H: Health, Nutrition & Food Variability in Saponin Content, Cancer Antiproliferative Activity and Physicochemical Properties of Concentrated Agave Sap Liliana Santos-Zea, Aratza Mireya Rosas-P´ erez, Ana Mar´ ıa Leal-D´ ıaz, and Janet A Guti´ errez-Uribe Abstract: Concentrated agave sap (CAS) has gained popularity as an unrefined sweetener. It is obtained by boiling “aguamiel” that contains phytochemicals with diverse bioactivities. Saponins have been the most widely studied agave phytochemicals due to their cancer antiproliferative effect but their concentration may vary due to maturity of the agave plant and collection site. In this study, 18 CAS samples produced in different states of Mexico were analyzed using multivariate methods to determine which physicochemical or phytochemical parameters were responsible for variation. Additionally, extracts with different saponin profiles were tested to determine possible correlations with antiproliferative activity. Total soluble solids, pH, and water activity were similar to those reported for other agave sweeteners. Antioxidant capacity of samples was correlated to browning index. Eleven steroidal saponins were found in CAS samples and they were the main source of variability. Magueyoside B, a kammogenin tetraglycoside, was the most abundant saponin in all samples. With respect to bioactivity, multivariate analysis indicated that magueyoside B and a gentrogenin tetraglycoside were compounds strongly related with bioactivity. CAS from Hidalgo, Puebla, and Veracruz had higher concentration of magueyoside B than from the other kamogenin tetraglycoside found in the samples from other Mexican states. These results could be used as a first approach to characterize and standardize CAS to validate the potential health benefits derived from its consumption. Keywords: anticancer, gentrogenin, kammogenin, magueyoside B, saponins, variability Practical Application: Concentrated agave sap (CAS) is a syrupy liquid obtained from agave plants and it is used as a unrefined sweetener. Compounds known as saponins have been detected in this product and some of them have anticancer activity. This research will help to observe the natural variation among these compounds in different samples of CAS and their relationship to anticancer effect. With these results, CAS can be classified according to the concentration of these compounds. Introduction In recent years sweeteners derived from agave have gained im- portance and popularity as an alternative to refined sugar, due to their natural origin and antioxidant capacity (Foster-Powell and others 2002; Phillips and others 2009; Santos-Zea and oth- ers 2016a) and health benefits such as anticancer and antidiabetic properties (Santos-Zea and others 2012). Among these products, there are 2 types to be recognized: agave nectars (also referred to as syrups), produced from the thermal or enzymatic hydrolysis of agave head fructans (Willems and Low 2012) and concentrated agave sap (CAS), a syrupy liquid obtained after boiling down agave sap or “aguamiel” (Gutierrez-Uribe and Serna-Saldivar 2013). Agave sap contains steroidal saponins (Ortiz-Basurto and oth- ers 2008; Leal-D´ ıaz and others 2015) and such compounds have shown strong anticancer activity (Man and others 2010; Santos- Zea and others 2012). Studies on diverse Agave utahensis glyco- sides, recognized that changes in hydroxylation patterns of the aglycone reduced spirostanol saponins cytotoxicity (Yokosukaand others 2009). The number of sugars attached to the sapogenin also impacts bioactivity, as seen in the antifungal properties of Agave MS 20160209 Submitted 2/4/2016, Accepted 5/26/2016. Authors are with Tec- nol´ ogico de Monterrey, Campus Monterrey, Centro de Biotecnolog´ ıa-FEMSA, Escuela de Ingenier´ ıa y Ciencias, Av. Eugenio Garza Sada 2501 Sur, C.P, 64849 Monterrey, N.L, Mexico. Direct inquiries to author Guti´ errez-Uribe (e-mail: jagu@itesm.mx). americana hecogenin glycosides, where carbohydrate moieties of 4 or 5 sugars had a stronger effect (Jin and others 2003). Maturity of the plant at the time of collection affect agave sap saponin con- centration (Leal-D´ ıaz and others 2015), therefore CAS saponin concentration may have significant variation among batches and production sites. It was noteworthy that after storing CAS during storing 20 weeks, no significant decrease in saponin content was observed (Santos-Zea and others 2016a). During storage, CAS can undergo changes in its physicochem- ical properties, pointing out nonenzymatic browning and an in- crease in antioxidant capacity (Santos-Zea and others 2016a). Sim- ilar studies on honey and maple syrup have found a link between darker color with a higher antioxidant capacity (Brudzynski and Miotto 2011; Singh and others 2014). Multivariate methods have served to detect adulteration in honey and agave nectars (Rios-Corripio and others 2012, Willems and Low 2012), discrimination of agave syrups among other sweeteners (Mellado-Mojica and L´ opez 2015) and recognition of possible bioactive compounds in honey and black bean extracts (Brudzynski and Miotto 2011; Guajardo-Flores and others 2013). In the case of recognition of agave syrup from other sweeteners or from adulterated samples, multivariate analysis determined that oligosaccharide and sugar profile were the parameters that allowed discriminating samples according to their origins (Willems and Low 2012; Mellado-Mojica and L´ opez 2015). Analysis of saponin concentration using tools, such as principal component analysis, C  2016 Institute of Food Technologists R  doi: 10.1111/1750-3841.13376 Vol. 00, Nr. 0, 2016  Journal of Food Science H1 Further reproduction without permission is prohibited