Antibacterial and Antioxidant Activities in Extracts of Fully Grown Cladodes of 8 Cultivars of Cactus Pear E. S´anchez, J. D´avila-Avi˜ na, S. L. Castillo, N. Heredia, R. V´azquez-Alvarado, and S. Garc´ıa
The antimicrobial and antioxidant activities of some cultivars of the nopal cactus have not been determined. In this study, 8 cultivars of nopal cacti from Mexico were assayed for phenolic content, antioxidant activities, and antimicrobial activities against Campylobacter Jejuni, Vibrio cholera, and Clostridium Perfringens. Plant material was washed, dried, and macerated in methanol. Minimum bactericidal concentrations (MBCs) were determined using the broth microdilution method. Antioxidant activities were quantitatively determined using spectrophotometric methods. The MCBs of the nopal cacti ranged from 1.1 to 12.5 mg/mL for c. jejuni, 4.4 to 30 mg/mL for V. cholera, and 0.8 to 16 mg/mL for C. perfringens in the cultivars Cardon Blanco, Real de Catorce, and Jalpa, respectively. High quantities of total phenols and total flavonoids were found in the Jalpa cacti (3.80 mg of gallic acid equivalent GAE/g dry weight [DW] and 36.64 mg of quercetin equivalents [QE]/g DW, respectively). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities (RSA) were correlated to bioactive compound contents. The Villanueva cacti had the highest %RSA at 42.31%, and the lowest activity was recorded in Copena V1 at 19.98%. In conclusion, we found that some of the 8 cactus pear cultivars studied may be used for their antioxidant compounds or antimicrobials to control or prevent the contamination of foods. Keywords: antimicrobial activity, ethanol, flavonoids, herbs, water
Mexico had a great diversity of cactus pear cultivars, which have various uses. This study compares the antimicrobial and antioxidant activities of various cultivars of cactus pear widely distributed in Mexico. Several of the cultivars were determined whether any were candidates for the inclusion in food systems as nutraceuticals or antimicrobial agents. Our data indicated that some cacti may be used for these purposes due to their antioxidant compounds or antimicrobial activity.
Practical Application:
Introduction Cactus pear, also known as prickly pear, is a group of succulent plants belonging to the Cactaceae family that grow principally in the arid and semiarid regions of the world (Hern´andez-Hern´andez and others 2011). However, due to their remarkable genetic variability, these plants show great adaptability and are found in many geographic areas and habitats (Stintzing and Carle, 2005). Opuntia, the most important genera of cactus pear plants, is used in many human applications (Flores-Valdez and Aranda-Osorio 1998). Reportedly, 104 species of Opuntia are present in M´exico, and Opuntia ficus-indica is the most widespread and economically important among these cactus crops (Bravo-Hollis 1978). At 3 to 4 wk of age, cladodes (nopalitos) from cactus pear can be eaten fresh or cooked in several ways. The fruits and young cladodes are also used to make products, including candy, liqueurs, body lotions, creams, and shampoos (Stintzing and Carle 2005). They are used in industry for the production of flour and nutraceuticals (Lee and others 2003). Mature cladodes are used to feed animals MS 20131444 Submitted 10/10/2013, Accepted 1/19/2014. Authors Sanchez, Davila-Avina, Castillo, Heredia, and Garcia are with Facultad de Ciencias Biol´ogicas, Univ. Aut´onoma de Nuevo Le´on, Nuevo Le´on CP 66451, Mexico. Author VazquezAlvarado is with Facultad de Agronom´ıa, Univ. Aut´onoma de Nuevo Le´on, Apdo. Postal 124-F, San Nicol´as, Nuevo Le´on CP 66451, Mexico. Direct inquiries to author Santos Garc´ıa (E-mail: [email protected]).
R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12416 Further reproduction without permission is prohibited
during droughts (L´opez-Garc´ıa and others 2001). Interestingly, antimicrobial, anti-inflammatory, and antihyperglycemic effects have also been attributed to these plants (Feugang and others 2006). The cactus pear reportedly shows medicinal properties, including antiulcer, antiinflammatory, diuretic, cardiac, and diabetic effects (Feugang and others 2006; Halmi and others 2012). Extracts from the prickly pear cactus, Opuntia ficus-indica var. saboten, showed antimicrobial activity against antibiotic-resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecium (Kim and others 2005), and inhibitory activity against Salmonella spp. and Escherichia coli O157:H7 (Kim and others 2002). Furthermore, extracts of Opuntia ficus-indica reduced the growth of Listeria monocytogenes on fresh-cut apples to nondetectable levels (YoungHo Seo and others 2012) Extracts of Opuntia ficus-indica var. Villanueva disturbed membrane integrity, membrane potential, cytoplasmic pH, and ATP synthesis in Vibrio cholerae (S´anchez and others 2010). These extracts were also shown to inhibit growth and to reduce the adhesion and cytotoxic activity of Campylobacter jejuni and Campylobacter coli against Vero cells (Castillo and others 2011). Extracts from another species, Opuntia dilleniial, exhibited antimicrobial properties against Bacillus subtillis, S. aureus, E. coli, and Salmonella typhi (Umar and others 2013). Opuntia species contain bioactive antioxidant compounds, including phenolic acids, flavonoids, flavonols, carotenes, ascorbic acid, phytosterols, and chlorophyll (Gallegos-Infante and
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Abstract:
Activities of cultivars of cactus pear . . .
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others 2009; Guevara-Figueroa and others 2010), and antioxidant properties have been reported (Jaramillo-Flores and others 2003). Although the antimicrobial activity and antioxidant properties of the genus Opuntia have been described for a few species, little information is available about cultivars of Opuntia ficus-indica. The current study was undertaken to determine the phenolic compound content, antioxidant activities, and antimicrobial activities in cladode extracts from 8 cultivars of O. ficus-indica.
each well. Cultures of C. perfringens and V. cholerae were incubated at 37 °C for 24 h in an anaerobic chamber (5% CO2 , 95% N2 ) (S´anchez and others 2010). Cultures of C. jejuni were incubated at 42 °C for 48 h under microaerobic conditions (5% O2 , 10% CO2 , 85% N2 ) (Castillo and others 2011). Antimicrobial activities were determined by measuring the inhibition zones surrounding the wells with calipers (Ncube and others 2008). Absolute methanol (100 μL) served as a control.
Materials and Methods
Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) MIC and MBC were determined using the broth microdilution method of Klanˇcnik and others (2010) with minor modifications. For MIC determination, sterile 96-well polystyrene U-shaped microtiter plates (Corning, Costar Cambridge, Mass., U.S.A.) were filled with 100 μL of each bacterial suspension (1 × 106 CFU/mL, final concentration) in suitable growth medium containing 100 μL of serially diluted plant extracts. Final concentrations of extracts ranged from 0.014 to 30 mg/mL. Wells with culture medium, with bacterial suspension, and with and without methanol were used as controls. MIC was defined as the lowest extract concentration that inhibited visible growth of the bacteria (Ncube and others 2008). To determine MBC, 25 μL from each well were drop-plated on appropriate culture media. Agar plates were incubated and examined for the presence of microbial growth. MBC was defined as the lowest extract concentration at which no microbial growth was detected in the plates (S´anchez and others 2010).
