Effect of Agave americana and Agave salmiana ... - ACS Publications

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Effect of Agave americana and Agave salmiana Ripeness on Saponin Content from Aguamiel (Agave Sap) Ana María Leal-Díaz,† Liliana Santos-Zea,† Hilda Cecilia Martínez-Escobedo,† Daniel Guajardo-Flores,† Janet Alejandra Gutiérrez-Uribe,*,† and Sergio Othón Serna-Saldivar† †

Centro de Biotecnología FEMSA, Tecnológico de Monterrey, Avenida Eugenio Garza Sada 2501 Sur, CP 64849, Monterrey, N.L. México S Supporting Information *

ABSTRACT: Steroidal saponins have shown beneficial health effects. Agave spp. leaves and rhizomes are sources of these compounds, but their presence has not been reported in the aguamiel. Aguamiel is the sweet edible sap from mature agave, and its quality is influenced by the plant ripening stage. The purpose of this research was to identify and quantitate saponins in aguamiel from Agave americana and Agave salmiana at two ripening stages. Saponins and sapogenins were identified with HPLC/ ESI-MS/TOF and quantitated with HPLC/ELSD. Results proved the presence of saponins derived from kammogenin, manogenin, gentrogenin, and hecogenin. The saponin content in aguamiel from immature A. salmiana was 2-fold higher (478.3 protodioscin equivalents (PE) μg/g aguamiel (DM)) compared with A. americana (179.0 PE μg/g aguamiel (DM)). In both species, saponin content decreased when plants reached sexual maturity. This should be considered before evaluating the effects of Agave spp. as a source of bioactive saponins. KEYWORDS: Agave americana, Agave salmiana, aguamiel, kammogenin, ripeness, sapogenin, saponin

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INTRODUCTION The genus Agave belongs to the Agavaceae family and includes more than 300 species. These plants are found in arid and tropical regions of the western hemisphere, particularly Mexico.1 The relationship between humans and agaves date as early as 7000 BCE.2 During the late postclassical (1150− 1521 C.E.) period, agave cultivation allowed life to be sustained in arid lands where no other food or water were available.3 These plants are rosette, monocotyledoneous, and flower only once toward the end of their life cycle.1,4 They are also succulent plants; therefore, they can store a vast amount of sap because of its rapid water uptake and minimum loss during drought.5 Anatomically, they have two main aerial parts: the long spiked leaves and the stem in which aguamiel is accumulated. Aguamiel can be defined as the edible agave sap collected from the stem of mature plants. This sweet and translucent liquid has been consumed since pre-Columbian times.6 Aguamiel may be consumed fresh as a beverage, fermented into the traditional Mexican alcoholic beverage called pulque, or concentrated.7 High fructose agave syrup has gained popularity because of its low glycemic index8 and is defined as the natural sweet substance produced by hydrolyzing agave oligosaccharides exclusively.9 To achieve this, the agave plant is harvested, the leaves are removed, and the remaining stem is processed.10 Aguamiel concentrate is different from agave syrup because the plant is not harvested; aguamiel is collected and concentrated by heat.7 The most suitable species to obtain aguamiel are Agave americana, Agave salmiana, Agave atrovirens, Agave mapisaga, and Agave ferox.4 Rather than plant age, the ripening stage is considered the most relevant factor affecting sugar accumulation in the stem,11 directly influencing the aguamiel © XXXX American Chemical Society

