids [11, 12]. Flavonoids, such as Kaempferol-3-O-rutinoside; isorhamnetin-3-O-rutinoside and isorhamnetin-3-O-glucoside have been identified in juices extracted by pressing whole prickly pears of Opuntia ficus indica [11]. Quercetin was the predominant flavonol, quantified as aglycone [13]. The different types of phenolics have been associated with color and sensory attributes [14]. The two main betalain pigments are the purple-red betanin and the unique yellow indicaxanthin [5]. Betalains are mainly responsible of the purple, red and orange colorations. Since high antioxidant capacity is a desired feature for fruits, the aim of this study was to screen the antioxidant capacity and chemical and phenolic composition of nine different types of prickly pears juices and

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determine their in vitro anticancer effect against hepatic, colon, prostate and hormone-dependent mammary cells.

Materials and Methods Prickly Pears Prickly pears from nine different types of cactus species (Table 1) were harvested at a place named El Peuelo in west Nuevo Leon, Mexico located at a Latitude of 24 56 21, longitude of 100 77 27 and an altitude of 1830 m above sea level. Almost all the species are indigenous to this region except Gavia and Pelon. The plants are grown without any agronomical inputs and fruits harvested during the months of August and September. Prickly pears were collected by a native experienced picker during these months of year 2007. The fruits were weighed and the peel and pulp manually removed as described by Stintzing et al. [12]. The pulp was homogenized and filtrated in a 20mesh filter, in order to separate the juice from the seeds and other insolubles. The resulting juices were preserved under frozen storage (85C) until analyses. Determination of Brix, pH, Acidity and Color Brix values were measured in the juices with a manual refractometer (ATAGO Model N-1). pH values were

determined with a pH-meter (Beckman model 50) previously calibrated with standards pH 7.00 and 4.01 buffers. Juice acidity, expressed as citric acid equivalents, was assayed following the AOAC procedure 942.15 based on titrating the juice with NaOH to a pH of 8.2 using phenolphthalein indicator [15]. Juice color was determined with a Minolta Color Meter (Model CR-300, Minolta Co., Ltd. Osaka, Japan). L, a, and b were obtained and hue and color index E determined by the following equations: hue = TAN1(b/ a), E = (L2 + a2 + b2)1/2, respectively. Determination of Total Phenolics, Flavonoids and Betalains The total phenolic content of juices was determined using the method described by Vinson et al. [16]. First, the appropriate juice dilutions were oxidized with FolinCiocalteu reagent and the absorbance of the resulting blue color was measured at 760 nm in a microplate reader (Synergy HT, Bio-Tek, Winooski, VM). Gallic acid was used as the standard and total phenols expressed as mg gallic acid equivalent/L juice. Total flavonoids, expressed as mg quercetin eq./L juice, were determined by the colorimetric method described by Zhishen et al. [17] whereas betalains by the assay described by Stintzing et al. (2005 [12]). The extinction coefficient of betain and indicaxanthin were obtained from Trezzini and Zrd [18] and Wyler and Meuer [19], respectively. All determinations were done by triplicate.

Table 1 Classification, pulp yield, acidity and sugar contents of juices extracted from nine Mexican prickly pearsa Scientific Name/Variety Fruit Characteristics Pulp Yieldb % Juice pH Acidity % citric ac. Brix Brix/ Acidity 163.714.0 222.28.0 266.47.5 470.00.0 431.012.0 41.718.5 33.51.7 64.815.7 67.35.5

Opuntia robusta Gavia Opuntia streptacantha Cardon Opuntia robusta Amarillo Opuntia ficus indica Peln Opuntia violaceae Moradillo Opuntia rastrera Rastrero Opuntia leucotricha Duraznillo Blanco Opuntia leucotricha Duraznillo Rojo Opuntia robusta Taponc
a

Spiny, green peel, green pulp. Spiny, red peel, purple pulp. Spiny, yellow peel, greenyellow pulp. Spiny, orange peel, orangered pulp. Spiny, purple peel, purplered pulp. Spineless, purple peel and pulp. Spineless, light green peel, white pulp. Spineless, red peel, purple pulp. Spineless, purple-red peel and pulp.

