Industrial Crops and Products 49 (2013) 143149

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Industrial Crops and Products

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Chemical characterization, antioxidant and antibacterial activities of six Agave species from Sinaloa, Mexico
Yesmi Patricia Ahumada-Santos a,1 , Julio Montes-Avila a,1 , Magdalena de Jess b, Uribe-Beltrn a , Sylvia Pz Daz-Camacho a , Gabriela Lpez-Angulo a , Rito Vega-Avina Jos ngel Lpez-Valenzuela a , Jos Basilio Heredia c , Francisco Delgado-Vargas a,
Faculty of Chemical and Biological Sciences of the Autonomous University of Sinaloa (UAS), University City 80010, Culiacan, Sinaloa, Mexico Faculty of Agronomy of the UAS, Culiacan-El Dorado Highway km 17.5, 80000, Culiacan, Sinaloa, Mexico c Research Center for Food and Development from Culiacan, Culiacan-El Dorado Highway km 5.5, 80129, Culiacan, Sinaloa, Mexico
b a

a r t i c l e

i n f o

a b s t r a c t
Mexico has the greatest diversity of Agave species in the world and considering their uses in traditional medicine, these plants could be a rich source of bioactive compounds. In this research, we studied ve wild Agave species from Sinaloa, Mxico (A. rzedowskiana, A. impressa, A. ornithobroma, A. schidigera and A. angustifolia) and one cultivated (A. tequilana). They were evaluated for antioxidant- and antibacterial activities and chemical composition. Statistical analysis consisted of a completely randomized design with one factor analysis of variance and the means were contrasted by the Tukey test (p 0.05). Agave tequilana showed the highest antibacterial activity with a Minimal Inhibitory Concentration (MIC) of 5 mg/mL, while A. rzedowskiana showed the highest antioxidant capacity by the DPPH method; both activities were higher than those reported for other Agave species. Agave ornithobroma had a higher content of the evaluated phytochemicals, mainly triterpenes and steroids. An activity based separation was carried out with the hexane extract of A. rzedowskiana; chromatographic separation and analysis by gas chromatographymass spectrometry (GCMS) showed 2-(3,4-dimethoxyphenil)N-methylethanolamine, 9-octadecenoic acid and -tocopherol (vitamin E) as the most abundant compounds. -Tocopherol was clearly associated with the hexane extract antioxidant activity. This research showed a variety of phytochemicals in the studied Agave species and some of these species showed the highest antibacterial and antioxidant activities published up to date for this genus. 2013 Elsevier B.V. All rights reserved.

Article history: Received 26 November 2012 Received in revised form 3 April 2013 Accepted 27 April 2013 Keywords: Agave species from Sinaloa Mexico Phytochemicals Antioxidant activity Antibacterial activity

1. Introduction In recent years, infectious and chronic degenerative diseases have been the main mortality causes in the world (OMS, 2009). Antibiotics have been the most common agents against bacterial infections; however, public health concern is focused on bacterial resistance and the emergence of new pathogens. In Mexico, 4.7% of deaths in children younger than 5 y in 2004 were associated with diarrheas and 9.3% with pneumonia; in 2008, prevalence increased up to 6% and 13%, respectively (OMS, 2009; WHO, 2010). Oxidative stress is an important mechanism in the development of chronic degenerative diseases such as cardiovascular diseases and cancer. Thus, the balance between pro-oxidant and

Corresponding author. Tel.: +52 667 713 6615; fax: +52 667 713 6615. E-mail addresses: fdelgado@uas.edu.mx, fcodelgadovargas@gmail.com (F. Delgado-Vargas). 1 Equal contribution. 0926-6690/$ see front matter 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.04.050

antioxidant agents is essential for homeostasis and health (Eberhardt and Jeffery, 2006). Based on these facts, many researches have focused on the establishment of the antioxidant potential of plant compounds (e.g. phenolics, vitamins, terpenoids) as well as other human endogenous metabolites (Kaneria et al., 2009; Lim et al., 2007; Liu et al., 2009; Ou et al., 2002). Drugs commonly used for the treatment of infectious and chronic degenerative diseases are costly and not 100% effective; therefore, new and better therapeutic alternatives are required. Since the origin of the human being, the knowledge of medical traditional uses of plants has been accumulated, whereas scientists look for the chemicals associated with such uses. Plants are big reservoirs of biologically active compounds (Chea et al., 2007; Vaghasiya and Chanda, 2007; Verastegui et al., 2008) but only 2030% of the plants have been characterized for their secondary metabolites (Wink, 2010). Sinaloa has a great diversity of plants with about 3000 out of the 25,000 vascular plants registered for Mexico (Semarnat, and Villasenor-Ros, 2008), and there is 2008; Vega-Avina

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and Villasenor evidence of more than 3500 species (Vega-Avina Ros, Unpublished results), many of them threatened mainly by deforestation (WWF, 2012; Zulueta Rodrguez et al., 2006). The conservation and rational use of this oristic richness requires of scientic based knowledge. The Agave genus is endemic of the Americas and is distributed from the Southwest United States and South of Florida (US) to the tropical area of South America, including the Caribbean Islands (Garca-Mendoza, 2007; Garca Mendoza and Lott, 1994; Reveal and Hodgson, 2012). For North America, Gentry (1982) reported 197 taxa constituted by 136 species and 61 intraspecies. Mexico is the center of origin for the Agave species with 186 taxa (150 species and 36 intraspecies) (Garca-Mendoza, 2007), but 39 of them are in danger of extinction (Conabio, 1998). Oaxaca has the highest number of registered species with 37 and Sinaloa has 21 (Garca-Mendoza, 2007). Agave species have many traditional uses around the world and some of these are supported by scientic information. In ethnopharmacology, Agave species have been used for the treatment of diseases of bacterial etiology (e.g. gastrointestinal and wound infections, urologic disorders, dysentery) and against those associated with oxidative stress (e.g. cancer, diabetes and hypertension) (Cornara et al., 2009; Instituto Nacional Indigenista, 2009; Montesano et al., 2012; Semenya et al., 2012). On the other hand, anti-inammatory (Da Silva et al., 2002), antihypertensive (Duncan et al., 1999), immunomodulatory (Chen et al., 2009), antiparasitary (Orestes Guerra et al., 2008), and antifungal (Verastegui et al., 2008) activities have been demonstrated for Agave species. Our research group is interested in the chemical- and biological characterization of wild plant species from Sinaloa to establish their potential utility. We are involved in the generation of scientic information to support strategies for conservation and rational use of the regional/national oristic biodiversity. Thus, the objective of this research was the chemical characterization and evaluation of the antioxidant and antibacterial properties of ve wild Agave species and one cultivated from Sinaloa, Mexico.