Bacterial strains and culture conditions Clostridium perfringens FD-1041 and FD-1 were obtained from S. Harmon US-FDA, and R. Labbe from the Univ. of Massachusetts at Amherst, respectively. V. cholerae 569-B and 1837 were obtained from Elisa Elliot, US-FDA, Wash., U.S.A. C. jejuni 5653 and 11168 were obtained from Irene Wesley, USDA-ARS-NADC, Ames, Iowa, U.S.A. and from the ATCC culture collection in Manassas, Va., U.S.A., respectively. Strains of C. perfringens were maintained in cooked meat medium (Difco Laboratories, Sparks, Md., U.S.A.) at –20 °C. Fresh cultures were obtained by inoculating 5 μL of thawed culture into 5 mL of thioglycollate broth (Difco Laboratories). Cultures were grown for 18 h at 37 °C. V. cholerae was maintained in Luria Bertani slants (LB) (Difco Laboratories) at room temperature. To obtain fresh cultures, loops from the slants were inoculated into 5 mL of LB broth and grown for 18 h at 37 °C. C. jejuni were stored at –80 °C in 20% glycerol brain heart infusion (BHI) broth (Difco Laboratories), supplemented with 0.6% yeast extract. Fresh cultures were obtained by inoculating samples in Mueller–Hinton Phenol and flavonoid determination broth (Difco Laboratories) and incubating at 42 °C under miTo determine the total phenol and flavonoid contents, fresh croaerobic conditions (5% O2 , 10% CO2 , 85% N2 ) in a Shel-Lab samples of all cultivars (1.25 g) were placed in vials and lyophilized CO2 incubator (Cornelius, Oreg., U.S.A.). (Labconco, Kansas City, Mo., U.S.A.). Dried samples were macerated (100 mL methanol:water, 7:3), filtered, dried, and adjusted Plant collection and extract preparation to a final concentration of 12.5 mg/mL (w/v). Total phenols were O. ficus-indica (L), Jalpa (JL), Villanueva (VN), Copena V1 determined using the Folin–Ciocalteu reagent with gallic acid as (CV1), Cristalino (CR), Real de Catorce (R14), Copena a standard (Singleton and Rossi 1965). Briefly, 50 μL of O. ficusF1 (CF1), Forrajero Mina (FM), and O. streptacantha Cardon indica extract were added to 3 mL of deionized water plus 250 μL Blanco (CB) were used in this study. For antimicrobial evaluation, of Folin–Ciocalteu reagent. After 5 min, 750 μL of 20% Na2 CO3 extracts of the 8 cultivars were made using fully grown (6-mo-old) solution were added. The mixture was brought to 5 mL by adding cladodes, which were obtained from the experimental field of the deionized water. Phenols were measured at 760 nm after 30 min School of Agronomy, at our university. Cladodes were washed with using a 6405 UV-vis Jenwey spectrophotometer (Staffordshire, tap water and disinfected using 70% ethanol. Thorns were removed OSA, U.K.). Results were reported as mg of gallic acid equivalent manually, and cladodes were sliced to facilitate drying in a forced air (GAE) per g of dry weight. oven at 45 °C. Dried cladodes were ground in a grain mill. A total Flavonoid content was determined based on the methods deof 150 g of milled dried plant material was placed in a screw capped scribed by Zhishen and others (1999). Extracts (1 mL) were mixed glass flask and macerated with 500 mL of absolute methanol at with 4 mL of deionized H2 O and 300 μL of 5% NaNO2 and equi25 °C overnight. Macerated extracts were filtered using Whatman librated for 5 min. Following equilibrium, 300 μL of 10% AlCl3 No.1 paper, and the methanol was evaporated in a forced air oven were added. Extracts were left to stand for 1 min, and 2 mL of 1 M at 45 °C. Dried extracts were resuspended in 10 mL of methanol. NaOH were added. The volume was brought to 10 mL with An aliquot was used to determine the dry weight of each extract. deionized water while stirring, and the absorbance (415 nm) was All extracts were stored at 4 °C in amber vials until needed. determined. Total flavonoids were expressed as milligrams (dry weight) of quercetin equivalents/g dry weight (mg QE/g DW). Preliminary antibacterial assay Flavonoid compounds were determined by calculating the differPreliminary analyses of the antimicrobial effects were conducted ence between the total phenol concentration and flavonoids. The using agar well diffusion assays (Das and others 2010). A 100-μL results were expressed as percent of phenolics. aliquot of a fresh culture (106 CFU/mL) was homogeneously spread on the surface of Mueller–Hinton agar (C. perfringens), Radical scavenging activity 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was determined acLB agar (V. cholerae), or MH agar supplemented with 5% (v/v) defibrinated horse blood (C. jejuni). Wells were made in each cording to the methods in Brand-Williams and others (1995), of the seeded agar plates, using an inverted sterile Durham tube with minor modifications. A stock solution of DPPH was pre(6-mm diameter), and 100 μL of each extract was deposited into pared by mixing 2.5 mg of the DPPH radical with 100 mL of M2 Journal of Food Science r Vol. 00, Nr. 0, 2014
Activities of cultivars of cactus pear . . . Table 1–Inhibition zones (cm) of extracts of 8 cultivars of O. Ficus-indica against growth of C. jejuni, V. cholerae, and C. perfringens. C. jejuni Cactus pear cultivar CB R14 JL FM VN CV1 CF1 CR
V. cholerae
5653
11168
1.8 ± 0.1∗b 1.9 1.6 1.6 1.4 1.4 1.5 1.5
± ± ± ± ± ± ±
2.0 1.7 1.5 1.6 1.3 1.4 1.5 1.4
0.1b 0.2ab 0.1ab 0.1a 0.2b 0.2a 0.1a
± 0.1d ± ± ± ± ± ± ±
0.1c 0.1abc 0.1bc 0.1a 0.1ab 0.1ab 0.1a
C. perfringens
1837 2.1 1.6 1.7 1.7 1.6 1.5 1.7 1.2
± ± ± ± ± ± ± ±
569
0.1c
1.7 1.5 1.3 1.5 1.4 1.4 1.2 1.3
0.1b 0.1b 0.1b 0.1b 0.1b 0.1b 0.1b
± ± ± ± ± ± ± ±
0.1d 0.1ab 0.1ab 0.1bc 0.1ab 0.1ab 0.1a 0.1a
FD-1041 2.2 2.1 2.1 2.0 2.1 2.1 1.9 1.7
± ± ± ± ± ± ± ±
0.1b 0.2b 0.1b 0.1b 0.1b 0.2b 0.2ab 0.2a
FD-1 2.5 ± 0.2b 2.1 ± 0.1a 2.0 ± 0.1a 2.0 ± 0.1a 2.2 ± 0.1a 2.1 ± 0.1a 1.9 ± 0.1a 2.0 ± 0.2a
∗
Standard deviation. Different letters represents significant differences (P < 0.05) between cultivars. Each observation is a mean ± SD of three replicates.
Table 2–Minimum bactericidal concentrations (mg/mL) of extracts of 8 cultivars of O. ficus-indica against growth of C. jejuni, V. cholerae, and C. perfringens.