sweetness. Nevertheless, it may be extracted earlier to meet the market demand, but this practice compromises its quality and yield. During the long plant vegetative period, fructans are synthesized and accumulated in the stem;11−13 however, when the plant enters the reproductive stage, usually after 6−8 years, fructans are then hydrolyzed into fructose and glucose to meet the great energy demand required to generate and maintain the flower stalk.11 At this point, the agave sap will have a sweeter taste and may be consumed as aguamiel. To prepare the plant for aguamiel extraction, at the onset of the reproductive stage (prior to generation of the flower stalk), the apical meristem is manually removed. Inside the created cavity in the stem, the sweet sap will be accumulated and collected twice a day for 2−6 months.14,15 The volume extracted is affected by the species, plant ripeness, soil nutrients, and water availability.16 Most of the recent research on the agave stem and sap has been focused on fructan analysis.11,13,15,17,18 A previous study reported that aguamiel from A. mapisaga contained 11.5% of dry matter, mainly composed of sugars (75%; among them, 10% fructans.15 Agave has also been studied with regard to its health properties as a food or plant extract related to certain secondary metabolites.7 In the leaves and rhizomes, the occurrence of steroidal saponins is well documented.7,19,20 The saponin structure consists of a nonpolar aglycone called sapogenin, coupled to one or more sugar moieties, which may be attached as one, two, or three side chains. Saponins have great structural diversity because of the variability in the aglycone, sugar composition, and location.21,22 These compounds contribute to Received: February 16, 2015 Revised: March 26, 2015 Accepted: March 26, 2015

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DOI: 10.1021/acs.jafc.5b00883 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

mL of n-butanol, and the mixture was shaken for 20 min at 250 rpm and 37 °C. Samples were then centrifuged for 5 min (3000 rpm and 4 °C). The organic phase was collected and dried under vacuum at 40 °C for 2 h. This extract was used to evaluate the saponin content. To release the sapogenin, all the crude saponin extract was hydrolyzed by adding 5 mL of 2 N HCl and left for 60 min in a water bath at 100 °C. After hydrolysis, samples were neutralized (pH 7.0 ± 0.1) with 2 N NaOH. The neutralized samples were brought to a volume of 25 mL with distilled water, and this solution was added to 25 mL of n-butanol. The mixture was sonicated for 1 min and then centrifuged for 5 min (3000 rpm and 4 °C). The organic phase rich in sapogenins was collected and dried under vacuum (2 h at 40 °C). This extract was used to evaluate the sapogenin content. Prior to analysis, all the samples were resuspended in 2 mL of methanol/water (1:1) HPLC grade and filtered through a syringe filter, pore size 0.45 μm, made of hydrophilic polypropylene (Pall Life Science, Ann Arbor, MI). All extractions were performed in triplicate. Saponin and Sapogenin Analysis. Saponins and sapogenins were analyzed using a model 1100 high performance liquid chromatography coupled to electrospray ionization mass spectrometer equipped with a model G1969A time-offlight (HPLC/ESI-MS/TOF) detector (Agilent Technologies, Santa Clara, CA). Conditions were adapted from a methodology to analyze steroidal saponins from Taiwanese yam with some modifications detailed below.38 Separation was performed at 25 °C. The column used was a 150 mm × 4.6 mm i.d., 5 μm, Zorbax Eclipse XDB-C18 with a 12.5 mm × 4.6 mm i.d. guard column of the same material (Agilent Technologies, Santa Clara, CA). The sample (20 μL) was eluted at 0.8 mL/min flow rate with a gradient consisting of solvent A, HPLC-grade water containing 0.1% formic acid; and solvent B, HPLC-grade acetonitrile containing 0.1% formic acid. The gradient used was as follows: 0→8 min isocratic at 28% B; 8→28 min, increased to 55% B; 28→31 min, increased to 100% B; 31→40 min, isocratic at 100% B. Mass spectra were collected using an electrospray source in positive mode (ESI+) under the following conditions: m/z 300−1500 range; nitrogen gas at 350 °C and 9.0 L/min flow rate; 45 psig nebulizer pressure; 4000 V capillary voltage; fragment voltage 145 V; and skimmer at 40 V. Sapogenin identifications were based on the accurate mass previously reported for agave except for hecogenin, which was also confirmed with a commercial standard. For the identification of the glycosylated forms, ionization tests were performed to fragment the molecular ion and obtain the corresponding fragmentation pattern, which was compared with that previously reported for agave saponins. Extracted ion chromatograms were analyzed considering the saponins and sapogenin accurate mass (±0.05 units) using Analyst QS 1.1 software (Applied Biosystems, Carlsbad, CA). Saponin and Sapogenin Quantitation. Quantitative analysis was performed with a 1200 Series HPLC coupled to an evaporative light scattering detector (HPLC/ELSD) (Agilent Technologies, Santa Clara, CA). Chromatographic conditions were the same as described above. ELSD conditions were adapted from a methodology to analyze steroidal saponins from Taiwanese yam.38 Detection was accomplished using nitrogen as the drying gas, a pressure of 3.4 bar and tube temperature of 45 °C, gain of 9, and sampling time of 33 ms. All the saponins from the same aglycone were quantitated together. Sapogenin concentrations were obtained as hecogenin

