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Steroidal saponins isolation of Agave americana subsp. andina by enzymatic hydrolysis: a new approach.
1  Instituto Superior Tecnológico "17 de Julio"


Penco azul (Agave americana subsp. andina) is part of the landscape of the Ecuadorian Andean region, in the same way, of its culture and tradition. Moreover, it is interesting from the agricultural perspective, due to its type of asexual reproduction (of which the genus Agave or Agavaceae is characterized), since it does so through its rhizomes (underground stems that form the young or genetically identical replicas). Thus, it is common in Latin American countries, such as Mexico and Ecuador, that there are crops taking advantage of this property. However, the advantages are also observed from a biotechnological perspective, due to the possible separation of steroidal saponins (SE) such as hecogenin and tigogenin, from the rhizomes by enzymatic hydrolysis. Among the properties of these molecules, the one described by Santos, Fajardo, Romo & Uribe (2015), who concluded in their work with Agave Salmiana, that the anticancer effect of SE is verified by their first evaluation of the apoptotic potential of kammogenins and mangenins found in mead . Therefore, thanks to these SE, "programmed cell death" can be caused against colon cancer.
Steroidal saponins are glycosides, that is, they are compounds that by hydrolysis (acidic or diastatic) give one or more sugar molecules (glucones) and other non-glycidic substances or aglucones (for example, acids, phenols, alcohols or aldehydes). In samples of A. americana has been found that the content of hecogenin and tigogenin increases by a ratio of 12: 1 (respectively) during plant growth. For this reason, the author in this research proposes to separate steroidal saponins in the most adult specimens, whose approximate age is 12 years (determined empirically). This is due to the high content of hecogenin, the most active steroidal saponin of the genus Agave.
On the other hand, at the University of Manchester, Cambridge and Bahuguna Garhwal, special glycosides such as steroidal saponin were found in the structure of the rhizomes. However, the traditional method for extraction consists in the following treatment of the sample: it starts with drying at 40 ° C; macerate with ethanol and water (at room temperature and in the absence of light); the extract obtained is evaporated in vacuo; 10 g of this content are dissolved in water, and extracted again in separatory funnels with hexane and butanol; for hydrolysis, 5 g of the saponin extract was dissolved in 75 mL of ethanol and 75 mL of 2M hydrochloric acid was added; finally, it was extracted with ethyl acetate.
To conclude, hydrolysis is a water solvolysis, which allows a solvent (nucleophile) to break one or more bonds in the solute. The method proposed with the present study is through enzymatic hydrolysis. In this way, we seek to adapt a technique made by García et al. (2009), who experimented with potato dextrose agar. From this culture medium, the extract of the hydrolyzing enzyme was obtained, inoculating yeasts that ferment the water-honey of the Agave tequilana. In Ecuador, "penco azul" mead is known as mishki or chaguarmishki, so it is sought to replicate the experiment on a laboratory scale, with this native drink. As for the treatment of the sample, the traditional method will be used.

Keywords: Agave americana subsp. andina; steroidal saponins; enzymatic hydrolysis; rhizomes.