regions of Mexico, and in Central America (Gentry, 1982), it was collected in the municipality of Elota, Sinaloa (74 masl, 23 51 22 N, R., 10142); Agave tequilana Weber grows 106 47 29 O; Vega-Avina wild from Jalisco to Oaxaca and Puebla (Gentry, 1982), and currently, it is found along all the states of the Pacic Coast of Mexico (McVaugh, 1989), it was collected from a commercial plantation in the municipality of Elota, Sinaloa (60 masl, 23 51 42.18 N, 106 50 44.7 O; Po-Len J.F., without number). The bacteriological culture media used were trypticase soy agar (TSA) and Mueller Hinton broth (Becton Dickinson). The reagents were analytical grade: 2,2-diphenyl-1-pycrilhydrazyl (DPPH), 2,2 -azino-bis(3-ethylbenzothiazolin)-6-sulfonic acid (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxilic acid (Trolox), 3,4,5-trihydroxybenzoic acid (gallic acid), 2,2 -azo-bis(2amidino propane) dihydrochloride (AAPH), 3 ,6 -dihydroxyspiro [isobenzofuran-1[3H],9 [9H]-xanthen]-3-one) (uorescein), linoleic acid, -carotene, Tween 80, Tween 40 and butylated hydroxytoluene (BHT) (Sigma-Aldrich). 2.2. Preparation of the methanol extracts of Agave species Leaves were washed, cut in small parts and freeze dried (The Virtis Company, New York, USA). Dried samples were milled in a waring blender (Osterizer, Mxico) to obtain meals which pass through a number 40-mesh sieve. Then, Agave meal (1 g) was mixed with methanol (10 mL), sonicated (15 min) (Sonicator FS30H Fisher Scientic, USA), centrifuged (28,620 g/15 min/20 C) and the supernatant was recovered. Extraction was carried out one more time with the residual meal. The recovered supernatants were mixed and the solvent was eliminated under vacuum (40 C) with a rotary evaporator (BCHI Labortechnick AG, Switzerland), followed by removal of any residual solvent in a vacuum oven at 40 C. Three methanol extracts were prepared for each Agave species and stored at 4 C in darkness until their use. 2.3. Preparation of the methanol and hexane extracts of Agave rzedowskiana A. rzedowskiana was chosen for the antioxidant bioguided strategy of plant compound purication; i.e., in the chromatographic separations only the fractions with the highest antioxidant activities were selected for the next step of purication/analysis. Agave meal extracts were obtained by Soxhlet extraction using a 1:21 (p/v) meal-solvent ratio; extraction temperature was 6772 C; meal was rst extracted with hexane (4 h) and then with methanol (12 h). The hexane and methanol extracts were obtained by elimination of the extraction solvents under vacuum (40 C); both extracts were stored at 4 C in darkness until their use. 2.4. Phytochemical analyses Secondary metabolites (i.e. triterpenes/steroids, avonoids, tannins, saponins, volatile coumarins, free anthracenic derivatives, alkaloids, reducing sugars and cardiotonics) were determined by tube and thin layer chromatography (TLC) tests as reported by Harborne (1973). The evaluation was done by direct visual observation and the content was estimated based on a relative scale of high, medium, low and trace. 2.5. Determination of the antibacterial activity Antibacterial activity was evaluated against Gram negative and Gram positive human pathogenic bacteria, four ATCC control strains (Staphylococcus aureus 29213, Enterococcus faecalis 29212, Escherichia coli 25922 and Pseudomonas aeruginosa 27853) and ve strains isolated from clinical samples (Streptococcus group A-4,

2. Materials and methods 2.1. Materials A minimum of ve adult plants were sampled for each of the wild Agave species from Sinaloa, Mexico during September 2010; samples were representative of the Agave species in the geographical area of collection. Two or more leaves were collected depending on the size of the plant, taking special care to avoid plant damage. The following description of each Agave species includes in parentheses the coordinates, the name of the collector, and assigned number in the herbarium of the Agronomy Faculty of the Autonomous University of Sinaloa (UAS). Agave impressa Gentry is distributed in the states of Jalisco, Nayarit and Sinaloa, it was collected in the low mountain range area of Tecualilla, Escuinapa, Sinaloa (300 meters above sea level, masl, 22 45 36 R., 10076); Agave ornithobroma GenN, 105 38 24 O; Vega-Avina try is distributed in Nayarit and Sinaloa, and it was collected from the Copala region, Concordia, Sinaloa (200 masl, 23 20 24 N, R., 10750); Agave rzedowskiana P. Car105 56 06 O; Vega-Avina rillo, R. Vega & R. Delgadillo is found in Jalisco and Sinaloa, and collected near La Petaca town, Concordia, Sinaloa (1700 masl, R., 11671); Agave schidig23 23 52 N, 105 48 37 O; Vega-Avina era Lem. is widely distributed in the states of the central and north regions of Mexico (Gentry, 1982), and it was collected from the low mountain range of the Baila town, Culiacan, Sinaloa (120 masl, R., 7814); Agave angusti24 11 30 N, 106 58 54 O; Vega-Avina folia Hawis distributed in the States of the coast, central and north