Cactus pear cultivar CB R14 JL FM VN CV1 CF1 CR
5653 1.1 1.2 7.5 4.5 7.4 12.5 10.5 11.2
± ± ± ± ± ± ± ±
0.8∗a 0.2a 0.6c 0.6b 0.5c 0.6e 0.6d 1.0d
V. cholerae 11168 5.2 2.7 5.7 5.8 5.8 8.2 12 9.2
± ± ± ± ± ± ± ±
0.9b 0.5a 0.9b 0.4b 0.4b 0.9c 0.9d 0.5c
C. perfringens
1837 4.4 4.4 7.4 9.5 11 13 12 30
± ± ± ± ± ± ± ±
0.9a 0.5a 0.5b 0.5c 0.8d 0.8e 0.4de 0h
569 5.8 4.4 7.5 9.7 11 15 13 30
± ± ± ± ± ± ± ±
0.4b 0.5a 0.5c 0.5d 0.9e 0.8g 0.5f 0h
FD-1041 1.6 1.6 3.6 4.4 4.7 6.5 12 15
± ± ± ± ± ± ± ±
0.5a 0.9a 0.5b 0.5bc 0.5c 0.5d 0.5e 0.5f
FD-1 0.9 0.8 1.5 5 5.5 5.5 10 16
± ± ± ± ± ± ± ±
0.2a 0.3a 0.5a 0.6b 0.5b 0.5b 0.4c 0.8d
∗
Standard deviation. Different letters represents significant differences (P < 0.05) between cultivars. Each observation is a mean ± SD of three replicates.
pure methanol. The solution was adjusted with methanol to an absorbance of 0.7 ± 0.02 at 515 nm. DPPH radicals (3.9 mL) were placed in a test tube, and 100 μL aliquots of the extracts were added. The mixture was vortexed and kept in the dark for 30 min. Absorbance at 515 nm was determined. The percentage of radical scavenging activity (%RSA) was determined using the following equation: %RSA = [(control Abs − sample Abs)/control Abs)] × 100
Statistical analyses Data were analyzed using the general linear model (GLM) procedure from the Number Cruncher Statistical System version 6.0 software (NCSS, LLC). Differences between cultivars were determined using Tukey´s comparison test. The P ࣘ 0.05 was considered significant. Each experiment was performed in triplicate.
Results
the lowest MBCs (0.8 and 1.1 mg/mL, respectively, for the CB cultivar and 0.9 and 1.2 mg/mL, respectively, for the R14 cultivar). V. cholerae 569-B was the most resistant to the extracts (MBC of 5.8 for the CB cultivar and 4.4 for the R14 cultivar) (Table 2).
Total phenols and flavonoids The total extractable phenols and flavonoids were different between the cactus pear cultivars (P < 0.05) (Figure 1A and 1B). Total phenolic content was greatest in the CF1 cultivar (4.27 mg GAE/ g DW), followed by JL, CV1, CB, VN, R14, CR, and FM with contents of 3.80, 3.61, 2.77, 2.73, 2.37, 2.05, and 1.49 GAE/100 g DW, respectively (Figure 1B). Phenol measurements in extracts from the FM cultivar were approximately 65% lower than extracts from the CF1 cultivar. In general, the total phenolic content varied between cultivars of the cactus pear; however, the differences between the CF1, JL, and CV1, and the CB, VN, and R14 cultivars were not significant. Total flavonoid content in cultivars of the cactus pear ranged from 15.4 to 36.6 mg QE/g DW. The greatest and smallest values were recorded in the JL and R14 cultivars, respectively. The following trend was found in total flavonoid content: JL > VN > CR > FM > CF1 > CB > CV1 > R14 (36.6, 24.7, 22.5, 18.9, 18.1, 17.9, 16.0, and 15.3 mg QE/g DW, respectively) (Figure 1A). Extracts from the R14 cultivar contained 58% less flavonoids than JL. The flavonoid content of the JL cultivar was significantly higher than the other cultivars; however, no significant differences were observed between the R14, CV1, CB, CF1, and FM cultivars.
Antimicrobial activity Preliminary analyses of the antibacterial activities of extracts from the 8 cultivars of cactus pear indicated that they had inhibitory activities against all strains of C. jejuni/Coli, C. perfringens, and V. cholerae. Extracts of cultivars used for prickly pear production, CB and R14, showed the largest inhibition zones, which ranged from 1.7 to 2.5 cm and 1.5 to 2.1 cm, respectively. The other cultivars showed inhibition zones from 1.2 to 2.2 cm (Table 1). The cultivars CB and R14 also showed the lowest MBCs, which ranged from 0.8 to 5.8 mg/mL. The cultivars JL, FM, VN, CV1, CF1, and CR presented MBC values in the ranges of 1.5 to 7.5, 4.4 Radical scavenging activity to 9.7, 4.7 to 11.2, 5.5 to 15, 10.2 to 13.4, and 9.2 to >30 mg/mL, Significant differences in %RSA were found among the cultirespectively, depending on the strain tested (Table 2). C. perfringens vars. Cultivar VN had the greatest %RSA (40.31%), followed by FD-1 and C. jejuni 5653 were the most sensitive isolates, with FM, CF1, CR, JL, R14, CB, and CV1 (38.70%, 34.64%, 31.94%, Vol. 00, Nr. 0, 2014 r Journal of Food Science M3
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C. jejuni
Activities of cultivars of cactus pear . . .
A
d
ab
20
ab a
ab
a
10
% Polyphenolic groups
100
c bc
0
B
c
c
60
40
20
40
c
4 (mg GAE/g DW)
80
0
b 3
b
b
ab a
2
B
e
e d cd
DPPH. (%RSA)
5
% Non-Flavonoids % Flavonoids
A
30 (mg QE/g DW)
Total Flavonoid Content
40
Total Phenolic Content
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29.61%, 26.40%, 25.09%, and 19.98%, respectively) (Figure 2B). methanol extracts contained the best antimicrobial activities against Extracts from the CV1 cultivar had 48% lower DPPH radical- select microorganisms. MBCs of the extracts, which ranged from scavenging activity than the VN cultivar. 45.1 to 153 mg/mL, were higher than the MBCs of O. ficus-indica determined in this study. In a different study, chloroform extracts of O. dillenii were more active than those made with methanol to Discussion The cultivars of Opuntia used in this study have different uses, inhibit the growth of Gram-positive and Gram-negative microorcultivars Jalpa (JL), Villanueva (VN), and Copena V1 (CV1) are ganisms (Umar and others 2013). Ethanol extracts and fractions used as young tender pads for human consumption; Cristalino obtained by sequential ethanol fractionation of a mixture of 3 (CR), Real de Catorce (R14), Cardon Blanco (CB), are used for different cultivars of O. ficus-indica (Surfarina, Muscaredda, and fruit production; and Copena F1 (CF1), and Forrajero Mina (FM) Sanguigna) did not present antimicrobial activities against the miare used as forage. Cactus pear cladodes possess remarkable medic- croorganisms studied (Ginestra and others 2009). Similarly Kim inal properties (Stintzing and Carle 2005). Studies of O. ficus-indica and others (2002) reported that, although bactericidal effects of have focused on its potential antihyperglycemic (Halmi and oth- aqueous extracts of O. ficus-indica var. Saboten were not observed, ers 2012), analgesic, antiinflammatory, antiulcer, and cholesterol- there were inhibitory activities against Salmonella spp. and E. Coli lowering activities (Feugang and others 2006). However, although O157 (Kim and others 2002). Polyphenols, which have varied molecular structures, are comthe chemical compositions (Ginestra and others 2009; S´aenz and others 2010; Hern´andez-Urbiola and others 2011) and antioxi- mon constituents of plants. Plant phenolics include phenolic acids, dant properties (Bari and others 2012; Dhaouadi and others 2013) flavonoids, tannins, and, less common stilbenes and lignans (Dai of cactus pear cladodes are known, few studies have compared the and Mumper 2010). Phenols and flavonoids are bioactive compounds that have been related to restorative processes by their antioxidant properties of different cultivars. Comparisons of antimicrobial activities of different cactus pear antimicrobial properties and abilities to reduce free radical formacultivars are also scarce or not available. Among the available stud- tion and to scavenge free radicals (Daglia 2012). Analyses of various commercial powders and commercial vaies, Kim and others (2002) reported that extracts of O. ficus-indica var. Saboten contained antimicrobial agents that were effective rieties of cactus pear exhibited total phenol contents of 1.7 to against pathogenic bacteria, including 2 Vibrio species; however, 11.7 mg GAE/g (Guevara-Figueroa and others 2010). These reno information about bactericidal effects were provided (Kim sults were consistent with those found in this study. On the other and others 2005). Bari and others (2012) used solvents to extract the antimicrobial agents from O. monacantha, concluding that
30
c b
b
a 20
10
1
0 CF1
CB
CR
JL
FM
R14
Cultivars
VN
CV1
0 CF1
CB
CR
JL
FM
R14
VN
CV1
Cultivars
Figure 1–(A) Total phenol and (B) flavonoid content of extracts from 8 Figure 2–(A) Percentage of polyphenolic groups. (B) Radical scavenging O. ficus-indica cultivars. Different letters represent significant differences activities. Different letters represent significant differences (P < 0.05) (P < 0.05) between cultivars. between cultivars.
