Validation of an HPLC method for direct measurement of steviol equivalents in foods.

Steviol glycosides are intense natural sweeteners used in foods and beverages. Their acceptable daily intake, expressed as steviol equivalents, is set at 0-4 mg/kg body weight. We report the development and validation of a RP-HPLC method with fluorometric detection of derivatized isosteviol, formed by acid hydrolysis of steviol glycosides. Dihydroisosteviol was used as an internal standard. Using this method, the amount of steviol equivalents in commercial steviol glycoside mixtures and different foods can be directly quantified. The method was successfully tested on strawberry jam, low-fat milk, soft drink, yogurt and a commercial mixture of steviol glycosides. Calibration curves were linear between 0.01 and 1.61 mM steviol equivalents, with a quantification limit of 0.2 nmol. The % RSD of intra-day precision varied between 0.4% and 4%, whereas inter-day precision varied between 0.4% and 5%, for high and medium concentrations, and between 3% and 8% for low concentrations. Accuracy of the analysis varied between 99% and 115%.

Diterpene glycosides from Stevia phlebophylla A. Gray.

The rare Mexican species Stevia phlebophylla A. Gray was long considered to be the only known Stevia species, beside the well-known S. rebaudiana, containing the highly sweet diterpenoid steviol glycosides. We report a re-evaluation of this claim after phytochemically screening leaves obtained from two herbarium specimens of S. phlebophylla for the presence of steviol glycosides. Despite extensive MS analyses, no steviol glycosides could be unambiguously verified. Instead, the main chromatographic peak eluting at retention times similar to those of steviol glycosides was identified as a new compound, namely 16ß-hydroxy-17-acetoxy-ent-kauran-19-oic acid-(6-O-ß-D-xylopyranosyl-ß-D-glucopyranosyl) ester (1) on the basis of extensive NMR and MS data as well as the characterization of its acid hydrolysate. Seven more compounds were detected by ESIMS which are possibly structurally related to 1. It can therefore be concluded that S. phlebophylla is unlikely to contain significant amounts of steviol glycosides, if any.

Steviol glycosides: chemical diversity, metabolism, and function.

Steviol glycosides are a group of highly sweet diterpene glycosides discovered in only a few plant species, most notably the Paraguayan shrub Stevia rebaudiana. During the past few decades, the nutritional and pharmacological benefits of these secondary metabolites have become increasingly apparent. While these properties are now widely recognized, many aspects related to their in vivo biochemistry and metabolism and their relationship to the overall plant physiology of S. rebaudiana are not yet understood. Furthermore, the large size of the steviol glycoside pool commonly found within S. rebaudiana leaves implies a significant metabolic investment and poses questions regarding the benefits S. rebaudiana might gain from their accumulation. The current review intends to thoroughly discuss the available knowledge on these issues.

Spatio-temporal variation of the diterpene steviol in Stevia rebaudiana grown under different photoperiods.

As part of an ongoing study on the effects of photoperiodism on the metabolism of steviol glycosides (SVglys) in Stevia rebaudiana, the spatio-temporal variations of free steviol (SV) have now been evaluated. For its quantitation, an internal standard method was used, based upon a specific fluorometric detection of SV as its methoxycoumarinyl derivative. The level of free SV in leaves did not exceed 30 µg/g dry wt and was at least 1000-fold smaller than that of its glycosidic conjugates. In other organs, free SV was mainly measured in stem tissue and apices, with relatively large amounts measured in the latter. Similarly to SVglys, the content of free SV was influenced by photoperiod and genotype. In plants grown under long-days (LD) of 16 h, more spatial variations were seen compared to those under short-days (SD) of 8h. In the former, upper leaves contained almost four times more free SV compared to lower ones near the end of vegetative growth. In addition, the correlation between SV and its glycosidic conjugates was more linear under SD. Despite the variability of SV levels, a decrease was noted in all conditions after flower opening, which can be related a decreased transcription of the biosynthetic genes involved.

Influence of photoperiodism on the spatio-temporal accumulation of steviol glycosides in Stevia rebaudiana (Bertoni).

The effect of photoperiodism on steviol glycoside (SVgly) accumulation was investigated in Stevia rebaudiana. Topped plants were cultivated to develop new branches under a 16h or 8h photoperiod. During different ontogenetic phases, leaves, stems, lateral shoots, roots and reproductive organs were collected and analysed for nine SVglys. Long-day (LD) conditions prolonged vegetative growth, significantly increasing leaf biomass and total SVgly content. In both photoperiods, declines in SVglys were observed during reproductive development, occurring mainly in mature leaves under LDs or young leaves under SDs. When lateral shoots were included in plants under LDs, total leaf and SVgly yield per branch significantly increased, indicating a harvest during flowering is possible. The ratio of rebaudioside A (Reb A) to stevioside (ST) amounts was influenced by ontogeny and daylength, with larger ratios during vegetative growth under SDs. Linear correlations were observed between dry matter and total SVglys and between the major SVglys individually. Minor SVglys showed larger fluctuations, especially under SDs. Under LDs, the Reb A to ST ratio was inversely correlated with both leaf dry matter and total SVglys. The highly dynamic nature of the observed patterns suggests a complex regulation of SVgly metabolism on molecular and biochemical level.

Stimulation of steviol glycoside accumulation in Stevia rebaudiana by red LED light.

The aim of this study was to determine whether steviol glycoside accumulation is under phytochrome control. The results indicate that Stevia rebaudiana Bertoni plants grown under short-day conditions showed precocious flowering and stagnation of steviol glycoside accumulation. Long night interruption by red LED light stimulated and sustained the vegetative growth as well as the accumulation of steviol glycosides in the leaves. After 7 weeks of treatment, steviol glycoside content was about two-fold higher in LED-treated plants than in the short-day control group. The effects of red LED light were measured both in a greenhouse and in a phytotron, irrespective of cultivar-specific differences. Therefore, it can be concluded that a mid-night interruption by red LED light during short photoperiods provides an easy and inexpensive method to increase vegetative leaf biomass production with an increased steviol glycoside yield.

UDP-dependent glycosyltransferases involved in the biosynthesis of steviol glycosides.

A short-term experiment was designed to measure the transcript levels of downstream genes contributing to the biosynthesis of steviol glycosides. Stevia rebaudiana plants were subjected to long- and short-day conditions for different time intervals. Samples from both lower and upper leaves were collected. Using quantitative real-time polymerase chain reaction, the transcript levels of three UDP-dependent glycosyltransferases, UGT85C2, UGT74G1 and UGT76G1, were studied. The results were compared with the steviol glycoside contents measured in the leaves, which were quantified by reversed phase HPLC. In the same daylength condition, steviol glycoside concentration and the transcript levels of the three UGT genes were higher in upper leaves than in lower leaves. Steviol glycosides accumulated more in plants under short-day conditions. Under these conditions, a highly significant correlation was found between UGT85C2 transcription and total steviol glycoside accumulation in the upper leaves. This suggests that the glycosylation of steviol to form steviolmonoside is the rate-limiting step in the glycosylation pathway of steviol glycosides. In these upper leaves, a relatively high accumulation of rebaudioside A compared to stevioside was also observed, however, without correlation with the transcription of UGT76G1.