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145

S. aureus 3, E. coli A011, Salmonella enterica Typhi and Shigella dysenteriae), which were donated by the Laboratory of Bacteriology of the National Institute of Pediatrics, D.F., Mexico. Evaluation was carried out by the broth microdilution method as described by the CLSI (Clinical and Laboratory Standards Institute, 2009). Bacteria was incubated at 37 C for 1820 h; an inoculum suspension (1 108 UFC/mL) was prepared in saline solution (0.85%p/v) and Mueller Hinton broth was added to get 1x106 UFC/mL. U-bottom 96 microwell plates were used, 50 L of inoculum, 50 L of Mueller Hinton broth and 50 L of the extracts to be evaluated were dissolved in Tween 80 (10%v/v); the same mixture without the extract was used as negative control, while the positive control contained gentamicin (0.2516 g/mL) instead of the extract. Microwell plate was incubated (37 C/1820 h) and the Minimal Inhibitory Concentration (MIC) was determined by visual examination (absence of turbidity or button of growth at the bottom of the well). The Minimal Bactericidal Concentration (MBC) was determined based on the results of the MIC assay, TSA plates were inoculated with aliquots of every well of the MIC assay where bacterial growth was not observed, including that of the MIC value, and incubated (1820 h/37 C). The MBC value corresponded to the well with the minimal extract concentration that prevented bacterial growth in the TSA plates. Assays were carried out by triplicate. 2.6. Determination of the antioxidant activity of A. rzedowskiana The Agave species with the highest antioxidant activity (AA) was chosen based on the results obtained using the DPPH method. Hexane and methanol extracts were obtained from the selected Agave species and the AA was evaluated by the DPPH (2,2-diphenil-1-pycrilhydrazyl), ABTS (2,2 -azinobis(3-ethylbenzothiazolin)-6-sulfonic acid), ORAC (Oxygen Radical Absorbance Capacity), and -carotene bleaching (BCBM) methods; the results were expressed as TEAC (Trolox Equivalent Antioxidant Capacity; M Trolox equivalents/g d.w.) for the rst three methods and as percentage of antioxidant activity (%AA) relative to the control used for the last one. To determine the TEAC values, a calibration curve was prepared using Trolox as antioxidant (0800 M) and following the procedure established for the corresponding method of AA. 2.6.1. DPPH method It was determined as described by Brand-Williams et al. (1995). Briey, 0.2 mL of sample, or methanol for blank, were mixed with 1.8 mL of 150 M DPPH in a tube; the mixture was allowed to stand for 20 min in darkness and the absorbance was measured at 515 nm (Spectronic 20 Genesis, Spectronic Instruments, USA). The calibration curve for quantitation as TEAC was y = 0.2444x + 1.2819 (r2 = 0.9912), where y is the % of inhibition of the radical inactivation and x is the Trolox concentration. 2.6.2. ABTS method It was carried out as described by Re et al. (1999). The sample (0.05 mL) was mixed with 1.95 mL of ABTS + radical (5 mL of 7 mM ABTS and 88 L of 140 mM sodium persulfate), allowed to stand (10 min/37 C) and the absorbance was measured at 734 nm. The calibration curve for quantitation as TEAC was y = 0.1364x 0.6415 (r2 = 0.9989), where y is the % of inhibition of the radical inactivation and x is the Trolox concentration. 2.6.3. ORAC method The methanol and hexane extracts were dissolved in methanol and DMSO, respectively. The wells of a microwell plate were added with 25 L of the sample to be evaluated, 25 L of phosphate buffer for the blank or 25 L of Trolox (6.25100 M) to construct the standard curve. The microwell plate was placed in the uorescence

equipment (Synergy 2 SL, BioTek Instruments, USA), where 200 L of uorescein and 75 L of AAPH were automatically added. The uorescence intensity (485 nm (ex)/525 nm (em)) was measured for 75 min (37 C) with 1 min intervals (Huang et al., 2002). The calibration curve for quantitation as TEAC was y = x 0.0003 (r2 = 0.9912), where y is the net area under the curve and x is the Trolox concentration. 2.6.4. -Carotene bleaching method Determination was based in the method described by Velioglu et al. (1998). The reaction mixture was prepared with 50 mg of Tween 40, 6.25 L of linoleic acid, 500 L of -carotene (2 mg/mL in CH2 Cl2 ); solvent was eliminated under N2 (g) and 25 mL of H2 O2 were added. A blank mixture was prepared with the same procedure but without -carotene. Using 96 microwell plates, a blank was prepared by mixing 50 L of DMSO and 250 L of the blank mixture; 50 L of the sample and 250 L of the reaction mixture were used for sample evaluation; negative and positive controls contained 50 L of DMSO or BHT (at the same concentrations used for the samples), respectively. Plates were incubated at 50 C and the absorbance was measured (Multiskan Bichromatic, Fisher Scientic, USA) at 492 nm every 15 min during 2 h. The rate of the -carotene bleaching was calculated as R = [lna/b)]/t and the antioxidant activity as % Antioxidant Activity = [(Rcontrol Rsample )/Rcontrol ] 100; where a is the absorbance at time 0 and b at time t for t = 15, 30, 45, 60, 75, 90, 105 and 120 min. 2.7. Determination of the total phenolics content The total phenolics content (TPC) was determined by the FolinCiocalteu method as described by Singleton and Rossi (1965) with slight modications. Briey, 0.2 mL of the sample to be evaluated were mixed with 1.58 mL of distilled water and 0.1 mL of the FolinCiocalteu reagent; the components were mixed (2 min/40 C) in darkness and 0.3 mL of a sodium carbonate saturated solution were added; the mixture was allowed to stand for 30 min/40 C and the absorbance was read at 765 nm (Spectronic 20 Genesis, Spectronic Instruments, USA); water was used as blank. Quantication was carried out by using a gallic acid standard curve (0500 g/mL), which was prepared by the procedure described for the samples. TPC was expressed as mg of gallic acid equivalents/g d.w. (mg GAE/g d.w.) 2.8. Fractionation of the A. rzedowskiana hexane extract The A. rzedowskiana hexane extract (282 mg) was separated by thin layer radial chromatography (Chromatotron 7924T, T-Squared Technology, Inc. USA). The thin layer plate in the equipment (630 rpm) was prepared by passing 100 mL of hexane; hexane extract was injected and elution started with hexane (100 mL), followed by hexane: ethyl acetate mixtures of increasing concentration v/v in the second component (98:02, 600 mL; 97:03, 200 mL; 95:05, 100 mL; 90:10, 500 mL; 85:15, 1250 mL; 80:20, 150 mL). Fractions (5 mL each) were collected and analyzed by TLC on aluminum silica covered plates (Silica gel 60 GF254 Merck) developed with hexane: ethyl acetate (9:1 v/v); similar chromatographic fractions were mixed. 2.9. Analysis by Gas ChromatographyMass Spectrometry (GCMS) of the A. rzedowskiana fraction with the highest antioxidant activity The fraction with the highest antioxidant activity (5 mg/mL) was ltered (0.45 m PVDF, Titan, USA) and injected (5 L) to a GCMS HP 6890 equipment (Agilent Technologies, USA). Separation was

