*2.1. Chemical and Enzymatic Reactions of Diterpene Glycosides* 2.1.1. Alkaline Hydrolysis

*Stevia* DGs are thermostable natural compounds in a neutral solution, although in an alkaline medium with some increase in temperature can be hydrolyzed and yield the free acid compounds at C-19 [16,26].

DGs with one sugar and few examples with two sugars (less hindered as rebaudiosides I and U) attached at position C-19 cleave using mild alkaline conditions. However, DGs with a hindered disaccharide or longer oligomers at C-19 only cleave using stronger alkaline conditions. This difference in saponification conditions for DGs together with the preparation and characterization of some saponified DGs has been useful for the development of a simple and rapid reversed-phase C-18 high-performance liquid chromatography method (HPLC) to identify known and detect novel C-13 oligomer arrangements, see Section 4.1 [26]. In Table 1 are shown all the DGs from *S. rebaudiana* that have been modified by using alkaline conditions.

### 2.1.2. Acid Hydrolysis

The DGs under acidic conditions may undergo double bond isomerization, sugar cleavage from the glycosides, and Wagner-Meerwein rearrangement of the diterpene aglycone portion depending on temperature. The acid hydrolysis of DGs has been studied since several soft drinks and juices prepared with DGs from *S. rebaudiana* as sweeteners can produce undesirable products under inappropriate temperature storage that may affect the taste of these acidic beverages. Even under different mild acid conditions a double bond isomerization process occurs yielding some by-products that have been isolated and characterized [33–39]. However, proper high-resolution analytical methods need to be developed to differentiate natural DGs from their endocyclic isomer compounds since they elute very closely in HPLC [40]. Additionally, a cleavage of all the sugars in DGs occurs under strong acid conditions producing a mixture of at least three main aglycones from this reaction: isosteviol (major), the endo steviol isomer and steviol (as very minor component). Hence, acid hydrolysis is not a good method to produce steviol in quantities. Recently, these three structurally close aglycones have been purified in their intact form by a two-step gradient high-performance silica gel chromatography [38]. In Table 1 are shown all the DGs formed under basic or acidic conditions while all the aglycones produced from steviol glycosides under acidic conditions and others reported from *S. rebaudiana* are compiled in Figure 1.

**Figure 1.** Aglycone cores reported from *S. rebaudiana* as natural forms, degradation products or forming part of a glycoside compound (R1 and R2). steviol (**I**); *ent*-atisene (**II**); isosteviol (**III**); endo steviol (**IV**); CH3 and OH at C-16 (**V**); CHO at C-16 (**VI**); CH2OH at C-16 (**VII**); C-12 linkage (**VIII**); 15-α-hydroxy-rebaudioside M (**IX**).

**Table 1.** Compounds reported as degradation products from natural diterpene glycosides by alkaline or acid conditions. **a** 13- [(2-*O*-*β*-D-xylopyranosyl-*β*-D-glucopyranosyl-)oxy]*ent*-kaur-16-en-19-oic acid *β*-D-glucopyranosyl ester; **b** 13-[(2-*O*-6-deoxy*β*-D-glucopyranosyl-3-*O*-*β*-D-glucopyranosyl-*β*-D-glucopyranosyl)oxy]*ent*-kaur-16-en-19-oic acid *β*-D-glucopyranosyl ester.



**Table 1.** *Cont.*
