Revista Brasileira de Farmacognosia 27 (2017) 510–518 ww w.elsevier.com/locate/bjp Original Article Turbo-extraction of glycosides from Stevia rebaudiana using a fractional factorial design a c b c,∗ Paula M. Martins , Aurea D. Lanchote , Bhaskar N. Thorat , Luis A.P. Freitas a Faculdade de Ceilândia, Universidade de Brasília, Brasília, DF, Brazil b Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai, India c Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil a b s t r a c t a r t i c l e i n f o Article history: Stevia rebaudiana (Bertoni) Bertoni, Asteraceae, leaf extract has recently called the attention of food Received 29 November 2016 industry as a proposal for natural sweetener. The sweet flavor is attributed to the glycosides, in especial Accepted 24 February 2017 stevioside and rebaudioside A, which are the plant main chemical markers. The aim of the work reported Available online 1 April 2017 here was to optimize the turbo-extraction of stevia leaves using water, ethanol 70% and 90% (w/w) as 5-2 green solvents. A 2 factorial design was applied to study the linear effects of the drug size, solvent to Keywords: drug ratio, temperature, time and also the turbolysis speed on the extraction of glycosides. The glycosides Design of experiments exhaustive extraction showed that ethanol 70% gave better results and was used for turbo-extraction. Stevioside The stevioside and rebaudioside A contents were quantified by a validated method by high performance Rebaudoside A liquid chromatographic with photodiode array detector. The contents of stevioside and rebaudioside A in Hydroethanolic extract Desirability fluid extract increased with the drug size, but decreased at high shearing speeds and solvent to drug ratio, while their yields decreased at higher temperature and were not affected by turbo speed. An increase in solvent to drug ratio reduced significantly the glycosides percent in dried extract. Optimal solution for S. rebaudiana leaves turbo-extraction was determined by desirability functions. The optimal extraction condition corresponded to drug size of 780 ␮m, solvent to drug ratio of 10, extraction time of 18 min; ◦ temperature of 23 C and turbo speed of 20,000 rpm, resulting in yields of 4.98% and 2.70%, for stevioside and rebaudioside A, respectively. These yields are comparable to the ones recently published for dynamic maceration, but with the advantage of shorter extraction times. This work demonstrates that turbolysis is promising for S. rebaudiana glycosides extraction and stimulate new research on the purification of these extracts, which may become an interesting source of income for developing countries such as India and Brazil. © 2017 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Introduction surrounded by epicalyces housing, color wine, light and pentamer- ous. The fruit is striated (Madan et al., 2010). The “estevia” is a shrub popularly known worldwide and Stevia leaves contain eight terpene glycosides, identified as ste- belongs to the 261 members of genus in the family Asteraceae and vioside, rebaudoside A, B, C, D and E, dulcoside A and C. The main has the botanical name of Stevia rebaudiana (Bertoni) Bertoni. It glycosides are stevioside and rebaudoside A, summing up to 5–10% originates from the Valley of Amambay, which extends from north- of the drug (Singh and Rao, 2005; Abou-Arab et al., 2010; Yadav east Paraguay to south Brazil and southern Argentina. The plant is et al., 2011) and give a pronounced sweet flavor. Stevia is described a perennial shrub height up to 30 cm. Its leaves are sessile, 3–4 cm in the Brazilian Pharmacopoeia 5th Ed. (Farmacopeia Brasileira, long, shaped as spatula or long blade with sawn edges. The adaxial 2010) indicating dried leaves as the part used and must contain surface is slightly glandular. The branches are fragile and the roots at least 12% of total carbohydrates and 4% steviosides. are rhizome type, slightly branched. The flowers are composite, According to Madan et al. (2010), the species has been used for centuries by the Guarani Indians as a sweetener in some drinks, especially the mate tea, and the active component that has the highest sweetness index is rebaudoside A while stevioside is ∗ the main component and gives a post digestive bitter taste. The Corresponding author. E-mail: [email protected] (L.A. Freitas). http://dx.doi.org/10.1016/j.bjp.2017.02.007 0102-695X/© 2017 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). P.M. Martins et al. / Revista Brasileira de Farmacognosia 27 (2017) 510–518 511 concentration of these diterpene glycosides may vary depending Martins et al., 2013) this process could give a promising and opti- on cultivars, soil and climate, and agronomic techniques (Yadav mized method for stevia extraction. et al., 2011). Thus, the aim of this work was to study the turbo-extraction of The medicinal use of the species is