146 Table 1 Phytochemical analysis of Agave species. a Agave species Compounds Extractb A. impressa M E A C M E A C M E A C M E A C M E A C M E A C

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Triterpenes and/or steroids ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++

Flavonoids * * * * ++ * * * * * * * *

Tannins * ++ ++ ++ + * *

Saponins + * ++ + + +

Volatile coumarins +

Reducing sugars

A. ornithobroma

A. rzedowskiana

A. tequilana

A. schidigera

A. angustifolia

+ *

a The content was estimated as: ++, medium; +, low; *, trace; and , absence. An empty cell corresponds with non determined value. All the evaluated Agave species/extracts were negative for free anthracenics and alkaloids. b Extracts: M, methanol; E, ethanol; A, aqueous; and C, chloroform.

carried out in a QUADREX 007 column (30 m 0.25 mm i.d. 0.25 m) (Quadrex Corp., Woodbridge, CT, USA) using helium as the carrier gas; the ow started at 0.6 mL/min and then increased up to 0.9 mL/min with increments of 0.2 mL/min. The temperatures used were: injector, 250 C; oven temperature, 60 C, gradient of 5 C/min up to 200 C and 10 C/min up to 275 C, constant temperature until the end of the run (60.5 min). Mass detector was operated in the electron impact mode with 70 eV of energy. Temperatures of detector and quadrupole were maintained at 245 and 150 C, respectively (Fiorentino et al., 2009). 2.10. Purication of the main compound of the antioxidant fraction of A. rzedowskiana The A. rzedowskiana hexane extract (620 mg) was fractionated by preparative thin layer chromatography (20 20 cm Preparative TLC Glass Plates Si60 F254 2 mm) (155 mg/plate) using hexane: ethyl acetate (9:1 v/v) as mobile phase. The main band of the hexane extract was recovered, extracted with hexane, concentrated and analyzed by GCMS as described above. The identity of -tocopherol was corroborated by co-chromatography with a commercial pure standard. 2.11. Quantication of tocopherol A standard curve was prepared with -tocopherol acetate in hexane. GCMS analysis was performed as described for the analysis of the chosen fraction from A. rzedowskiana hexane extract (2.5 mg/mL). 2.12. Statistical analysis A completely randomized design with a single factor analysis of variance was used. For the rst part of the study, the factor was the Agave species with six levels (A. rzedowskiana, A. impressa, A. ornithobroma, A. schidigera, A. angustifolia and A. tequilana). For the second part, the factor was the type of extract for

A. rzedowskiana with two levels (methanol or hexane). Signicant differences were established by contrast of means using the Tukey HDS test (p 0.05). All statistical analyses were carried out with the software STAGRAPHICS Centurion XVI (Statpoint Inc., Warrenton, VA, USA). 3. Results and discussion 3.1. Phytochemical analysis The Agave species showed different compound families (Table 1). The results were similar to those reported for Agave intermixta, Agave vera and Agave sisalana (Garcia et al., 1999; Hammuel et al., 2011; Vaghasiya and Chanda, 2007). Chemical compounds belonging to the phytochemicals registered for the Agave species used in this research have been characterized and quantitated for other Agave species: free reducing sugars (Arrizon et al., 2010), ter penes (Pena-Alvarez et al., 2004), tannins (Castillo et al., 2010), avonoids (Morales-Serna et al., 2010) and saponins (Eskander et al., 2010). The variety of compounds in the studied Agave spp. could be associated with biological activities (e.g. antibacterial, antioxidant) previously established for compounds of every group of the phytochemicals determined. 3.2. Antibacterial activity Antibacterial activity was observed against ve of the nine evaluated bacteria. The MIC values obtained for four out of the six studied plants (Table 2) were similar to those reported for other Agave species. Remarkably, the antibacterial activities of A. tequilana and A. schidigera (MIC 5 mg/mL) were higher than those reported previously for Agave species, e.g. A. picta and A. sisalana showed MIC values in the range of 6 to 20 mg/mL, depending of the bacteria (Ade-Ajayi et al., 2011; Hammuel et al., 2011; Verastegui et al., 2008). The MIC values of ATCC strains to Gentamicin (Table 2) corresponded with published data (Lennette et al., 1987); thus, the antibacterial activities of the studied Agave species could be

Y.P. Ahumada-Santos et al. / Industrial Crops and Products 49 (2013) 143149 Table 2 Minimal Inhibitory Concentration (MIC, mg/mL) and Minimal Bactericidal Concentration (MBC, mg/mL) of methanol extracts from Agave species.a Bacterial strains Agave speciesb A. impressa MIC Streptococcus group A-4 Salmonella enterica Typhi Shigella dysenteriae Escherichia coli 25922 Pseudomonas aeruginosa 27853 Enterococcus faecalis 29212 Staphylococcus aureus 3 Escherichia coli A011 Staphylococcus aureus 29213
a b c