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Activities of cultivars of cactus pear . . . effects of the active compounds on the normal human microbial flora must be determined. Finally, the risks and benefits of potential applications must be determined. Work is in progress in our laboratory on the isolation and identification of the antimicrobial compounds and their mechanism of action against these pathogens.
Conclusions This study determined the antimicrobial and antioxidant activities of various full-grown cultivars of cactus pear. The CB and R14 cultivars contained the highest antimicrobial activities, and the VN and FN cultivars contained the highest antioxidant activities. This vegetable, which is distributed throughout the world, may be an excellent candidate for use as a nutraceutical or as a source of antioxidant compounds.
Acknowledgments This research was supported by the Consejo Nacional de Ciencia y Tecnolog´ıa de M´exico (CONACYT, grant # 105389). Eduardo S´anchez and Sandra Castillo were supported by a scholarship from CONACYT. Jorge D´avila-Avi˜na was supported by a posdoctoral fellowship from CONACYT.
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hand, the total phenol content of young cladodes of cactus pear (O. ficus-indica) were reported to be in the range of 17 to 40 mg/g sample (dry basis) (Guevara-Figueroa and others 2010; Medina-Torres and others 2011). Those values were higher than our findings. The variation may be due to differences in the cladode maturity, cultivation region, climate, and quantification methodologies (Guevara-Figueroa and others 2010; Kim and others 2013). Flavonoids are the most abundant polyphenols in the human diet. There are 6 subgroups of flavonoids, including flavones, flavonols, flavanols, flavanones, isoflavones, and anthocyanins, which are distinguished based on the oxidation state of the central C ring (Dai and Mumper 2010). Cho and others (2006) suggested that quercetin and its derivatives play major roles in the antioxidant effects of the cactus. Medina-Torres and others (2011) evaluated the bioactive compounds of cladodes after dehydration. They reported that the total flavonoid content ranged from 10 to 20 mg/g sample, using rutin as a reference compound. These values were slightly different than those found in this study, which ranged from 15.4 to 36.6 mg QE/g DW. On the other hand, GuevaraFigueroa and others (2010) reported that, in general, flavonoids were found in smaller amounts than phenolic acids, and that the Blanco and Manso (commercial varieties) contained the greatest flavonoid content (9.8 and 5.9 mg QE/g, respectively). These results also differed from the findings in this study, which determined that phenols were less abundant than flavonoids. Differences in the physicochemical compositions may be attributed to the maturity of cladodes and the environmental conditions (Rodr´ıguez-Garcia and others 2007; Guevara-Figueroa and others 2010). The DPPH assay was used to measure the hydrogen atomdonating activity and to evaluate free radical scavenging. Significant differences in %RSA were found among the cultivars, and activities ranged from 19% to 40% (Figure 2B). Similar results were obtained by Gallegos-Infante and others (2009), who reported a 42.5% RSA in extracts of O. ficus indica. In general, polyphenols contributed to increased radical scavenging activity in the cactus (Kim and others 2013), and values could be associated with the type of phenols present (Gallegos-Infante and others 2009). The low radical scavenging activity (%RSA) shown by Opuntia extracts may be explained by the abundance of monohydroxylated phenolic compounds, which have relatively low activities (Gallegos-Infante and others 2009). Several plant polyphenols possess antibacterial activities (Kurek and others 2011). In fact, phenolic acids such as gallic acid, coumaric acid, 3,4-dihydroxy-benzoic acid, 4-hydroxy benzoic acid, ferulic acid, and salicylic acid and flavonoids like iso-quercitrin, isorharmnetin 3-O-glucoside, nicotiflorin, rutin, narcissin, have been reported in the cladodes of Opuntia varieties, and many of these exhibited antimicrobial activity (GuevaraFigueroa and others 2010). The antimicrobial activity of these compounds rely in the ability to disturb the bacterial membranes of microorganisms, and affect the expression of various virulence factors (Ayala-Zavala and others 2011; Daglia 2012). Although in this study no significant correlation between the antibacterial activity and total phenolics was observed, the antibacterial activity could be related to a particular or a mixture of compounds. Medina and others (2006) reported similar results, indicating that the content of polyphenols does not correlate with antimicrobial activities in all cases. The antimicrobial activities of Opuntia extracts may be used to control foodborne pathogens, especially in food production. However, prior to use in this industry, appropriate methods for nontoxic extraction of the active compounds must be developed. Also, the
Activities of cultivars of cactus pear . . . Kim JH, Lee H-J, Park Y, Ra KS, Shin K-S, Yu K-W, Suh HJ. 2013. Mucilage removal from cactus cladodes (Opuntia humifusa Raf.) by enzymatic treatment to improve extraction efficiency and radical scavenging activity. LWT-Food Sci Technol 51(1):337–42. Kim S, Kwon N, Kim J, Lim J, Bae W, Kim J, Noh K, Hur J, Jung W, Park K. 2002. Antimicrobial activity of natural product made by Opuntia ficus-indica var. saboten against Salmonella spp. and Escherichia coli O157: H7. J Food Hyg Safety 17:71–8. Klanˇcnik A, Piskernik S, Jerˇsek B, Moˇzina SS. 2010. Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts. J Microbiol Meth 81(2):121–6. Kurek A, Grudniak AM, Kraczkiewicz-Dowjat A, Wolska KI. 2011. New antibacterial therapeutics and strategies. Pol J Microbiol 60(1):3–12. Lee EH, Kim HJ, Song YS, Jin C, Lee K-T, Cho J, Lee YS. 2003. Constituents of the stems and fruits of Opuntia ficus-indica var. saboten. Arch Pharmacal Res 26(12):1018–23 L´opez-Garc´ıa J, Fuentes-Rodr´ıguez J, Rodr´ıguez R. 2001. Production and use of Opuntia as forage in northern Mexico. In: Mongrag´on-Jacobo C, P´erez-Gonz´alez S, editors. Cactus (Opuntia spp.) as forage. Rome: FAO Plant Production and Protection Paper. p 29–36. Medina-Torres L, Vernon-Carter EJ, Gallegos-Infante JA, Rocha-Guzm´an NE, HerreraValencia E, Calderas F, Jim´enez-Alvarado R. 2011. Study of the antoxidant properties of extracts obtained from nopla cactus (Opuntia ficus-indica) cladodes after convective drying. J Sci Food Agric. 91(6):1001–5. Medina E, de Castro A, Romero C, Brenes M. 2006. Comparison of the concentrations of phenolic compounds in olive oils and other plant oils: correlation with antimicrobial activity. J Agric Food Chem 54(14):4954–61. Ncube N, Afolayan A, Okoh A. 2008. Assessment techniques of antimicrobial properties of natural compounds of plant origin: current methods and future trends. Afr J Biotechnol 7(12):1797–806.