the plant’s defense systems to protect it from insect attack, and many are known to have antimicrobial and antifungal properties.22,23 Among the beneficial health properties attributed to saponins are hypocholesterolemic, anti-inflammatory, immunostimulant, antiobesity, and antiparasitic; all are proven on animal models.23−27 Cell-based assays have also proven cytotoxic and antimicrobial effects.28−30 It has been suggested that the saponin content in agave species is affected by the ripening stage. The sapogenin structure and content are also affected by the agave species. Blunden et al.19 evaluated 34 agave species and identified 12 sapogenins, with hecogenin and gentrogenin the most commonly found. In addition, hecogenin has been widely studied because of its use as a raw material to produce cortical hormones31 and has been reported in dried leaves from 22 agave species.19,28,32−34 Furthermore, Pinos-Rodriguez et al.35,36 concluded in two studies with A. salmiana, that the total saponin content was lower in ripe plants compared with immature ones. All these studies have been performed in agave leaves or rhizomes. To the best of our knowledge, there is no study performed to characterize the saponin content in aguamiel. Thus, the aim of this research was to identify and quantitate saponins in aguamiel from A. americana and A. salmiana at two ripening stages. It is important to evaluate the effect of species and maturity to standardize the sap and characterize the saponin content for further functional activity correlations. This would be a starting point for the development of functional foods derived from agave sap.

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MATERIALS AND METHODS Chemical Reagents. HPLC grade water, ethanol, and acetonitrile were obtained from BDH (Poole, UK). The reactive grade n-butanol solvent used for extraction was ́ obtained from Desarrollo de Especialidades Quimicas (Monterrey, Mexico). Hecogenin standard was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), and protodioscin, from Sigma-Aldrich (St. Louis, MO). Biological Material. Aguamiel samples were donated by AGMEL S.A de C.V. and collected at General Cepeda County, in Coahuila, Mexico (25° 22N, 101° 28W) at an altitude of 1410 m above sea level. The plant was identified by Dr. Marcela González Á lvarez from the Universidad Autónoma de Nuevo León (UANL) in Mexico. A voucher specimen was deposited in the herbarium of the UANL under the numbers 025618 for A. americana and 025619 for A. salmiana. Eight-year-old plants from A. americana and A. salmiana were used in two ripeness stages: immature plants prior to entering into the reproductive stage and mature plants at the onset of reproductive stage. When the agave enters the reproductive stage, its shoot apex is thinned, displaying black, shiny spines.11 Agaves were prepared following the traditional process to collect the aguamiel.14 Aguamiel was collected during the month of May 2013. Samples were immediately placed on dry ice, transported to the laboratory, and stored at −20 °C until analysis. A pool of eight agaves per ripeness stage and species were used, for a total of 32 agaves. Dry matter (DM) was determined by gravimetric means (AOAC 425.45).37 Sample (2 g) was mixed with 0.5 g of dry silicon dioxide (DEQ, Monterrey, Mexico) and dried at 60 °C for 24 h. Weight was recorded before and after drying to obtain the percentage of DM. Saponin Extraction and Sapogenin Release. To obtain the saponin crude extract, 50 mL of aguamiel was added to 50 B


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Table 1. Sapogenin Quantitative Analysis in the Hydrolyzed Aguamiel from A. americana and A. salmiana at Two Ripening Stages and Total Dry Matter (DM) A. salmiana sapogenin (HE μg/g aguamiel (DM))a,b,c kammogenin manogenin gentrogenin hecogenin total sapogenin fresh aguamiel dry matter