147

A. ornithobroma MIC 15 15 MBC

A. rzedowskiana MIC 15 MBC

A. tequilana MIC 5 5 5 10 5 MBC 10 15

A. schidigera MIC 5 10 5 10 MBC 10 15

G A. angustifoliac MIC 15 15 MBC

Gc MIC 0.5 0.25 4 1 1 4 0.5 0.5 1

MBC

15 15

Evaluated extract concentrations were 15, 10, 5 and 2.5 mg/mL, extracts were dissolved in 10% Tween 80 (v/v). The character stands for no activity up to the maximal tested concentration (15 mg/mL). G stands for gentamicin and evaluated concentrations were in the range 0.2516 g/mL. MIC values were in g/mL.

better contrasted with those generated in future studies with other plants using the same controls (ATTC strains/gentamicin). As it can be observed, the MIC values for the Agave species extracts, mixtures of compounds, were at least three orders of magnitude higher than that for pure gentamicin. Vaghasiya and Chanda (2007) studied the antibacterial activity of the methanol extract of A. vera leaves; their extract was not active against S. aureus ATCC 25923, P. aeruginosa ATCC 27853 and E. coli ATCC 25922, while in our study the same P. aeruginosa and E. coli ATCC strains were inhibited by the methanol extracts of A. tequilana and A. schidigera (Table 2). Considering the bactericidal activities (Table 2), the studied Agave species showed higher activity than that reported for A. sisalana (Ade-Ajayi et al., 2011; Hammuel et al., 2011); the results obtained with A. schidigera against S. enterica Typhi were very remarkable (Table 2). The antibacterial activity of Agave species has been associated with the presence of tannins, alkaloids, avonoids and saponins (Ade-Ajayi et al., 2011; Hammuel et al., 2011; Vaghasiya and Chanda, 2007). In this research, the phytochemical composition of the methanol extracts of the Agave species with the higher and lower antibacterial activities did not show great differences, except for the tannins content (Table 1); thus, further studies are required to identify the antibacterial compounds. The antibacterial activity of the Agave species used in this study (Table 2) was the highest reported up to date; however, this activity could be considered as weak (MIC > 1.6 mg/mL) (Aligiannis et al., 2001) and it was not considered for the bioguided fractionation.

Table 3 DPPH antioxidant activity and total phenolics content of methanol extracts of Agave species. Agave species Antioxidant activity (M TE/g d.w.)a 9.86 19.93 8.00 27.41 15.79 6.46 1.51w 0.49y 1.32w 3.35z 3.72x 1.24w Total phenolics content (TPC) (mg GAE/g d.w.)b 5.22 12.37 3.57 7.72 4.04 2.06 0.81x 2.35z 0.49w,x 0.55y 1.22w,x 0.25w

A. tequilana A. ornithobroma A. impressa A. rzedowskiana A. schidigera A. angustifolia

Results were expressed as: a M of Trolox Equivalents/g d.w. (M TE/g d.w.). b mg of Gallic Acid Equivalents/g d.w. (mg GAE/g d.w.). Values were the mean of three independent measurements standard deviation. Different letters in the same column (w, x, y, z) showed signicant differences (p 0.05).

3.4. Antioxidant activity of the hydrophobic and hydrophilic components of A. rzedowskiana The hexane and methanol extracts of A. rzedowskiana were obtained as the rst stage of chromatographic separation of components with AA. The methanol extract showed the highest AA but in the BCBM it was a pro-oxidant (Table 4). The hexane extract showed the highest inhibition of DPPH and ABTS radicals; however, when the results were expressed as M TE/g d.w., the values were higher for the methanol extract, which was associated with its higher extraction yield. The Agave AA has been scarcely studied; it was reported that an aqueous extract of a non-specied Agave

3.3. Antioxidant activity of the studied Agave species and their total phenolics content The methanol extract of A. rzedowskiana showed the highest AA by the DPPH method (Table 3) and it was chosen to carry on the bioguided fractionation. The methanol extract of A. ornithobroma had the highest TPC (Table 3), which was similar to the value reported by Wu et al. (2004) for a non-dened Agave sp. Taking into consideration the phytochemical analysis (Table 1), the TPC differences found among the studied Agave species could be associated with differences in the tannins content. The AA and TPC did not show signicant correlation (r = 0.74, P 0.05). This result could be explained by the presence of phenolics with higher AA or more reactive in the evaluated system, even though their TPC contents were lower than other samples; another explanation could be the participation of non-phenolic antioxidants. Scientic reports about the AA-TPC correlation have showed contrasting results, being positive, negative and in other cases null.
Table 4 Antioxidant activity of hexane and methanol extracts of A. rzedowskiana. Evaluation method Extract [Concentration, mg/mL] Hexane [1] Methanol [4] Hexane [1] Methanol [4] Hexane [1] Methanol [4] Hexane [1] Methanol [4] Antioxidant activity (M TE/g d.w.)a 7.76 2.18x 15.63 4.31y 9.76 1.82x 212.12 1.65y 46.27 10.10x 862.62 18.75y 71.55% 5.00%x 86.53%y

DPPH ABTS ORAC -carotene bleaching

Different letters in the column (x, y) showed signicant differences among extracts for each method. a Antioxidant activity was the mean standard deviation of three independent measurements. Values are expressed as M of Trolox Equivalents/g d.w. (M TE/g d.w.) but for the -carotene bleaching method as percentage of antioxidant activity.