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Rodr´ıguez-Garcia ME, Lira C, Hern´andez-Becerra E, Cornejo-Villegas MA, Palacios-Fonseca AJ, Rojas-Molina I, Reynoso R, Quintero LC, Del-Real A, Zepeda TA, Mu˜noz-Torres C. 2007. Physicochemical characterization of nopal pads (Opuntia ficus indica) and Dry vacuum nopal powders as a function of the maturation. Plant Foods Hum Nutr 62(3): 107–12. S´aenz C, Sep´ulveda E, Pak N, Lecaros M. 2010. Chemical and physical characterization of cactus cladodes (Opuntia ficus-indica) powder. Ital J Food Sci 22(4):416–22. S´anchez E, Garc´ıa S, Heredia N. 2010. Extracts of edible and medicinal plants damage membranes of Vibrio cholerae. Appl Environ Microbiol 76(20):6888–94. Singleton V, Rossi JA. 1965. Colorimetry of total phenolics with phosphomolybdicphosphotungstic acid reagents. Am J Enol Viticult 16(3):144–58. Stintzing FC, Carle R. 2005. Cactus stems (Opuntia spp.): a review on their chemistry, technology, and uses. Mol Nutr Food Res 49(2):175–94. Umar MI, Javeed A, Ashraf M, Riaz A, Mukhtar MM, Afzal S, Altaf R. 2013. Polarity-based solvents extraction of opuntia dillenii and zingiber officinale for in vitro antimicrobial activities. Intl J Food Prop 16(1):114–24. Young-Ho Seo, Chang-Ho Han, Jeong-Mi Lee, Sung-Min Choi, Moon K-D. 2012. Effects of Opuntia ficus-indica extracts on inactivation of Escherichia coli O157:H7 and Listeria monocytogenes on fresh-cut apples. J Korean Soc Food Sci Nutr 41(7):1009–13. Zhishen J,Mengcheng T,Jianming W. 1999. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem 64(4): 555–9.
The antimicrobial and antioxidant activities of some cultivars of the nopal cactus have not been determined. In this study, 8 cultivars of nopal cacti from Mexico were assayed for phenolic content, antioxidant activities, and antimicrobial activities against Campylobacter Jejuni, Vibrio cholera, and Clostridium Perfringens. Plant material was washed, dried, and macerated in methanol. Minimum bactericidal concentrations (MBCs) were determined using the broth microdilution method. Antioxidant activities were quantitatively determined using spectrophotometric methods. The MCBs of the nopal cacti ranged from 1.1 to 12.5 mg/mL for c. jejuni, 4.4 to 30 mg/mL for V. cholera, and 0.8 to 16 mg/mL for C. perfringens in the cultivars Cardon Blanco, Real de Catorce, and Jalpa, respectively. High quantities of total phenols and total flavonoids were found in the Jalpa cacti (3.80 mg of gallic acid equivalent GAE/g dry weight [DW] and 36.64 mg of quercetin equivalents [QE]/g DW, respectively). 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities (RSA) were correlated to bioactive compound contents. The Villanueva cacti had the highest %RSA at 42.31%, and the lowest activity was recorded in Copena V1 at 19.98%. In conclusion, we found that some of the 8 cactus pear cultivars studied may be used for their antioxidant compounds or antimicrobials to control or prevent the contamination of foods. Keywords: antimicrobial activity, ethanol, flavonoids, herbs, water
Mexico had a great diversity of cactus pear cultivars, which have various uses. This study compares the antimicrobial and antioxidant activities of various cultivars of cactus pear widely distributed in Mexico. Several of the cultivars were determined whether any were candidates for the inclusion in food systems as nutraceuticals or antimicrobial agents. Our data indicated that some cacti may be used for these purposes due to their antioxidant compounds or antimicrobial activity.
Practical Application:
Introduction Cactus pear, also known as prickly pear, is a group of succulent plants belonging to the Cactaceae family that grow principally in the arid and semiarid regions of the world (Hern´andez-Hern´andez and others 2011). However, due to their remarkable genetic variability, these plants show great adaptability and are found in many geographic areas and habitats (Stintzing and Carle, 2005). Opuntia, the most important genera of cactus pear plants, is used in many human applications (Flores-Valdez and Aranda-Osorio 1998). Reportedly, 104 species of Opuntia are present in M´exico, and Opuntia ficus-indica is the most widespread and economically important among these cactus crops (Bravo-Hollis 1978). At 3 to 4 wk of age, cladodes (nopalitos) from cactus pear can be eaten fresh or cooked in several ways. The fruits and young cladodes are also used to make products, including candy, liqueurs, body lotions, creams, and shampoos (Stintzing and Carle 2005). They are used in industry for the production of flour and nutraceuticals (Lee and others 2003). Mature cladodes are used to feed animals MS 20131444 Submitted 10/10/2013, Accepted 1/19/2014. Authors Sanchez, Davila-Avina, Castillo, Heredia, and Garcia are with Facultad de Ciencias Biol´ogicas, Univ. Aut´onoma de Nuevo Le´on, Nuevo Le´on CP 66451, Mexico. Author VazquezAlvarado is with Facultad de Agronom´ıa, Univ. Aut´onoma de Nuevo Le´on, Apdo. Postal 124-F, San Nicol´as, Nuevo Le´on CP 66451, Mexico. Direct inquiries to author Santos Garc´ıa (E-mail: [email protected]).
R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12416 Further reproduction without permission is prohibited
during droughts (L´opez-Garc´ıa and others 2001). Interestingly, antimicrobial, anti-inflammatory, and antihyperglycemic effects have also been attributed to these plants (Feugang and others 2006). The cactus pear reportedly shows medicinal properties, including antiulcer, antiinflammatory, diuretic, cardiac, and diabetic effects (Feugang and others 2006; Halmi and others 2012). Extracts from the prickly pear cactus, Opuntia ficus-indica var. saboten, showed antimicrobial activity against antibiotic-resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecium (Kim and others 2005), and inhibitory activity against Salmonella spp. and Escherichia coli O157:H7 (Kim and others 2002). Furthermore, extracts of Opuntia ficus-indica reduced the growth of Listeria monocytogenes on fresh-cut apples to nondetectable levels (YoungHo Seo and others 2012) Extracts of Opuntia ficus-indica var. Villanueva disturbed membrane integrity, membrane potential, cytoplasmic pH, and ATP synthesis in Vibrio cholerae (S´anchez and others 2010). These extracts were also shown to inhibit growth and to reduce the adhesion and cytotoxic activity of Campylobacter jejuni and Campylobacter coli against Vero cells (Castillo and others 2011). Extracts from another species, Opuntia dilleniial, exhibited antimicrobial properties against Bacillus subtillis, S. aureus, E. coli, and Salmonella typhi (Umar and others 2013). Opuntia species contain bioactive antioxidant compounds, including phenolic acids, flavonoids, flavonols, carotenes, ascorbic acid, phytosterols, and chlorophyll (Gallegos-Infante and
Vol. 00, Nr. 0, 2014 r Journal of Food Science M1
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Abstract:
Activities of cultivars of cactus pear . . .
