**1. Introduction**

*Stevia rebaudiana*, a native plant species to Brazil and Paraguay, was discovered by Mosè Giacomo Bertoni in 1899, an Italian-speaking Swiss naturalist and described as *Eupatorium rebaudianum* Bertoni. In 1905 the plant was grouped in a different genus and named as *Stevia rebaudiana* (Bertoni) Bertoni. *S. rebaudiana* has been widely used to sweeten teas and potions for centuries by Guarani indigenous due to the high content in diterpene glycosides (DGs) in their leaves [1]. Essential oils have been reported from *S. rebaudiana*, being spathulenol (13.4–40.9%), caryophyllene oxide (1.3–18.7%), β-caryophyllene (2.1–16.0%) and β-pinene (5.5–21.5%) the major volatile compounds [2]. Non-glycosidic labdane-type diterpenes have also been isolated e.g., sterebins I -N [3] and several hydroxycinnamate and flavonoid derivatives have been identified [4,5]. There is no doubt, that the DGs is the most significant group of secondary metabolites from *S. rebaudiana* due to their applications in the Sweeteners Industry. Stevioside was the first steviol glycoside isolated from this Asteraceae in 1931, reported by M. Bridel and R. Lavielle, two French Chemist [6]. Then, rebaudiosides A, B and steviolbioside were discovered by a Japanese group 45 years later, followed by dulcosides A, B and rebaudiosides C (same chemical structure as dulcoside B), D and E in 1977 [7–10]. In 2002 rebaudioside F and its saponification products were reported [11]. In 2008, the United States Food and Drug Administration granted generally

J.D. Approaches toward the Separation, Modification, Identification and Scale up Purification of Tetracyclic Diterpene Glycosides from *Stevia rebaudiana* (Bertoni) Bertoni. *Molecules* **2021**, *26*, 1915. https://doi.org/10.3390/ molecules26071915

**Citation:** Perera, W.H.; McChesney,

Academic Editor: Muhammad Ilias

Received: 3 March 2021 Accepted: 24 March 2021 Published: 29 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

recognized as safe (GRAS) regulatory acceptance of rebaudioside A while Food Standards Australia New Zeeland accepted the use of steviol glycosides [12,13]. Other steviol glycosides were recognized as safe in USA in 2010, and the European Union approved DGs from *S. rebaudiana* for marketing in November 2011 [14,15]. A great scientific contribution was made by Ohta et al. [16], where 21 steviol glycosides were isolated and characterized. Since that time, multiple groups have been working intensively on searching for new steviol glycosides. Rebaudioside A and stevioside, the major steviol glycosides (accounting for more than 40% of the DGs present in leaf extract) have shown more potent natural sweetness than sucrose which make them good candidates as non-caloric sugar-substitutes [1]. Hence, *S. rebaudiana* is being widely commercialized in mainland China, Japan, Korea, India, and elsewhere (Midmore and Rank, 2012) and several rebaudioside A-based products as well as table-top sweeteners are commercially available in the US consumer market.

It was recently discovered that rebaudioside A and stevioside, potentiate the activity of TRPM5 (a Ca2+-activated cation channel expressed in type II taste receptor cells and pancreatic β-cells) enhancing glucose-induced insulin secretion in a TRPM5-dependent manner. TRPM5 has been suggested as a potential target to prevent and treat type 2 diabetes [17]. It has also been described that steviol glycosides stimulates insulin secretion in a dose- and glucose-dependent manner from isolated mouse islets of Langerhans. Steviol glucuronide has been identified in human urine, thus steviol is the main active metabolite after oral intake of steviol glycosides [18,19]. The antihyperglycemic and blood pressurereducing effects of stevioside in the diabetic Goto-Kakizaki rat and the mechanism of the hypoglycemic effect of stevioside has been described [20,21].

Despite the potential positive human health benefits of rebaudioside A and the relatively easy access to this steviol glycoside, its lingering aftertaste makes it less desirable as a sweetener. Therefore, several efforts have been dedicated to preparing commercial *Stevia* extracts rich in DGs to find next generation non-caloric DGs with improved organoleptic properties and thus, attract consumers to a better health lifestyle.

Important advances have been made over the last several years in the methods of analysis and purification of these complex mixtures of glycosides. All these developments have allowed the discovery of several new natural and slightly modified DGs with remarkable potential as sugar substitutes e.g., several rebaudioside A isomers, a stevioside like-compound with a disaccharide linked at position C-12 of the aglycone [22], rebaudioside R (unique example with a non-glucose monosaccharide linked directly at position C-13 (xylose) [23], rebaudioside U and similar compounds, the only steviol glycosides with an arabinopyranosyl moiety [24,25], rebaudioside W (unique hexa-glycoside from *S. rebaudiana* with three different sugar monosaccharides attached (glucose, xylose and rhamnose) [26]. Additionally, rebaudioside IX series are the largest natural DGs composed of nine glucose units linked to steviol [27–29], only one natural example of rebaudioside M isomer has been reported [30] and a few new compounds with an *ent*-atisene core have also been reported [31,32]. Nowadays, many natural, chemically, and enzymatically modified diterpene glycosides with diverse sugar interlinkages and cores have been discovered, hence, a wide spectrum of structures are now available to evaluate for their sweetener potential and to understand better the relationship between structure-sweetness/lingering aftertaste. Rebaudioside M, has been pointed out as the next generation non-caloric sweetener but in our non-professional tasters experience there are other DGs with interesting organoleptic properties that should be explored e.g., rebaudiosides U and Y.

Herein, we summarize the most noteworthy chemical approaches for the separation, identification and structural elucidation of diterpene glycosides from *Stevia rebaudiana*: High-performance Thin-Layer Chromatography, High-Performance Liquid Chromatography, High-Resolution Electrospray Ionization Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy. The modification of diterpene glycosides by chemical and enzymatic reactions together with some strategies to achieve the scale up of the purification process economically sustainable are also discussed. An updated listing of the natural and chemically modified diterpene glycosides has been included.