A. americana

immature 41.81 7.14 5.11 5.79 59.85 11.91

± ± ± ± ± ±

mature

12.83 a 1.34 a 0.14 a 0.27 a 14.58 a 0.04 a

16.37 5.63 4.87 5.39 32.26 11.73

± ± ± ± ± ±

2.31 0.50 0.30 0.31 3.42 0.07

immature b b a ab b a

4.46 3.77 3.59 3.75 15.57 13.67

± ± ± ± ± ±

0.39 0.19 0.20 0.02 0.80 1.77

mature c c b bc c a

2.14 2.01 1.95 1.99 8.09 11.57

± ± ± ± ± ±

0.04 0.03 0.01 0.04 0.12 0.07

c d c c c a

HE, hecogenin equivalents μg/g aguamiel dry matter. bData is expressed as mean ± SD of three independent extractions. cMean values in each row with different letter are significantly different (p < 0.05). a

Table 2. Saponin Quantitative Analysis in Aguamiel from A. americana and A. salmiana at Two Ripening Stages A. salmiana saponin (PE μg/g aguamiel (DM)) a

kammogenin glycosides manogenin glycosides gentrogenin glycosides hecogenin glycosides total saponin content

b,c,d

immature 321.9 55.7 51.4 49.3 478.4

± ± ± ± ±

8.0 0.7 2.0 1.2 5.6

A. americana mature

a a a a a

200.9 50.2 50.4 24.1 325.7

± ± ± ± ±

11.8 b 3.0 a 0.9 a 0.5 b 15.7 b

immature 97.9 23.2 29.5 28.3 179.0

± ± ± ± ±

1.3 0.8 1.2 1.2 2.2

mature c b b a c

22.6 12.2 13.2 12.8 60.5

± ± ± ± ±

0.8 0.2 0.2 0.3 1.1

d c c c d

Saponins sharing the same aglycone were quantitated together. bPE, protodioscin equivalents μg/g aguamiel dry matter. cData are expressed as mean ± SD of three independent extractions. dMean values in each row with different letter are significantly different (p < 0.01).

a

equivalents (HE) using a standard curve from 10 to 125 ppm. Results were reported in HE μg/g aguamiel (DM). Saponin concentrations were obtained as protodioscin equivalents (PE) using a standard curve from 10 to 500 ppm. Results were reported in PE μg/g aguamiel (DM). Statistical Analysis. All analyses were performed in triplicate, and results are expressed as mean ± standard deviation. Statistical analyses were conducted by one-way ANOVA, and differences among means were compared with the Tukey test using a level of significance of p < 0.01. The software used was GraphPad Prism version 5.0b.

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RESULTS AND DISCUSSION Aguamiel dry matter (DM) content ranged from 11.57 to 13.67%, and it was not affected by the agave species or ripening stage (Table 1). These results are in agreement with previous studies in aguamiel in which a DM of 11.5% was reported.15 Analysis and Quantitation of Sapogenins. Four sapogenins (kammogenin 1, manogenin 2, gentrogenin 3, and hecogenin 4) were released from their saponins by acid hydrolysis (Figures 1 and 2). These were detected across both agave species and ripening stages. These sapogenins have been previously reported in dried agave leaves after an acid hydrolysis treatment.19,32 Kammogenin and manogenin have also been reported in callus cultures from A. ameniensis.20 Even though kammogenin was the most abundant sapogenin found in aguamiel from both agave species, it has been previously quantitated as an aglycone only in callus cultures. These results also illustrate that the agave species and ripening stage did not affect the sapogenin diversity; however, the sapogenin content was, indeed, affected by both the species and the ripeness stage (Table 1). Aguamiel from immature A. salmiana contained more than twice the total amount of sapogenins compared with immature A. americana (59.85 ± 14.58 and 15.57 ± 0.80 μg HE/g aguamiel (DM), respectively). In both species, sapogenin content decreased almost half when the plant approached the reproductive stage. Among the four sapogenins identified,

Figure 1. Chemical structures of the sapogenins kammogenin, manogenin, gentrogenin, and hecogenin detected in the hydrolyzed aguamiel extract from mature and immature A. salmiana and A. americana.