M: Food Microbiology & Safety
others 2009; Guevara-Figueroa and others 2010), and antioxidant properties have been reported (Jaramillo-Flores and others 2003). Although the antimicrobial activity and antioxidant properties of the genus Opuntia have been described for a few species, little information is available about cultivars of Opuntia ficus-indica. The current study was undertaken to determine the phenolic compound content, antioxidant activities, and antimicrobial activities in cladode extracts from 8 cultivars of O. ficus-indica.
each well. Cultures of C. perfringens and V. cholerae were incubated at 37 °C for 24 h in an anaerobic chamber (5% CO2 , 95% N2 ) (S´anchez and others 2010). Cultures of C. jejuni were incubated at 42 °C for 48 h under microaerobic conditions (5% O2 , 10% CO2 , 85% N2 ) (Castillo and others 2011). Antimicrobial activities were determined by measuring the inhibition zones surrounding the wells with calipers (Ncube and others 2008). Absolute methanol (100 μL) served as a control.
Materials and Methods
Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) MIC and MBC were determined using the broth microdilution method of Klanˇcnik and others (2010) with minor modifications. For MIC determination, sterile 96-well polystyrene U-shaped microtiter plates (Corning, Costar Cambridge, Mass., U.S.A.) were filled with 100 μL of each bacterial suspension (1 × 106 CFU/mL, final concentration) in suitable growth medium containing 100 μL of serially diluted plant extracts. Final concentrations of extracts ranged from 0.014 to 30 mg/mL. Wells with culture medium, with bacterial suspension, and with and without methanol were used as controls. MIC was defined as the lowest extract concentration that inhibited visible growth of the bacteria (Ncube and others 2008). To determine MBC, 25 μL from each well were drop-plated on appropriate culture media. Agar plates were incubated and examined for the presence of microbial growth. MBC was defined as the lowest extract concentration at which no microbial growth was detected in the plates (S´anchez and others 2010).
Bacterial strains and culture conditions Clostridium perfringens FD-1041 and FD-1 were obtained from S. Harmon US-FDA, and R. Labbe from the Univ. of Massachusetts at Amherst, respectively. V. cholerae 569-B and 1837 were obtained from Elisa Elliot, US-FDA, Wash., U.S.A. C. jejuni 5653 and 11168 were obtained from Irene Wesley, USDA-ARS-NADC, Ames, Iowa, U.S.A. and from the ATCC culture collection in Manassas, Va., U.S.A., respectively. Strains of C. perfringens were maintained in cooked meat medium (Difco Laboratories, Sparks, Md., U.S.A.) at –20 °C. Fresh cultures were obtained by inoculating 5 μL of thawed culture into 5 mL of thioglycollate broth (Difco Laboratories). Cultures were grown for 18 h at 37 °C. V. cholerae was maintained in Luria Bertani slants (LB) (Difco Laboratories) at room temperature. To obtain fresh cultures, loops from the slants were inoculated into 5 mL of LB broth and grown for 18 h at 37 °C. C. jejuni were stored at –80 °C in 20% glycerol brain heart infusion (BHI) broth (Difco Laboratories), supplemented with 0.6% yeast extract. Fresh cultures were obtained by inoculating samples in Mueller–Hinton Phenol and flavonoid determination broth (Difco Laboratories) and incubating at 42 °C under miTo determine the total phenol and flavonoid contents, fresh croaerobic conditions (5% O2 , 10% CO2 , 85% N2 ) in a Shel-Lab samples of all cultivars (1.25 g) were placed in vials and lyophilized CO2 incubator (Cornelius, Oreg., U.S.A.). (Labconco, Kansas City, Mo., U.S.A.). Dried samples were macerated (100 mL methanol:water, 7:3), filtered, dried, and adjusted Plant collection and extract preparation to a final concentration of 12.5 mg/mL (w/v). Total phenols were O. ficus-indica (L), Jalpa (JL), Villanueva (VN), Copena V1 determined using the Folin–Ciocalteu reagent with gallic acid as (CV1), Cristalino (CR), Real de Catorce (R14), Copena a standard (Singleton and Rossi 1965). Briefly, 50 μL of O. ficusF1 (CF1), Forrajero Mina (FM), and O. streptacantha Cardon indica extract were added to 3 mL of deionized water plus 250 μL Blanco (CB) were used in this study. For antimicrobial evaluation, of Folin–Ciocalteu reagent. After 5 min, 750 μL of 20% Na2 CO3 extracts of the 8 cultivars were made using fully grown (6-mo-old) solution were added. The mixture was brought to 5 mL by adding cladodes, which were obtained from the experimental field of the deionized water. Phenols were measured at 760 nm after 30 min School of Agronomy, at our university. Cladodes were washed with using a 6405 UV-vis Jenwey spectrophotometer (Staffordshire, tap water and disinfected using 70% ethanol. Thorns were removed OSA, U.K.). Results were reported as mg of gallic acid equivalent manually, and cladodes were sliced to facilitate drying in a forced air (GAE) per g of dry weight. oven at 45 °C. Dried cladodes were ground in a grain mill. A total Flavonoid content was determined based on the methods deof 150 g of milled dried plant material was placed in a screw capped scribed by Zhishen and others (1999). Extracts (1 mL) were mixed glass flask and macerated with 500 mL of absolute methanol at with 4 mL of deionized H2 O and 300 μL of 5% NaNO2 and equi25 °C overnight. Macerated extracts were filtered using Whatman librated for 5 min. Following equilibrium, 300 μL of 10% AlCl3 No.1 paper, and the methanol was evaporated in a forced air oven were added. Extracts were left to stand for 1 min, and 2 mL of 1 M at 45 °C. Dried extracts were resuspended in 10 mL of methanol. NaOH were added. The volume was brought to 10 mL with An aliquot was used to determine the dry weight of each extract. deionized water while stirring, and the absorbance (415 nm) was All extracts were stored at 4 °C in amber vials until needed. determined. Total flavonoids were expressed as milligrams (dry weight) of quercetin equivalents/g dry weight (mg QE/g DW). Preliminary antibacterial assay Flavonoid compounds were determined by calculating the differPreliminary analyses of the antimicrobial effects were conducted ence between the total phenol concentration and flavonoids. The using agar well diffusion assays (Das and others 2010). A 100-μL results were expressed as percent of phenolics. aliquot of a fresh culture (106 CFU/mL) was homogeneously spread on the surface of Mueller–Hinton agar (C. perfringens), Radical scavenging activity 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was determined acLB agar (V. cholerae), or MH agar supplemented with 5% (v/v) defibrinated horse blood (C. jejuni). Wells were made in each cording to the methods in Brand-Williams and others (1995), of the seeded agar plates, using an inverted sterile Durham tube with minor modifications. A stock solution of DPPH was pre(6-mm diameter), and 100 μL of each extract was deposited into pared by mixing 2.5 mg of the DPPH radical with 100 mL of M2 Journal of Food Science r Vol. 00, Nr. 0, 2014
Activities of cultivars of cactus pear . . . Table 1–Inhibition zones (cm) of extracts of 8 cultivars of O. Ficus-indica against growth of C. jejuni, V. cholerae, and C. perfringens. C. jejuni Cactus pear cultivar CB R14 JL FM VN CV1 CF1 CR
V. cholerae
5653
11168
1.8 ± 0.1∗b 1.9 1.6 1.6 1.4 1.4 1.5 1.5
± ± ± ± ± ± ±
2.0 1.7 1.5 1.6 1.3 1.4 1.5 1.4
0.1b 0.2ab 0.1ab 0.1a 0.2b 0.2a 0.1a
± 0.1d ± ± ± ± ± ± ±
0.1c 0.1abc 0.1bc 0.1a 0.1ab 0.1ab 0.1a
C. perfringens
1837 2.1 1.6 1.7 1.7 1.6 1.5 1.7 1.2
± ± ± ± ± ± ± ±
569
0.1c
1.7 1.5 1.3 1.5 1.4 1.4 1.2 1.3
0.1b 0.1b 0.1b 0.1b 0.1b 0.1b 0.1b
± ± ± ± ± ± ± ±
0.1d 0.1ab 0.1ab 0.1bc 0.1ab 0.1ab 0.1a 0.1a
FD-1041 2.2 2.1 2.1 2.0 2.1 2.1 1.9 1.7
± ± ± ± ± ± ± ±
0.1b 0.2b 0.1b 0.1b 0.1b 0.2b 0.2ab 0.2a
FD-1 2.5 ± 0.2b 2.1 ± 0.1a 2.0 ± 0.1a 2.0 ± 0.1a 2.2 ± 0.1a 2.1 ± 0.1a 1.9 ± 0.1a 2.0 ± 0.2a
∗
Standard deviation. Different letters represents significant differences (P < 0.05) between cultivars. Each observation is a mean ± SD of three replicates.
