**6. Conclusions**

*Stevia rebaudiana* and its steviol glycosides have been widely studied, and in the continuous search for non-caloric sugar substitutes with improved taste, multiple new tetracyclic diterpene glycosides have been isolated from leaf of *S. rebaudiana* or have been prepared by chemical or enzymatic reactions from selected steviol glycosides. Herein, an updated list of natural DGs isolated from *S. rebaudiana* has been compiled, along with some chemically modified DGs. Some approaches for the rapid detection of new DG structures in fractions rich in DGs have also been presented. Thus, the fragmentation pattern for DGs by High-Resolution Electrospray Ionization Mass Spectrometry has been presented. The modification of DGs by a simple saponification reaction with further analysis of the products by Reverse-Phase High-Performance Liquid Chromatography for the detection of new moieties at position C-13 is another approach summarized in this review. Several HPLC methods with diverse stationary phases, but with a unique mobile phase used for the analysis of fractions rich of specific DGs were discussed. However, a major drawback in the search of novel structures in natural products, is the isolation of quantities that are insufficient to allow bioassays or in the case of DGs, conducting tasting assays to better understand the relationship structure-sweetness/bitterness of the DGs, and in turn followed by toxicological evaluation. Some strategies to save cost in the scale-up of the purification process were also shared. With the described strategies, several very minor DGs were purified in quantities of hundreds of milligrams to multiple grams. Currently, several DGs with different aglycone cores, numbers and types of sugar units, and arrangements have been well documented so far. Hence, the number of DG structures available is vast, although the reported tasting results are infrequent or at least not visible in the scientific literature. Further studies need to be pointed out for the tasting evaluation of the DGs already discovered. With the availability of this information, adequate strategies could be followed to overexpress specific groups of DGs in *S. rebaudiana* and/or to use appropriate blends of DGs with improved taste.

**Author Contributions:** Both W.H.P. and J.D.M. contributed to writing the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds described in this review are available from the authors.