59.44 41.53 58.47 62.84 46.50 66.28 56.52 52.54 79.04

4.850.01 5.100.00 4.940.01 5.460.00 5.440.00 4.940.00 4.270.00 4.580.01 5.330.00

0.090.01bc 0.060.03bc 0.050.00c 0.030.01c 0.030.01c 0.220.06a 0.270.06a 0.130.03b 0.120.02b

14.730.14a 13.330.24b 13.320.15b 14.100.10b 12.930.12b 9.181.11c 9.050.10c 8.420.47c 8.080.11c

Each value is the average of at least 3 observations. Means SEM with different letters within column are significant different (P<0.05), b The pulp yield was calculated after peeling at least one kilogram of each prickly pear, c It was difficult to differentiate pulp and peel tissues of prickly pears from Tapon. The removal of the pulp was performed with a knife. Therefore, the pulp yield was probably overestimated and juice contained peel tissue.

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Determination of Antioxidant Capacity The antioxidant capacity (ORAC) was determined according to the method described by Huang et al. [20]. Analyses were performed at 37C using a pH 7.4 phosphate buffer. The peroxide radicals were produced by 2,2-Azobis(2amidinopropane) dihydrochloride (AAPH), using fluorescein as substrate and Trolox as standard. Fluorescence was measured every 2 min during one hour and a calibration curve of Trolox at different concentrations (10 to 100M) was used in each plate read. Determination of In Vitro Cancer Cell Viability Four different mammalian cancer cell lines were used: mammary (MCF-7), prostate (PC3), colon (Caco2) and hepatic (HepG2). Normal fibroblast (NIH 3 T3) were used as control. Cells were maintained in DMEM-F12 medium containing 10% FBS (Fetal Bovine Serum) (Gibco, Grand Island, NY) and grown in an incubator (Fisher 1168751, Pittsburg, PA) set at 37C, 80% relative humidity and 5% CO2. Plates of 96-wells were prepared with 100l of a suspension containing 5 x 105 cells/ml of cancer cells at least 12 h before adding the various sorts of juices. Filtrated juice of each prickly pear was diluted 1:100 with DMEM-F12 medium containing 5% FBS, sterilized with 0.2m Nylon sterile filter and 100l were added to each well in the cell giving a final concentration of 0.5% of juice in the cell growth media. The background absorbance of the diluted juice in the cell growth media without cells was used as control for each sample. After 48 h incubation, 20l of CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) was used to determine viability. Absorbance was measured at 490 nm in a microplate reader (Synergy HT, Bio-Tek, Winooski, VM). Cell viability was computed using the average absorbance units obtained from the wells and expressed as percentage of the untreated cells. Statistical Analysis At least triplicate analyses were performed for each determination. Data was analyzed by ANOVA procedures and differences among means compared using Tukey tests with a level of significance of P<0.05. The computer software JMP 7.0 from the SAS Institute was used for all analyses.

Results and Discussion Prickly Pear Pulp Yield and Characteristics Tapon, Pelon and Rastrero yielded the highest amounts of pulp (Table 1). Pelon is an introduced species to Peuelos

Nuevo Len likely due to its high pulp yield. The yield of Tapon was probably overestimated since it was difficult to differentiate between peel and pulp tissues. Thus, its pulp and juice probably contained significant amounts of peel components that affected pH and the phytochemical profile. The 66.3 and 62.8% pulp yields obtained from Rastrero and Pelon, respectively, were higher and similar compared to yields previously reported by Felker et al. [21] for O. ficus indica clones. The same authors reported a large variability in pulp yields within the same species ranging from 38 to 62%. Gurrieri et al. [3] previously documented similar pulp yield for Sicilian cultivars. The Gavia cultivar was introduced to the Peuelos region because its utility to build natural and protective house fences. Prickly pears from Cardon, corresponding to O. streptacantha, yielded the lowest pulp. This fruit is mainly used for the production of queso de tuna, a traditional confection. Prickly pears from Cardon are susceptible to mechanical damage during postharvest management and transportation [22]. The large differences in yields were related to the prickly pear size, and the amount and thickness of the peels. The set of prickly pear juices had pHs ranging from 4.27 to 5.46 (Table 1). These values are less acidic than citrus juices (pH 3.35) [23]. As expected, the juices with the lowest pH values had the highest titritable acidities. Rastrero and Duraznillo Blanco contained the highest acidities followed by Duraznillo Rojo and Tapon. Both Duraznillo juices belong to the Opuntia leucotricha species. Rastrero and Duraznillo Blanco contained at least five times more acidity compared to Amarillo, Pelon and Moradillo. The pH (5.46) and acidity values (0.03%) of Pelon are within ranges determined by Gurrieri et al. [3] in prickly pear juices from Opuntia ficus indica. According to Felker et al. [21], the pH and acidity for ripen fruits is between 5.6 and 6.5 and 0.05% and 0.18%, respectively. Daz-Medina et al. [24], Viloria-Matos et al. [25] and Pimienta-Barrios et al. [26] reported more acidic pH in prickly fruits collected from Opuntia dillenni (pH 3.3), Opuntia boldinghii (pH 4.9) and Opuntia joconostle (pH 3.2). The sugar content of the prickly pear juices varied from 8 to 14.7Brix (Table 1). These values are within the range reported by Mullen et al. [27] for 13 commercially available fruit juices and drinks of the United Kingdom. The juice with the highest sugar content corresponded to Gavia with 14.7Brix. The Pelon juice also contained more than 14Brix. Stintzing et al. [12] reported 14.2 Brix for a juice extracted from the same species as Pelon. Prickly pears that yielded juices with high sugar contents tended to have lower acidities and as a sequence of the highest Brix/ Acidity ratios (Table 1).