Table 2–Minimum bactericidal concentrations (mg/mL) of extracts of 8 cultivars of O. ficus-indica against growth of C. jejuni, V. cholerae, and C. perfringens.
Cactus pear cultivar CB R14 JL FM VN CV1 CF1 CR
5653 1.1 1.2 7.5 4.5 7.4 12.5 10.5 11.2
± ± ± ± ± ± ± ±
0.8∗a 0.2a 0.6c 0.6b 0.5c 0.6e 0.6d 1.0d
V. cholerae 11168 5.2 2.7 5.7 5.8 5.8 8.2 12 9.2
± ± ± ± ± ± ± ±
0.9b 0.5a 0.9b 0.4b 0.4b 0.9c 0.9d 0.5c
C. perfringens
1837 4.4 4.4 7.4 9.5 11 13 12 30
± ± ± ± ± ± ± ±
0.9a 0.5a 0.5b 0.5c 0.8d 0.8e 0.4de 0h
569 5.8 4.4 7.5 9.7 11 15 13 30
± ± ± ± ± ± ± ±
0.4b 0.5a 0.5c 0.5d 0.9e 0.8g 0.5f 0h
FD-1041 1.6 1.6 3.6 4.4 4.7 6.5 12 15
± ± ± ± ± ± ± ±
0.5a 0.9a 0.5b 0.5bc 0.5c 0.5d 0.5e 0.5f
FD-1 0.9 0.8 1.5 5 5.5 5.5 10 16
± ± ± ± ± ± ± ±
0.2a 0.3a 0.5a 0.6b 0.5b 0.5b 0.4c 0.8d
∗
Standard deviation. Different letters represents significant differences (P < 0.05) between cultivars. Each observation is a mean ± SD of three replicates.
pure methanol. The solution was adjusted with methanol to an absorbance of 0.7 ± 0.02 at 515 nm. DPPH radicals (3.9 mL) were placed in a test tube, and 100 μL aliquots of the extracts were added. The mixture was vortexed and kept in the dark for 30 min. Absorbance at 515 nm was determined. The percentage of radical scavenging activity (%RSA) was determined using the following equation: %RSA = [(control Abs − sample Abs)/control Abs)] × 100
Statistical analyses Data were analyzed using the general linear model (GLM) procedure from the Number Cruncher Statistical System version 6.0 software (NCSS, LLC). Differences between cultivars were determined using Tukey´s comparison test. The P ࣘ 0.05 was considered significant. Each experiment was performed in triplicate.
Results
the lowest MBCs (0.8 and 1.1 mg/mL, respectively, for the CB cultivar and 0.9 and 1.2 mg/mL, respectively, for the R14 cultivar). V. cholerae 569-B was the most resistant to the extracts (MBC of 5.8 for the CB cultivar and 4.4 for the R14 cultivar) (Table 2).
Total phenols and flavonoids The total extractable phenols and flavonoids were different between the cactus pear cultivars (P < 0.05) (Figure 1A and 1B). Total phenolic content was greatest in the CF1 cultivar (4.27 mg GAE/ g DW), followed by JL, CV1, CB, VN, R14, CR, and FM with contents of 3.80, 3.61, 2.77, 2.73, 2.37, 2.05, and 1.49 GAE/100 g DW, respectively (Figure 1B). Phenol measurements in extracts from the FM cultivar were approximately 65% lower than extracts from the CF1 cultivar. In general, the total phenolic content varied between cultivars of the cactus pear; however, the differences between the CF1, JL, and CV1, and the CB, VN, and R14 cultivars were not significant. Total flavonoid content in cultivars of the cactus pear ranged from 15.4 to 36.6 mg QE/g DW. The greatest and smallest values were recorded in the JL and R14 cultivars, respectively. The following trend was found in total flavonoid content: JL > VN > CR > FM > CF1 > CB > CV1 > R14 (36.6, 24.7, 22.5, 18.9, 18.1, 17.9, 16.0, and 15.3 mg QE/g DW, respectively) (Figure 1A). Extracts from the R14 cultivar contained 58% less flavonoids than JL. The flavonoid content of the JL cultivar was significantly higher than the other cultivars; however, no significant differences were observed between the R14, CV1, CB, CF1, and FM cultivars.
Antimicrobial activity Preliminary analyses of the antibacterial activities of extracts from the 8 cultivars of cactus pear indicated that they had inhibitory activities against all strains of C. jejuni/Coli, C. perfringens, and V. cholerae. Extracts of cultivars used for prickly pear production, CB and R14, showed the largest inhibition zones, which ranged from 1.7 to 2.5 cm and 1.5 to 2.1 cm, respectively. The other cultivars showed inhibition zones from 1.2 to 2.2 cm (Table 1). The cultivars CB and R14 also showed the lowest MBCs, which ranged from 0.8 to 5.8 mg/mL. The cultivars JL, FM, VN, CV1, CF1, and CR presented MBC values in the ranges of 1.5 to 7.5, 4.4 Radical scavenging activity to 9.7, 4.7 to 11.2, 5.5 to 15, 10.2 to 13.4, and 9.2 to >30 mg/mL, Significant differences in %RSA were found among the cultirespectively, depending on the strain tested (Table 2). C. perfringens vars. Cultivar VN had the greatest %RSA (40.31%), followed by FD-1 and C. jejuni 5653 were the most sensitive isolates, with FM, CF1, CR, JL, R14, CB, and CV1 (38.70%, 34.64%, 31.94%, Vol. 00, Nr. 0, 2014 r Journal of Food Science M3
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C. jejuni
Activities of cultivars of cactus pear . . .
