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Jan M C Geuns - One of the best experts on this subject based on the ideXlab platform.
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synthesis of an is and Steviol Glycoside analysis by a validated internal standard method
American Journal of Analytical Chemistry, 2018Co-Authors: Jan M C Geuns, Tom Struyf, Stijn Ceunen, Gert Steurs, Wim M De BorggraeveAbstract:The internal standard (IS) method is the best method for the analysis of samples, as it is independent of errors in injection volume, changes in sample volumes, and changes in sensitivity of the detector, etc. Use of an internal standard allows for the correction of losses due to sample clean-up of complex samples. An ideal IS is a compound that has properties very similar to, and that behaves as the compounds to be analysed. Ideally, only in the last step of analysis (HPLC), the IS should be well separated from the compounds of the mixture to be analysed. After testing several existing compounds with negative results, we decided to synthesise the 19-O-β-D-galactopyranosyl-13-O-β-D-glucopyranosyl-Steviol as IS. This is the 19-galactosyl ester of Steviolmonoside (13-O-β-D-glucopyranosyl-Steviol). The IS was made according to published methods. Steviolmonoside (SM) was made from purified commercial rubusoside (Rub) by refluxing it in 10% KOH for 2 h. SM was precipitated and crystallized from MeOH. The hydroxyls of the glucose unit of SM were protected by acetylation. The acetylated SM was crystallized from acetone and dissolved in 1,2-dichloroethane. Then Ag2CO3 on Celite and tetra-acetylated galactopyranosyl bromide were added and the mixture was refluxed for 2 h. After cooling, BaO in MeOH was added to remove the acetyl groups. The 1,2-dichloroethane fraction was then extracted three times with equal volumes of water and the water fraction containing the IS was further purified on a C18 flash chromatography column. Traces of unreacted SM were removed by preparative HPLC on an Alltima C18 column (250 mm × 22 mm, particle size 10 μm) with AcCN:water (35:65, 20 ml/min). Detection was at 210 nm (KNAUER, “Smartline” UV detector 2500). The collected IS fraction from the HPLC was completely dried. Mixtures of Steviol Glycosides (SVglys) containing IS could be purified over SPE cartridges without change of the SVgly over IS ratio. The calibration curves for rebaudioside A (RebA) and stevioside (ST) were linear between 0.012 and 0.95 and between 0.013 and 1.13 mM for RebA and ST, respectively. The accuracy was checked by the standard addition method. It was concluded that the IS method gives an excellent precision and accuracy.
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Validation of an HPLC method for direct measurement of Steviol equivalents in foods.
Food chemistry, 2015Co-Authors: Uria Bartholomees, Tom Struyf, Olivier Lauwers, Stijn Ceunen, Jan M C GeunsAbstract: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%.
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glucose sucrose and Steviol Glycoside accumulation in stevia rebaudiana grown under different photoperiods
Biologia Plantarum, 2013Co-Authors: Stijn Ceunen, Jan M C GeunsAbstract:The amount of free glucose and sucrose within the leaves of Stevia rebaudiana has been evaluated under different photoperiods and related to Steviol Glycoside (SVgly) accumulation. The diurnal dynamics of glucose and sucrose in leaves, internodes, apices, and roots revealed considerable fluctuations. Within leaves, glucose and sucrose content decreased up to three-fold during the night whereas SVgly amounts did not show any significant diurnal fluctuations. Ontogenetic variations in glucose content was markedly different under long-day (LD) and short-day (SD) regimes. Under LD, glucose content increased two-fold after the onset of flower bud formation, whereas under SD, a stagnation or small decrease was observed. This increase under LD was already noticed in the upper sink leaves at the end of the vegetative stage, possibly acting as a metabolic trigger for the phase transition to reproductive development. Under SD, glucose content appeared more controlled resulting in a better linear relationship with dry matter and SVgly content. The increased sink demands of the transitional apex led to a rapid decline in leaf sucrose which showed little or no correlation with SVgly amounts. From our results, it becomes clear that the correlation between glucose or sucrose as substrates and SVglys as products is very dynamic and is significantly influenced by day-length and ontogeny.
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Steviol Glycosides: chemical diversity, metabolism, and function.
Journal of natural products, 2013Co-Authors: Stijn Ceunen, Jan M C GeunsAbstract: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.
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influence of photoperiodism on the spatio temporal accumulation of Steviol Glycosides in stevia rebaudiana bertoni
Plant Science, 2013Co-Authors: Stijn Ceunen, Jan M C GeunsAbstract:Abstract The effect of photoperiodism on Steviol Glycoside (SVgly) accumulation was investigated in Stevia rebaudiana . Topped plants were cultivated to develop new branches under a 16 h or 8 h 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.
Eugenia Jacira Bolacel Braga - One of the best experts on this subject based on the ideXlab platform.