148
-6

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x 10

12 8 6 4 2 10 14 18 22 26 30 34 38 42 46 50 54

Abundance

10

Table 5 Retention times and areas for the main components identied by GCMS in the fraction with the highest antioxidant activity (HE-F5) of the hexane extract of A. rzedowskiana.a Retention time (min) 30.546 33.068 33.123 33.374 36.956 39.328 41.613 42.458 45.855 47.078 48.646 49.294 Component n-Hexadecanoic acid 9-Octadecenoic acid 9-Octadecenoic acid Octadecanoic acid Bis(2-ethylhexyl) phthalate 3,4-Dimethoxy-N-methylbenzeneethanamine 4-Hexadecylbenzoic acid 2-(3,4-dimethoxyphenil)N-methylethanolamine -Tocopherol (vitamin E) 3,4-Dimethoxy-N-methylbenzeneethanamine Cholest-4-en-3-one (3)-Spiro[1,3ciclobutancholestane]-2 one -Sitosterol Pollinastanol 9,19-Ciclo-9-lanost-24en-3-ol 4-Stigmasten-3-one (S)-24-methyl-(3)-9,19ciclolanost-25-en-3-ol Area (%) 4.09 7.98 7.28 4.41 1.36 2.50 3.17 19.77 7.85 3.46 4.61 1.46

58

62

x 10

-6

14 12 10 8 6 4 2 10 15 20 25 30 35 40 45 50 55

B
c

Abundance

60

65

Time (min)
Fig. 1. GCMS chromatograms of the A. rzedowskiana samples. (A) Fraction with the highest antioxidant activity (HE-F5) obtained by radial chromatography from the hexane extract; and (B) band recovered by preparative TLC of the hexane extract. The main components were: (a) 9-octadecenoic acid; (b) 2-(3,4-dimethoxyphenil)N-methylethanolamine; and (c) -tocopherol. 51.148 52.189 53.600 55.857 56.089
a

1.64 2.61 7.00 4.98 1.69

In bold types are presented the compounds in the highest concentration.

sp., as well as extracts of phenolics and avonoids of A. americana, showed inhibition of the DPPH radical (Ben Hamissa et al., 2012; Rodrguez-Hernndez et al., 2000). Based on the results with the ORAC method, lipophilic components (hexane extract) showed lower AA values than those of higher polarity (methanol extract) (Table 4), a pattern similar to that reported by Wu et al. (2004). The differences registered between extracts and AA methods could be associated with differences in the composition of each extract, antioxidant compounds whose reactivity depends on the environment of the reaction mixture, as well as with the type of radical used; e.g. it has been reported that some antioxidants react fast with peroxyl radicals but they react slow, or are inactive against the DPPH radical, probably associated with steric hindrance (Prior et al., 2005). Our results suggested that the hexane extract of A. rzedowskiana had components with higher AA than those in the methanol extract, although these components are in low concentration in the meal; thus, the bioguided fractionation was carried out with the hexane extract of A. rzedowskiana.

3.6. Isolation and identication of the main compound in the antioxidant fraction of A. rzedowskiana Preparative TLC of the A. rzedowskiana hexane extract was used to obtain the main separated band. It was analyzed by GCMS (Fig. 1B) and demonstrated the presence of -tocopherol; moreover, 2-(3,4-dimethoxyphenil)-N-methylethanolamine was also identied after the purication process; this compound has been previously reported as a plant natural product, particularly, it has been isolated from peyote (Lophophora williamsii), a cactaceae with the same type of metabolism of the Agave species (Batis and RojasArchiga, 2002). 3.7. -Tocopherol analysis The -tocopherol concentration of the band recovered by preparative TLC of the A. rzedowskiana hexane extract was 413.63 g/mL. The antioxidant activity of the puried fraction was contrasted with a solution of commercially-pure -tocopherol (413.63 g/mL); the antioxidant activities by the DPPH method of these two samples did not show signicant differences (p < 0.05), showing values of 93.7% and 91.60%, respectively. Consequently, it was conrmed that antioxidant activity of the studied A. rzedowskiana fraction was mainly associated with its -tocopherol content. The -tocopherol is a well-known natural antioxidant, it is a liposoluble molecule that can inactivate free radicals, property probably associated with its structural phenolic group (AsensiFabado and Munn-Bosch, 2010). 4. Conclusions This paper had showed that the antioxidant activity of A. rzedowskiana and the antibacterial activity of A. tequilana were higher than those previously reported for other Agave species. Moreover, A. ornithobroma showed a more diverse range of phytochemicals among the studied Agave species. The bioguided characterization showed that the A. rzedowskiana antioxidant activity was

3.5. Fractionation of the hexane extract of A. rzedowskiana and analysis of the fraction with the highest antioxidant activity Seventeen fractions were collected from the radial chromatography of the A. rzedowskiana hexane extract; their antioxidant activity (DPPH method) showed radical inhibition in the range of 0.22% to 60.34%; fraction 5 of the hexane extract (HE-F5) was the best and it represented about 3.8% (w/w) of the methanol extract. GCMS analysis of HE-F5 (Fig. 1A) showed the presence of 2-(3,4-dimethoxyphenil)-N-methylethanolamine, 9-octadecanoic acid and -tocopherol as the compounds with the highest concentration. Peaks not numbered correspond to fatty acids, amines, carboxylic acids and sterols, ordered from low to high retention times (Table 5); taking into consideration the TLC and GCMS analysis, these are in lower concentrations. The established composition was similar to that reported for other Agave species (Abdel-Gawad et al., 2004). et al., 1999; Gutirrez et al., 2008; Pena-Alvarez