described in the con- stevioside and rebaudosídeo A from S. rebaudiana dried and pow- trol of obesity, high blood pressure, digestive problems and dered leaves by applying a fractional factorial design that allow the contraceptive by indigenous peoples in many countries of Latin evaluation of the main effects of drug powder size, weight ratio sol- America (Sardhara, 2015; Madan et al., 2010). Preclinical and clini- vent to drug, temperature, agitation and time on the yield of these cal studies have been conducted to evaluate the antioxidant activity glycosides. (Sharma et al., 2012), the reproductive system, kidney function, blood pressure, the effect on blood glucose and anti-viral activity, Material and methods as well as studies on pharmacokinetic (Madan et al., 2010), toxicity, side effects and allergenic (Urban et al., 2015). Stevia drug characterization In many countries stevia has its use regulated as a dietary sup- plement and thus is not used as a sweetener, such as in Europe. The leaves of Stevia rebaudiana (Bertoni) Bertoni, Asteraceae, But in 2008, the FDA has categorized the species as Generally Rec- ◦ came from a plantation located at latitude 22 79 81.059 S and lon- ognized as Safe (GRAS) after publication by the Joint Committee of ◦ gitude 47 11 56.138 W, grown by the CPQBA – Multidisciplinary Experts on the FAO Food Additives/WHO – JECFA, which established Center for Chemical, Biological and Agricultural Sciences from State the monograph of glycosides of steviol (WHO, 2004). The product University of Campinas (Campinas – SP, Brazil), with a voucher approved for industrial and commercial use is a powder with min- deposited at the CPQBA herbarium with file number 273. The plant imum of 95% of steviol glycosides and the sum of stevioside and was supplied to CPQBA by Cenargen (Brasília, DF, Brazil) in 1998, rebaudosideo A not less than 70%. according to Dr Illio Montanari Jr. Stevia products are considered healthy and natural foods and The pharmacognostic characterization of drug was performed have gained attention in international markets (Gasmalla et al., th according to the Brazilian Pharmacopoeia 5 Edition (Farmacopeia 2014) with China, India, Brazil, Korea, Mexico, United States, ◦ Brasileira, 2010) after drying at 45 ± 2 C in a circulating air oven Indonesia, Tanzania and Canada as producers (Ahmad et al., 2014). and milling. The physicochemical parameters determined were the Recently, the commercial giant Coca Cola Company has launched moisture content, total ash, swelling index and solvent uptake. the “green” product “Coke Life” and is expected to highly increase the stevia world sales (Ausfood, 2015). The separation of the glycosides from the plant extract is Turbolysis extraction study hampered by several factors including impurities such as resins, proteins, organic acids and especially pigments (chlorophyll, In order to determine the stevioside and rebaudioside A content carotene and xanthophyll). The aqueous extraction of the pow- in the drug, a preliminary exhaustive extraction study was per- dered leaves was the most indicated choice until recently and formed by using water, alcohol 70% and 90% as solvents. Fine drug presents the highest yields of stevioside and rebaudosídeo A in powder (5 g) was added to 50 ml of solvent and stirred at room tem- ◦ crude extract (Abou-Arab et al., 2010; Chhaya et al., 2012a,b; perature (25 C) for 2 h. The drug powder as filtered and then the Mondal and Chhaya, 2012; Rao et al., 2012; Periche et al., 2014; procedure was repeated three-fold with 50 ml of fresh solvent. The Sardhara, 2015). fourth and final extraction step was run for 12 h at same conditions ◦ The use of methanol was also proposed to give selective extrac- (50 ml fresh solvent under stirring and 25 C). The extraction runs tion and easy purification (Rajab et al., 2009), but the end product were carried out in triplicate and the stevioside content (CEST), is no longer classified as GRAS. Because of the blooming interest rebaudoside A content (CREB) and the total solids content (TSOL) in stevia products, there is a large number of publications and were determined. patents on the extraction and purification of stevioside using dif- The turbo-extraction or turbolysis was performed using a high ferent methods (Periche et al., 2015; Jentzer et al., 2015), such shear stirrer Ultraturrax T-25 (Ika Ltda, Sao Paulo Brazil). Grounded as enzymatic extraction (Puri et al., 2012), bleaching agents and stevia leaves together with 50 ml of ethanol 70% as extracting sol- selective precipitation processes using metal ions (Kienle, 2010; vent were poured into a 50 ml beaker and submitted to the high Abou-Arab et al., 2010), selective extraction by supercritical fluid shear stirring according to temperature, velocity and time pre extraction and separation processes for adsorptive methods using determined by the experimental design.