Plant Foods Hum Nutr (2009) 64:146152 Table 2 Color of juices extracted from nine Mexican prickly pearsa Cactus Common Name Pulp Color Juice Color L Gavia Cardon Amarillo Peln Moradillo Rastrero Duraznillo Blanco Duraznillo Rojo Tapn
a b

149

a 6.00.7f 9.11.1cd -4.80.1f 14.61.5ab 11.31.7bc 6.60.8e 0.00.9e 16.80.1a 8.92.0 cd

b 19.61.6a 4.10.4d 21.02.8a 7.30.5c 4.71.4d 3.10.5d 9.80.8b 9.50.3bc 3.60.8d

Eb 35.11.2a 11.30.8ef 31.80.6ab 18.10.5d 13.31.7e 8.30.4f 27.40.3bc 23.80.2cd 11.12.1ef

Hue 107.01.2a 24.21.9c 103.01.8a 26.61.4c 23.04.2c 25.34.9c 89.35.4b 29.40.6c 24.36.0c

Green Purple Green-yellow Orange-red Purple-red Purple White Purple Purple

28.43.4a 5.02.2de 23.30.7b 7.81.1d 5.11.1de 3.91.1e 25.50.6ab 14.00.5c 4.83.6de

Each value is the average of at least 3 observations. Means SEM with different letters within column are significant different (P<0.05) Color index E = (L2 + a
2

+ b2 )1/2

Color The nine juices varied in color from white to purple (Table 2). Gavia and Amarillo, which had a yellow-green coloration, showed the highest hue and b values and were the only juices that had negative a values (yellow coloration). Duraznillo Blanco yielded a white juice which had a relatively high L, b and hue values. As expected, the purple and the orange-red colored juices had the highest a values, and the lowest L and b values. Cardon, Moradillo, Rastrero and Tapon, with the lowest L, E and hue values (Table 2), had the highest amounts of betalains (Table 3). As a consequence, there was a significant correlation between the color index E and betaxanthins (0.705) and betacyanins (0.773). Stintzing et al. (2005 [12]) obtained a correlation value of 0.928 between the content of betalains and betaxanthins. Juices from Gavia

and Amarillo had the highest E and L values or lightest coloration followed by Duraznillo Blanco and Duraznillo Rojo. Rastrero had the lowest E value and was among the juices that contained more betacyanins than betaxanthins explaining its purple coloration. As expected, the yellowgreen varieties had higher contents of yellow betaxanthins than red-purple betacyanins [12]. Phenolics, Flavonoids and Betalains The total phenolics varied among species from 22.3 to 226.3g/g or g gallic acid equivalents (GAE)/g (Table 3). The juice from Tapon contained the lowest amounts. The other two varieties (Gavia and Amarillo) of the species O. robusta contained comparable amounts to Moradillo, Rastrera and Duraznillo Blanco. Duraznillo Rojo, with red peels and purple pulp, contained the highest amount of total

Table 3 Total phenols, flavonoids, betaxanthins, betacyanins and antioxidant capacities (ORAC) of juices extracted from nine Mexican prickly pearsa Cactus Common Name Gavia Cardon Amarillo Peln Moradillo Rastrero Duraznillo Blanco Duraznillo Rojo Tapon
a

Total Phenols (g GAE /g) 114.323.2c 195.524.8b 55.49.2c 172.114.3b 72.83.8c 121.914.3bc 109.227.5c 226.326.4a 22.30.2d

Flavonoids (g quercetin eq./g) 209.625.9b 321.423.5a 193.116.2c 276.99.2a 374.330.3a 236.016.8b 238.022.4c 95.82.0c 338.121.5a