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mainly associated with non-polar compounds (hexane extract) and specically with the content of -tocopherol. This information provides the basis for a knowledge-based preservation strategy of the studied Agave species, which have very restricted geographic distribution and are endangered. Acknowledgments Authors acknowledge the nancial support of CONACYT, PROFAPI Universidad Autnoma de Sinaloa and COECYT, as well as the technical assistance of Laura Angulo Contreras of the Centro de Investigacin en Alimentacin y Desarrollo de Culiacn. References
Abdel-Gawad, M.M., El-Sayed, M.M., Abdel-Hameed, E.S., 1999. Molluscicidal steroidal saponins and lipid content of Agave decipiens. Fitoterapia 70, 371381. Ade-Ajayi, A.F., Hammuel, C., Ezeayanaso, C., Ogabiela, E.E., Udiba, U.U., Anyim, B., Olabanji, O., 2011. Preliminary phytochemical and antimicrobial screening of Agave sisalana Perrine juice (waste). J. Environ. Chem. Ecotoxicol. 3, 180183. Aligiannis, N., Kalpoutzakis, E., Mitaku, S., Chinou, I.B., 2001. Composition and antimicrobial activity of the essential oils of two origanum species. J. Agric. Food Chem. 49, 41684170. Arrizon, J., Morel, S., Gschaedler, A., Monsan, P., 2010. Comparison of the watersoluble carbohydrate composition and fructan structures of Agave tequilana plants of different ages. Food Chem. 122, 123130. Asensi-Fabado, M.A., Munn-Bosch, S., 2010. Vitamins in plants: occurrence, biosynthesis and antioxidant function. Trends Plant Sci. 15, 582592. Batis, A.I., Rojas-Archiga, M., 2002. El peyote y otros cactos alucingenos de Mxico. Biodiversitas 6, 1217. Ben Hamissa, A.M., Seffen, M., Aliakbarian, B., Casazza, A.A., Perego, P., Converti, A., 2012. Phenolics extraction from Agave americana (L.) leaves using hightemperature, high-pressure reactor. Food Bioprod. Process. 90, 1721. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. LWT Food Sci. Technol. 28, 2530. Castillo, F., Hernndez, D., Gallegos, G., Mendez, M., Rodrguez, R., Reyes, A., Aguilar, C.N., 2010. In vitro antifungal activity of plant extracts obtained with alternative organic solvents against Rhizoctonia solani Khn. Ind. Crops Prod. 32, 324328. Chea, A., Jonville, M.C., Bun, S.S., Laget, M., Elias, R., Dumenil, G., Balansard, G., 2007. In vitro antimicrobial activity of plants used in Cambodian traditional medicine. Am. J. Chin. Med. 35, 867873. Chen, P.Y., Kuo, Y.C., Chen, C.H., Kuo, Y.H., Lee, C.K., 2009. Isolation and immunomodulatory effect of homoisoavones and avones from Agave sisalana Perrine ex Engelm. Molecules 14, 17891795. Clinical and Laboratory Standards Institute, 2009. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard, 8th ed. PA, USA. Conabio, 1998. La Diversidad Biolgica de Mxico: Estudio de Pais, 1998. Comisin Nacional para el Conocimiento y Uso de la Biodiversidad, Mxico. Cornara, L., La Rocca, A., Marsili, S., Mariotti, M.G., 2009. Traditional uses of plants in the Eastern Riviera (Liguria, Italy). J. Ethnopharmacol. 125, 1630. Da Silva, B.P., De Sousa, A.C., Silva, G.M., Mendes, T.P., Parente, J.P., 2002. A new bioactive steroidal saponin from Agave attenuata. Z. Naturforsch. C: Biochem. Biophys. Biol. Virol. 57, 423428. Duncan, A.C., Jger, A.K., van Staden, J., 1999. Screening of Zulu medicinal plants for angiotensin converting enzyme (ACE) inhibitors. J. Ethnopharmacol. 68, 6370. Eberhardt, M.V., Jeffery, E.H., 2006. When dietary antioxidants perturb the thiol redox. J. Sci. Food Agric. 86, 19961998. Eskander, J., Lavaud, C., Harakat, D., 2010. Steroidal saponins from the leaves of Agave macroacantha. Fitoterapia 81, 371374. Fiorentino, A., Mastellone, C., DAbrosca, B., Pacico, S., Scognamiglio, M., Cefarelli, G., Caputo, R., Monaco, P., 2009. [delta]-Tocomonoenol: a new vitamin E from kiwi (Actinidia chinensis) fruits. Food Chem. 115, 187192. Garcia, M.D., Saenz, M.T., Puerta, R., Quilez, A., Fernandez, M.A., 1999. Antibacterial activity of Agave intermixta and Cissus sicyoides. Fitoterapia 70, 7173. Garca Mendoza, A., Lott, E.J., 1994. Agave. In: Davidse, G., Sousa Snchez, M., Chater, A.O. (Eds.), Flora Mesoamericana, Vol. 6. Alismataceae a Cyperaceae. Universidad Nacional Autnoma de Mxico (Mexico) and Missouri Botanical Garden, London, pp. 4044. Garca-Mendoza, A.J., 2007. Los agaves de Mxico. Ciencias 87, 1423. Gentry, H.S., 1982. Agaves of Continental North America. The University of Arizona Press, Tucson, Arizona, USA. Gutirrez, A., Rodrguez, I.M., del Ro, J.C., 2008. Chemical composition of lipophilic extractives from sisal (Agave sisalana) bers. Ind. Crops Prod. 28, 8187. Hammuel, C., Yebpella, G.G., Shallangwa, G.A., Magomya, A.M., Agbaji, A.S., 2011. Phytochemical and antimicrobial screening of methanol and aqueous extracts of Agave sisalana. Acta Pol. Pharm. Drug Res. 68, 535539. Harborne, J., 1973. Phytochemical Methods. Chapman and Hall, London. Huang, D., Ou, B., Hampsch-Woodill, M., Flanagan, J.A., Prior, R.L., 2002. Highthroughput assay of oxygen radical absorbance capacity (ORAC) using a multichannel liquid handling system coupled with a microplate uorescence reader in 96-well format. J. Agric. Food Chem. 50, 44374444.