A
d
ab
20
ab a
ab
a
10
% Polyphenolic groups
100
c bc
0
B
c
c
60
40
20
40
c
4 (mg GAE/g DW)
80
0
b 3
b
b
ab a
2
B
e
e d cd
DPPH. (%RSA)
5
% Non-Flavonoids % Flavonoids
A
30 (mg QE/g DW)
Total Flavonoid Content
40
Total Phenolic Content
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29.61%, 26.40%, 25.09%, and 19.98%, respectively) (Figure 2B). methanol extracts contained the best antimicrobial activities against Extracts from the CV1 cultivar had 48% lower DPPH radical- select microorganisms. MBCs of the extracts, which ranged from scavenging activity than the VN cultivar. 45.1 to 153 mg/mL, were higher than the MBCs of O. ficus-indica determined in this study. In a different study, chloroform extracts of O. dillenii were more active than those made with methanol to Discussion The cultivars of Opuntia used in this study have different uses, inhibit the growth of Gram-positive and Gram-negative microorcultivars Jalpa (JL), Villanueva (VN), and Copena V1 (CV1) are ganisms (Umar and others 2013). Ethanol extracts and fractions used as young tender pads for human consumption; Cristalino obtained by sequential ethanol fractionation of a mixture of 3 (CR), Real de Catorce (R14), Cardon Blanco (CB), are used for different cultivars of O. ficus-indica (Surfarina, Muscaredda, and fruit production; and Copena F1 (CF1), and Forrajero Mina (FM) Sanguigna) did not present antimicrobial activities against the miare used as forage. Cactus pear cladodes possess remarkable medic- croorganisms studied (Ginestra and others 2009). Similarly Kim inal properties (Stintzing and Carle 2005). Studies of O. ficus-indica and others (2002) reported that, although bactericidal effects of have focused on its potential antihyperglycemic (Halmi and oth- aqueous extracts of O. ficus-indica var. Saboten were not observed, ers 2012), analgesic, antiinflammatory, antiulcer, and cholesterol- there were inhibitory activities against Salmonella spp. and E. Coli lowering activities (Feugang and others 2006). However, although O157 (Kim and others 2002). Polyphenols, which have varied molecular structures, are comthe chemical compositions (Ginestra and others 2009; S´aenz and others 2010; Hern´andez-Urbiola and others 2011) and antioxi- mon constituents of plants. Plant phenolics include phenolic acids, dant properties (Bari and others 2012; Dhaouadi and others 2013) flavonoids, tannins, and, less common stilbenes and lignans (Dai of cactus pear cladodes are known, few studies have compared the and Mumper 2010). Phenols and flavonoids are bioactive compounds that have been related to restorative processes by their antioxidant properties of different cultivars. Comparisons of antimicrobial activities of different cactus pear antimicrobial properties and abilities to reduce free radical formacultivars are also scarce or not available. Among the available stud- tion and to scavenge free radicals (Daglia 2012). Analyses of various commercial powders and commercial vaies, Kim and others (2002) reported that extracts of O. ficus-indica var. Saboten contained antimicrobial agents that were effective rieties of cactus pear exhibited total phenol contents of 1.7 to against pathogenic bacteria, including 2 Vibrio species; however, 11.7 mg GAE/g (Guevara-Figueroa and others 2010). These reno information about bactericidal effects were provided (Kim sults were consistent with those found in this study. On the other and others 2005). Bari and others (2012) used solvents to extract the antimicrobial agents from O. monacantha, concluding that
30
c b
b
a 20
10
1
0 CF1
CB
CR
JL
FM
R14
Cultivars
VN
CV1
0 CF1
CB
CR
JL
FM
R14
VN
CV1
Cultivars
Figure 1–(A) Total phenol and (B) flavonoid content of extracts from 8 Figure 2–(A) Percentage of polyphenolic groups. (B) Radical scavenging O. ficus-indica cultivars. Different letters represent significant differences activities. Different letters represent significant differences (P < 0.05) (P < 0.05) between cultivars. between cultivars.
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Activities of cultivars of cactus pear . . . effects of the active compounds on the normal human microbial flora must be determined. Finally, the risks and benefits of potential applications must be determined. Work is in progress in our laboratory on the isolation and identification of the antimicrobial compounds and their mechanism of action against these pathogens.
Conclusions This study determined the antimicrobial and antioxidant activities of various full-grown cultivars of cactus pear. The CB and R14 cultivars contained the highest antimicrobial activities, and the VN and FN cultivars contained the highest antioxidant activities. This vegetable, which is distributed throughout the world, may be an excellent candidate for use as a nutraceutical or as a source of antioxidant compounds.
Acknowledgments This research was supported by the Consejo Nacional de Ciencia y Tecnolog´ıa de M´exico (CONACYT, grant # 105389). Eduardo S´anchez and Sandra Castillo were supported by a scholarship from CONACYT. Jorge D´avila-Avi˜na was supported by a posdoctoral fellowship from CONACYT.
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hand, the total phenol content of young cladodes of cactus pear (O. ficus-indica) were reported to be in the range of 17 to 40 mg/g sample (dry basis) (Guevara-Figueroa and others 2010; Medina-Torres and others 2011). Those values were higher than our findings. The variation may be due to differences in the cladode maturity, cultivation region, climate, and quantification methodologies (Guevara-Figueroa and others 2010; Kim and others 2013). Flavonoids are the most abundant polyphenols in the human diet. There are 6 subgroups of flavonoids, including flavones, flavonols, flavanols, flavanones, isoflavones, and anthocyanins, which are distinguished based on the oxidation state of the central C ring (Dai and Mumper 2010). Cho and others (2006) suggested that quercetin and its derivatives play major roles in the antioxidant effects of the cactus. Medina-Torres and others (2011) evaluated the bioactive compounds of cladodes after dehydration. They reported that the total flavonoid content ranged from 10 to 20 mg/g sample, using rutin as a reference compound. These values were slightly different than those found in this study, which ranged from 15.4 to 36.6 mg QE/g DW. On the other hand, GuevaraFigueroa and others (2010) reported that, in general, flavonoids were found in smaller amounts than phenolic acids, and that the Blanco and Manso (commercial varieties) contained the greatest flavonoid content (9.8 and 5.9 mg QE/g, respectively). These results also differed from the findings in this study, which determined that phenols were less abundant than flavonoids. Differences in the physicochemical compositions may be attributed to the maturity of cladodes and the environmental conditions (Rodr´ıguez-Garcia and others 2007; Guevara-Figueroa and others 2010). The DPPH assay was used to measure the hydrogen atomdonating activity and to evaluate free radical scavenging. Significant differences in %RSA were found among the cultivars, and activities ranged from 19% to 40% (Figure 2B). Similar results were obtained by Gallegos-Infante and others (2009), who reported a 42.5% RSA in extracts of O. ficus indica. In general, polyphenols contributed to increased radical scavenging activity in the cactus (Kim and others 2013), and values could be associated with the type of phenols present (Gallegos-Infante and others 2009). The low radical scavenging activity (%RSA) shown by Opuntia extracts may be explained by the abundance of monohydroxylated phenolic compounds, which have relatively low activities (Gallegos-Infante and others 2009). Several plant polyphenols possess antibacterial activities (Kurek and others 2011). In fact, phenolic acids such as gallic acid, coumaric acid, 3,4-dihydroxy-benzoic acid, 4-hydroxy benzoic acid, ferulic acid, and salicylic acid and flavonoids like iso-quercitrin, isorharmnetin 3-O-glucoside, nicotiflorin, rutin, narcissin, have been reported in the cladodes of Opuntia varieties, and many of these exhibited antimicrobial activity (GuevaraFigueroa and others 2010). The antimicrobial activity of these compounds rely in the ability to disturb the bacterial membranes of microorganisms, and affect the expression of various virulence factors (Ayala-Zavala and others 2011; Daglia 2012). Although in this study no significant correlation between the antibacterial activity and total phenolics was observed, the antibacterial activity could be related to a particular or a mixture of compounds. Medina and others (2006) reported similar results, indicating that the content of polyphenols does not correlate with antimicrobial activities in all cases. The antimicrobial activities of Opuntia extracts may be used to control foodborne pathogens, especially in food production. However, prior to use in this industry, appropriate methods for nontoxic extraction of the active compounds must be developed. Also, the
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