Betaxanthins (Indicaxanthin, g/g) 4.70. 7c 66.32.5b 8.81.4c 46.04.7b 33.11.9b 86.222.3b 4.40.8c 3.10.6c 189.97.3a

Betacyanins (Betanin, g /g) 1.60.1d 112.85.9c 4.20.7d 53.64.1c 89.414.2c 152.65.4b 2.40.5d 13.84.9d 300.58.8a

ORAC (mmole TE /L) 24.21.2a 24.01.8 a 17.41.7c 21.01.8b 24.82.0 a 25.81.4 a 22.51.8 ab 23.41.4a 22.71.6ab

Each value is the average of at least 3 observations. Means SEM with different letters within column are significant different (P<0.05)

GAE = gallic acid equivalents, TE = trolox equivalents

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phenols (226.3g GAE/g) followed by Cardon and Pelon. Interestingly, Tapon corresponding to other prickly pear with purple pulp and purple-red peels contained one tenth of the phenolics observed in Duraznillo Rojo (Table 3). The apparent relationship between total phenols and color within the set of juices studied herein depended mainly on the species as it was previously reported by Stintzing et al. [28]. Ndhlala et al. [4] analyzed the total phenolics and flavonoids of ethanol extracts of pulp and peels of prickly pears belonging to Opuntia megacantha and found that the pulp contained approximately 180g/g GAE and 10g/g cathecin. The juices from Duraznillo Rojo and Cardon contained the highest phenolics. Interestingly, the Tapon juice only contained 10% of the total phenols of Duraznillo Rojo. Chang et al. (2008 [29]) reported 915g/g GAE and 292g quercetin/g in methanol extracts of fruits of Opuntia dillenii. Our results show that the studied juices contained at least 10 times more flavonoids compared to values found in Opuntia megacantha. Only Cardon, Moradillo and Tapon contained higher flavonoids compared to values reported for Opuntia dillenii [29]. Kuti [13] analyzed different types of prickly pears and concluded that purple skinned fruits contained the highest amounts of flavonoids. The flavonoid profile has not been reported for the prickly pears analyzed in this study. However quercetin, kaempferol and isorhamnetin were found in cactus pads known as nopalitos at concentrations of 700, 900 and 1500g/g dry weight and in the peel of a red commercial prickly pear variety at concentrations of 100, 0 and 2,900, respectively. Since the fruit peel develops from pads, these values are consistent. Nevertheless, none of the flavonoids were found in the edible pulp portion (Dr. P. Felker personal communication). Thus, it is important to continue research on this field to evaluate the flavonoid profile at different maturity

stages. In addition, the analysis during the ripening process after harvesting needs to be evaluated for the different varieties, especially because it has been reported that the maturity affects the concentration of carotenoids and xanthophylls [30]. Furthermore the glycosidic forms of the flavonoids need to be analyzed because the composition of the anatomical parts, especially the ethanol-soluble carbohydrates in the pulp is almost twice the one found in the skin [31]. A comparison between the two juices obtained from O. lecuotrichia indicated that the red juice of Duraznillo Rojo contained twice the amount observed in Duraznillo Blanco. The total phenols of some of the prickly pear juices analyzed are similar to clarified apple or white grape juices [27]. Other juices extracted from cranberries, pomegranate, tropical fruits, purple grape, and apple contained more than three times the total phenolics determined in the set of prickly pear juices analyzed. In accordance with Butera et al. [32], prickly pears are considered a rich source of yellow-orange betaxanthins and red-violet betacyanins. The contents of betalains of some varieties (Table 3) were similar to those previously reported by Stintzing et al. [28]. However, there were wide differences in terms of betaxanthins and betacyanins contents. Tapon contained at least 40 times more betaxanthins and betacyanins compared to light colored juices from Gavia, Amarillo and Duraznillo Blanco. Although variety Tapon contained high amounts of betaxanthins, the color exerted by the red violet betalains predominated in this and other purple pigmented juices. The prickly pear juices were categorized into three groups according to betaxanthins. Gavia, Amarillo, Duraznillo Blanco and Duraznillo Rojo contained the lowest amounts (<8.8g/g) followed by Cardon, Pelon, Moradillo and Rastrero (33 to 86g/g) and Tapon (189.9g/g). The Tapon