kammogenin was the most abundant, regardless the species and ripening stage. Contrary to what was observed in aguamiel, in callus cultures from A. amaniensis, kammogenin content (16 μg/g dry callus) was half the manogenin content (32 μg/g dry callus).20 According to a sapogenin screening in 34 agave species,19 the species with the highest hecogenin content was Agave franzosini Nissen, with 0.46% leaf dry weight, in which A. americana and A. salmiana had 0.4% and 0.12%, respectively. These results differ from ours because A. salmiana was a better source of hecogenin as compared with A. americana. It is important to mention that in the above analysis, the agave ripeness stage was not standardized, and the plant age was not reported. C


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Figure 2. (A−D) Positive-ion mass spectra of kammogenin (1), manogenin (2), gentrogenin (3), and hecogenin (4) detected in the hydrolyzed aguamiel extract and (E) HPLC/ELSD chromatograms of the hydrolyzed aguamiel from A. salmiana an A. americana at two ripening stages.

607.31 ([M − 2 hexose − 2 pentose]+), and m/z 447.31 ([M − 3 hexose −2 pentose]+). This saponin has been recently isolated from the flowers and leaves of Agave offoyana and identified as magueyoside A.39,40 Compound 7 showed characteristic ion mass spectra at m/z 1249.54 ([M + Na]+), m/z 1117.50 ([M − 1 pentose + Na]+), m/z 609.36 ([M − 3 hexose −1 pentose]+), and m/z 447.31 ([M − 4 hexose − 1 pentose]+). Compound 8 exhibited the presence of ions at m/z 1219.56 ([M + Na]+), m/z 1087.56 ([M − 1 pentose + Na]+), m/z 609.36 ([M − 2 hexose −2 pentose]+), and m/z 445.31 ([M − 3 hexose − 2 pentose]+) and has also been found in the flowers from A. offoyana and was identified as magueyoside D.40

Analysis and Quantitation of Saponins. Saponins from A. americana and A. salmiana were exhaustively extracted. A total of eight saponins derived from kammogenin (compounds 5 and 6), manogenin (compounds 7 and 8), gentrogenin (compounds 9 and 10), and hecogenin (compounds 11 and 12) were detected in aguamiel from both ripening stages of aguamiel (Figure 3A). Except for compounds 5, 7, and 10, all the detected saponins had been previously reported. Compound 5 mass spectra exhibited characteristic ions at m/ z 1247.56 ([M + Na]+), m/z 1115.51 ([M − 1 pentose + Na]+), m/z 607.31 ([M − 3 hexose − 1 pentose]+), and m/z 447.31 ([M − 4 hexose − 1 pentose]+) (Figure 3B). Compound 6 exhibited the presence of ions at m/z 1217.56 ([M + Na]+), m/z 1085.51 ([M − 1 pentose + Na]+), m/z D


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Figure 3. Positive ion mass spectra from compounds 1−8 and HPLC/ELSD chromatograms obtained from aguamiel from A. salmiana and A. americana at two ripening stages.

Compound 9 exhibited the presence of ions at m/z 1201.53 ([M + Na]+), m/z 591.35 ([M − 2 hexose − 2 pentose]+), and m/z 429.30 ([M − 3 hexose − 2 pentose]+) as magueyoside H previously reported in A. offoyana leaves.39 Compound 10 presented characteristic ion mass spectra with m/z 1069.47 ([M + Na]+), m/z 591.35 ([M − 2 hexose − 1 pentose]+), and m/z 429.30 ([M − 3 hexose − 1 pentose]+), and therefore, it was tentatively similar to magueyoside H without a pentose. Finally, both hecogenin glycosides have been identified in different agave species. Compound 11 presented ion mass at m/z 1203.56 ([M + Na]+), m/z 593.38 ([M − 2 hexose − 2 pentose]+), and m/z 431.32 ([M − 3 hexose − 2 pentose]+) characteristic of those reported for cantalasaponin 4 in flowers from A. offoyana and in dried rhizomes from A. cantalana.40,41 Compound 12 showed the presence of ions at m/z 1071.49 ([M+ Na]+), m/z 593.38 ([M − 2 hexose −1 pentose]+), and m/z 431.32 ([M − 3 hexose −1 pentose]+) similar to those reported for a saponin identified in A. americana leaves,42 Agave cantala rhizomes,41 Agave sisalana leaves,30 and A. offoyana.40 This compound has been reported under different names:

agavoside C′, agavesaponin C′, agaveside C′, and cantalasaponin 2.41,42 Among the aguamiel saponins that have been previously detected, many have shown beneficial properties. Compounds 6, 8, and 9 identified as magueyosides A, D, and H, respectively, showed greater phytotoxicity compared with the commercial herbicide Logran.39,40 In addition, compound 8 extracted from A. sisalana, proved to be cytotoxic to human cell lines for gioblastoma, lung cancer, and breast cancer cell lines.30 The saponin content was affected by the plant ripening stage and species. When the plant evolved from immature vegetative to the reproductive stage, the saponin content significantly decreased in both species. This reduction was more pronounced in A. americana (66%) compared to A. salmiana (32%). These results concur with two previous studies with A. salmiana leaves in which total saponin content decreased significantly upon plant ripening.35,36 The correlation of the physiological phase and saponin content has been demonstrated in different plants.43 In agreement with our results, the highest diosgenin concentration in the annual plant Trigonella foenum-graecum was observed in E



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young plants and decreased upon plant maturity.44 In addition, in Phytolacca dodecandra berries, researchers found that young racemes contained the highest saponin concentration that decreased in the mature ones without changing the saponin proportion.45 It has also been suggested that saponin content and distribution may vary depending on the protection needed.21 Decrease upon maturity may be explained by the fact that these compounds contribute to the plant’s defense systems, because many saponins are known to be antimicrobial or antifungal, and to protect from insect attack.22,23 A high saponin content in immature agave could serve as a protector to keep predators controlled, ensure full plant development, and guarantee the entrance to the reproductive stage. However, when the plant enters the reproductive stage, its pollination success is strongly correlated to nectar-feeding bats, birds, and insects.46 This could explain the decrease in the saponin content to decrease pollinators deterrence and guarantee pollination. Taking in total the presented information, the results of this investigation prove for the first time the presence of saponins in the edible sap or “aguamiel”, which decreased with plant ripening. Moreover, A. salmiana yielded a greater amount of saponins compared with A. americana. Kammogenin saponins were the most abundant, regardless of the agave species and ripening stage. Aguamiel and its byproducts should be considered an edible source of saponins for further studies because these important phytochemicals have potential positive health implications.

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ACKNOWLEDGMENTS

We are also grateful to AGMEL S.A. de C.V. for providing the aguamiel samples.

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ABBREVIATIONS USED DM, dry matter; ELSD, evaporative light scattering device; MS2, tandem MS scan; HE, hecogenin equivalents; PE, protodioscin equivalents; UANL, Universidad Autónoma de Nuevo León