Instituto Nacional Indigenista, 2009. Biblioteca digital de la medicina tradicional mexicana. Universidad Nacional Autnoma de Mxico, Mexico. http://www.medicinatradicionalmexicana.unam.mx/index.php (21.09.11). Kaneria, M., Baravalia, Y., Vaghasiya, Y.S.C., 2009. Determination of antibacterial and antioxidant potential of some medicinal plants from Saurashtra Region, India. Indian J. Pharm. Sci., 406412. Lennette, E.H., Balows, A., Hausler, W.L., Shadomy, H.J., 1987. Pruebas de susceptibilidad: tcnica de microdilucin y macrodilucin en caldo. In: Lennette, E.H., Balows, A., Hausler, W.L., Shadomy, H.J. (Eds.), Manual de Microbiologa Clnica. Editorial Mdica Panamericana S.A., Buenos Aires, Argentina. Lim, Y.Y., Lim, T.T., Tee, J.J., 2007. Antioxidant properties of several tropical fruits: a comparative study. Food Chem. 103, 10031008. Liu, L., Sun, Y., Laura, T., Liang, X., Ye, H., Zeng, X., 2009. Determination of polyphenolic content and antioxidant activity of kudingcha made from Ilex kudingcha C.J. Tseng. Food Chem. 112, 3541. McVaugh, R., 1989. Flora Novo-galiciana, Vol. 15: Bromeliaceae to Dioscoreaceae. The University of Michigan Herbarium, Ann Arbor. Montesano, V., Negro, D.S., Giulio, De Lisi, A., Laghetti, G., Hammer, K., 2012. Notes about the uses of plants by one of the last healers in the Basilicata Region (South Italy). J. Ethnobiol. Ethnomed. 8, http://www.ethnobiomed.com/content/8/1/15 Morales-Serna, J.A., Jimenez, A., Estrada-Reyes, R., Marquez, C., Cardenas, J., Salmon, M., 2010. Homoisoavanones from Agave tequilana Weber. Molecules 15, 32953301. OMS, 2009. Estadsticas Sanitarias Mundiales. Organizacin Mundial de la Salud. Suiza, ISBN 978 92 4 356381 7, www.who.int/evidence/bod Orestes Guerra, J., Meneses, A., Simonet, A.M., Macas, F.A., Nogueiras, C., Gmez, A., Escario, J.A., 2008. Saponinas esteroidales de la planta Agave brittoniana (Agavaceae) con actividad contra el parsito Trichomona vaginalis. Rev. Biol. Trop. 56, 16451652. Ou, B., Huang, D., Hampsch-Woodill, M., Flanagan, J.A., Deemer, E.K., 2002. Analysis of antioxidant activities of common vegetables employing oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays: a comparative study. J. Agric. Food Chem. 50, 31223128. A., Daz, L., Medina, A., Labastida, C., Capella, S., Vera, L.E., 2004. Pena-Alvarez, Characterization of three Agave species by gas chromatography and solidphase microextraction-gas chromatographymass spectrometry. J. Chromatogr. A 1027, 131136. Prior, R.L., Wu, X., Schaich, K., 2005. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food Chem. 53, 42904302. Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C., 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 26, 12311237. Reveal, J.L., Hodgson, W.C., 2012. Agavaceae.Agave.eFloras.org, United States. http://www.eoras.org/orataxon.aspx?ora id=1&taxon id=100796 (23.02.13). Rodrguez-Hernndez, H., Panduro-Cerda, A., Burciaga-Nava, J.A., Reyes-Romero, M.A., 2000. Antibrogenic effect of amole tuber (Agave sp) in experimental cirrhosis and its antioxidant and scavenging properties. J. Hepatol. 32, 139-139. Semarnat, 2008. Informe de la situacin del medio ambiente en Mxico. In: Edicin 2008. Compendio de estadsticas ambientales. Direccin General de Estadstica e Informacin Ambiental. Secretara del Medio Ambiente y Recursos Naturales, Mxico, D.F. Semenya, S., Potgieter, M., Tshisikhawe, M., Shava, S., Maroyi, A., 2012. Medicinal utilization of exotic plants by Bapedi traditional healers to treat human ailments in Limpopo province, South Africa. J. Ethnopharmacol. 144, 646655. Singleton, V., Rossi, J., 1965. Colorimetry of total phenolics with phosphomolybdicphosphotungstic acid reagent. Am. J. Enol. Viticult. 16, 144158. Vaghasiya, Y., Chanda, S.V., 2007. Screening of methanol and acetone extracts of fourteen Indian medicinal plants for antimicrobial activity. Turkish J. Biol. 31, 243248. R., Villasenor-Ros, Vega-Avina, J.L., 2008. Listados orsticos de Sinaloa I. Flora del municipio de Culiacn, Sinaloa, 1st ed. Universidad Autnoma de Sinaloa, Mxico. R., Villasenor-Ros, Vega-Avina, J.L., Unpublished results. Catlogo de la ora de Sinaloa, Culiacn, Sinaloa, Mexico. Velioglu, Y.S., Mazza, G., Gao, L., Oomah, B.D., 1998. Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. J. Agric. Food Chem. 46, 41134117. Verastegui, A., Verde, J., Garcia, S., Heredia, N., Oranday, A., Rivas, C., 2008. Species of Agave with antimicrobial activity against selected pathogenic bacteria and fungi. World J. Microbiol. Biotechnol. 24, 12491252. WHO, 2010. World Health Statistics 2010. World Health Organization, France. Wink, M., 2010. Biochemistry of Plant Secondary Metabolism, 2nd ed. John Wiley & Sons. Wu, X., Beecher, G.R., Holden, J.M., Haytowitz, D.B., Gebhardt, S.E., Prior, R.L., 2004. Lipophilic and hydrophilic antioxidant capacities of common foods in the United States. J. Agric. Food Chem. 52, 40264037. WWF, 2012. Bosques Mexicanos. The Worlds Wild Life Mexico, Mexico, D.F. http://www.wwf.org.mx/wwfmex/prog bosques.php (23.02.12). Zulueta Rodrguez, R., Trejo Aguilar, D., Lara Capistrn, L., Lpez Moctezuma, H., Moreira Arana, C.E., 2006. Es til la ora de la selva baja caducifolia? La Ciencia y el Hombre. Revista de Divulgacin Cientca y Tecnolgica de la Universidad Veracruzana 19, http://www.uv.mx/ cienciahombre/revistae/vol19num1/articulos/ora/index.htm