Table 4 Effect of prickly pear juices on cell viability of four cancer cell lines compared to regular fibroblasta Cactus Common Name Cell Viability Fibroblast NIH 3T3 Gavia Cardon Amarillo Peln Moradillo Rastrero Duraznillo Blanco Duraznillo Rojo Tapon
a

Colon Cancer Caco-2 52.512.6 d 81.41.7 b 99.66.0 a 101.71.9a 63.04.9cd 74.15.2 bc 96.14.2 a 100.76.7 a 100.74.7 a

Prostate Cancer PC-3 75.22.4bc 68.96.9d 100.92.5a 92.44.7ab 61.25.3d 68.73.8d 82.53.6bc 69.75.8cd 72.42.3bc

Hepatic Cancer HepG2 79.210.0c 99.85.3ab 85.57.1c 87.45.8bc 102.210.2a 78.99.0c 102.78.3a 105.310.2a 80.610.7c

Mammary Cancer MCF7 102.57.3a 100.69.4a 105.69.3a 94.911.1a 112.58.6a 75.48.2b 104.68.3a 119.75.3a 107.96.2a

93.511.1ab 106.09.3a 94.412.0ab 99.36.2a 73.511.9c 98.810.4a 86.86.4b 86.38.2bc 87.98.6ab

Each value is the average of at least 3 observations. Means SEM with different letters within column are significant different (P<0.05).

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juice also contained the highest betacyanins followed by the light-colored juices from Rastrero, Cardon, Pelon and Moradillo. Antioxidant Capacity All juices had ORAC values in the narrow range of 17 to 25mol Trolox Equivalents (TE)/mL despite the significant differences in total phenols, flavonoids and betalains (Table 3). The juices ORAC values were higher compared to values (5.4511.20 mmol/L) reported by Stintzing et al. [12] and similar (15.849.4 mmol/kg) to values obtained by Kuti [13]. According to Wu et al. [33] most of the antioxidant capacity associated to fruits is exerted by ascorbic acid and phenolic compounds. Only about 1% of the total antioxidant capacity is due to lipophilic compounds. In comparison with commercial juices, the prickly pear juices contained at least twice the ORAC of strawberry, plum, orange, grapefruit, red and white grape, kiwi, apple, pear and tomato [34] and comparable values compared to red wine and pomegranate, concord grape, blueberry and black cherry juices [35]. Amarillo was the prickly pear juice with the lowest antioxidant activity (17.391.65, Table 3). However, it contained a slightly higher and similar ORAC value compared to commercial purple grape (15 mmol TE/L) and blueberry juices [35]. However, it is important to consider that the processing conditions used for the production of commercial fruit juices may reduce their antioxidant activity in comparison with the fresh product. Then, the ORAC values presented in Table 3 are lower than the ones obtained for homogenized acetone extracts from berries, cherry, plum or pomegranate [36]. The ORAC values of the prickly pear juices studied herein are higher than values reported for apricot, grapes, grapefruit or orange [33]. In Vitro Cancer Cell Culture Among the four cancer cells tested, the viability of prostate and colon cancer cells were the most affected especially by the Moradillo juice (Table 4). However, the Moradillo juice also diminished the growth of normal fibroblasts used as control. Rastrero also diminished the growth of prostate cancer cells but did not affect normal fibroblast viability. In fact, this juice was effective against the four cancer cell lines tested. Interestingly, Rastrero had the highest antioxidant capacity (Table 3) which was comparable to pomegranate juice [35]. The Gavia juice was the most effective against colon cancer cells and to some extent also affected prostate and hepatic cancer cell growth. However, Gavia did not have any effect on hormonedependent mammary and fibroblast cells. Juice from Cardon affected the viability of prostate and colon cancer

cells almost to the same level observed in Rastrero. The juices obtained from prickly pears of O. robusta or O. rastrera were the only ones that diminished the viability of hepatic cancer cells.

Conclusions There was a wide variation in pulp yields, color and nutraceutical potential of the nine different juices extracted from prickly pears of the genus Opuntia. Moradillo had the best in vitro effect on diminishing cancer cell viability, particularly prostate cells. This research shows the potential of prickly pears as an important source of natural antioxidants and nutraceuticals. Further research is needed in order to find the most bioactive anticancer compounds and if the in vitro results correlate with animal studies.

Acknowledgements This research was supported by grants CAT005 from Tecnolgico de Monterrey and CONACYT SEP-2004-0145723. Authors would like to thank Ing. Jose A. Garza and Gerardo Rayas Tovar for kindly providing the set of prickly pears. The senior author would like to acknowledge financial support provided by Alma Bours de Antillon.

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