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REFERENCES

(1) Jin, J.-M.; Zhang, Y.-J.; Yang, C.-R. Four new steroid constituents from the waste residue of fibre separation from Agave americana leaves. Chem. Pharm. Bull. 2004, 52, 654−658. (2) Radding, C. The children of Mayahuel: agaves, human cultures, and desert. Landscapes in northern Mexico. Environ. Hist. 2012, 17, 84−115. (3) Evans, T. S. The productivity of maguey terrace agriculture in central Mexico during the aztec period. Lat. Am. Antiq. 1990, 1, 117− 132. (4) Escalante, A.; Giles-Gómez, M.; Hernández, G.; Córdova-Aguilar, M. S.; López-Munguía, A.; Gosset, G.; Bolívar, F. Analysis of bacterial community during the fermentation of pulque, a traditional Mexican alcoholic beverage, using a polyphasic approach. Int. J. Food Microbiol. 2008, 124, 126−134. (5) Linton, M. J.; Nobel, P. S. Hydraulic conductivity, xylem cavitation, and water potential for succulent leaves of Agave deserti and Agave tequilana. Int. J. Plant Sci. 2001, 162, 747−754. (6) Anderies, J. M.; Nelson, B. A.; Kinzig, A. P. Analyzing the impact of agave cultivation on famine risk in arid pre-hispanic northern Mexico. Hum. Ecol. 2008, 36, 409−422. (7) Santos-Zea, L.; Leal-Díaz, A.; Cortés-Ceballos, E.; GutierrezUribe, J. Agave (Agave spp.) and its traditional products as a source of bioactive compounds. Curr. Bioact. Compd. 2012, 8, 218−231. (8) Willems, J. L.; Low, N. H. Major carbohydrate, polyol, and oligosaccharide profiles of agave syrup. Application of this data to authenticity analysis. J. Agric. Food Chem. 2012, 60, 8745−8754. (9) Secretariá de Economia.́ NMX-FF-110-SCFI-2008: Productos alimenticios-Jarabe de Agave-Especificaciones y Métodos de prueba. Diario Of icial de la Federación, 2008. (10) García-Aguirre, M.; Sáenz-Á lvaro, V. A.; Rodríguez-Soto, M. a; Vicente-Magueyal, F. J.; Botello-Alvarez, E.; Jimenez-Islas, H.; Cárdenas-Manríquez, M.; Rico-Martínez, R.; Navarrete-Bolaños, J. L. Strategy for biotechnological process design applied to the enzymatic hydrolysis of agave fructo-oligosaccharides to obtain fructose-rich syrups. J. Agric. Food Chem. 2009, 57, 10205−10210. (11) Michel-Cuello, C.; Juárez-Flores, B. I.; Aguirre-Rivera, J. R.; Pinos-Rodríguez, J. M. Quantitative characterization of nonstructural carbohydrates of mezcal agave (Agave salmiana Otto ex Salm-Dick). J. Agric. Food Chem. 2008, 56, 5753−5757. (12) Arrizon, J.; Morel, S.; Gschaedler, A.; Monsan, P. Comparison of the water-soluble carbohydrate composition and fructan structures of Agave tequilana plants of different ages. Food Chem. 2010, 122, 123− 130. (13) Lopez, M. G.; Mancilla-Margalli, N. A.; Mendoza-Diaz, G. Molecular structures of fructans from Agave tequilana Weber var. azul. J. Agric. Food Chem. 2003, 51, 7835−7840. (14) Granados-Sanchez, D. Los Agaves En México, 1st ed.; Universidad Autónoma de Chapingo: Chapingo, México, 1991; pp 11−22. (15) Ortiz-Basurto, R. I.; Pourcelly, G.; Doco, T.; Williams, P.; Dornier, M.; Belleville, M.-P. Analysis of the main components of the aguamiel produced by the maguey-pulquero (Agave mapisaga) throughout the harvest period. J. Agric. Food Chem. 2008, 56, 3682− 3687. (16) Albino Vargas from AGMEL. Personal communication, 2012.

ASSOCIATED CONTENT

S Supporting Information *

Further experiments were performed to compare tentatively new saponins with those previously identified in the literature. Figure S1 shows a chromatogram and mass spectrum of compounds 5 and 6. Figure S2 includes a total ion chromatogram for the partially hydrolyzed kammogenin glycoside fraction with compound 5 and compound 6 and mass spectrum proposed for their glycosides and aglycone. Figure S3 includes a total ion chromatogram for 60 min hydrolyzed kammogenin glycoside fraction with compound 5 and compound 6 and mass spectrum proposed for their glycosides and aglycone. Figure S4 and Figure S5 show chromatograms of extracted ions, mass spectra, tentative structures, and fragmentation pattern for compounds 7 and 8 and for compounds 9 and 10, respectively. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 52-81-8328-4262. Fax: 52-81-8328-4262. E-mail: [email protected]. Funding

We would like to thank to the Nutrigenomic Research Chair Fund from Fundación FEMSA, CAT-005 and NutriOmics from Tecnológico de Monterrey, Monterrey Campus and CONACYT for financial support for graduate studies (CVU 388427). Notes

The authors declare no competing financial interest. F


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