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Salt Stress-Induced Changes in In Vitro Cultured Stevia rebaudiana Bertoni: Effect on Metabolite Contents, Antioxidant Capacity and Expression of Steviol Glycosides-Related Biosynthetic Genes
Journal of Plant Growth Regulation, 2019Co-Authors: Simone Ribeiro Lucho, Marcelo Nogueira Amaral, Priscila Ariane Auler, Valmor João Bianchi, María Ángeles Ferrer, Antonio Asensio Calderón, Eugenia Jacira Bolacel BragaAbstract:Stevia rebaudiana is an industrially and medicinally important herb, mainly due to its Steviol Glycoside content, which is a calorie-free natural sweetener. Moreover, it contains other phytochemicals that make this species potentially beneficial for health. Salt stress affects plant growth, development and plant metabolism. The present study was executed with the objective of evaluating the effect of different NaCl concentrations (0.0, 0.5, 1.0 and 1.5 g L^−1) on growth parameters, total soluble sugars, Steviol Glycosides and phenolic compounds (total soluble phenols, flavonoids and hydroxycinnamic acids), as well as antioxidant capacity (with a FRAP and DPPH assay). We also analysed in this study the expression levels of genes involved in the three stages of the Steviol Glycosides biosynthesis pathway in S. rebaudiana . Treatment with NaCl decreased root numbers and weights (dry and fresh), while increasing antioxidant capacity (according to the FRAP assay), hydroxycinnamic acid and total soluble sugar content. Furthermore, the up-regulation of several genes ( CMS , CMK , HDR and UGT76G1 ) encoding key enzymes of the Steviol Glycoside biosynthetic pathways was observed after NaCl treatments. The results provide new insights into the physiological, biochemical and molecular response mechanisms of in vitro-grown stevia plants to NaCl.
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Validation of reference genes for RT-qPCR studies in Stevia rebaudiana in response to elicitor agents
Physiology and Molecular Biology of Plants, 2018Co-Authors: Simone Ribeiro Lucho, Marcelo Nogueira Amaral, Valmor João Bianchi, Cristini Milech, Letícia Carvalho Benitez, Alitcia Moraes Kleinowski, Eugenia Jacira Bolacel BragaAbstract:Stevia rebaudiana is an important source of natural Steviol Glycosides and is of increasing interest in various fields of study. Therefore, understanding the molecular processes regulating its metabolism is of great importance. In this study, the stability of seven reference genes ( 18S ribosomal RNA, Actin, Aquaporin, Calmodulin, Eukaryote elongation factor 1 - α, Malate dehydrogenase, and Ubiquitin ) under the effect of three stress-related elicitors (methyl jasmonate, salicylic acid, and spermidine) was evaluated in stevia plants. We used RefFinder software, which makes use of the four main currently available algorithms for reference gene selection: geNorm, NormFinder, BestKeeper, and the Comparative ∆Ct method. The results indicated that Ubiquitin and Actin can be used as reference genes under all tested experimental conditions. The genes, 18S ribosomal RNA, traditionally used as a reference gene, along with Calmodulin , showed the lowest stability. The expression of Deoxyxylulose - 5 - phosphate synthase and Kaurenoic acid hydroxylase genes was used to confirm the validated reference genes, showing that inadequacy of the reference gene may lead to erroneous results. This is the first study on the stability of reference genes in Stevia rebaudiana plants, and is of great relevance for further analysis of the gene expression of the Steviol Glycoside biosynthetic pathway.
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elicitor induced transcriptional changes of genes of the Steviol Glycoside biosynthesis pathway in stevia rebaudiana bertoni
Journal of Plant Growth Regulation, 2018Co-Authors: Simone Ribeiro Lucho, Valmor João Bianchi, Marcelo Nogueira Do Amaral, Cristini Milech, M A Ferrer, A A Calderon, Eugenia Jacira Bolacel BragaAbstract:Stevia rebaudiana is a medicinal plant widely used as a sweetener, and the sweetening power comes from the diterpenoid Glycosides known as Steviol Glycosides (SGs). The studies of gene expression patterns in response to elicitors treatment are of extreme importance for biotechnological purposes. Methyl jasmonate (MeJa), spermidine (SPD), salicylic acid (SA), and paclobutrazol (PBZ) (100 µM) were applied to stevia plants growing in a hydroponic system to examine the effect of these elicitors along the time (24, 48, 72, and 96 h). SGs’ contents and transcription levels of fifteen genes involved in the three stages comprising their biosynthesis pathway were evaluated. The results provide direct evidence that HDR, GGDPS, CDPS, KS, KO, and KAH are elicitor-responsive genes and they can be effectively regulated at least at the transcriptional level. MeJa and SPD treatments resulted in positive effects on the transcription of SGs biosynthetic genes, with SPD exhibiting the highest number of regulated genes at 48 h of exposure. PBZ treatment down-regulated genes encoding kaurenoid enzymes. SA treatment did not affect transcription of UGT85C2, UGT74G1, and UGT76G1 and decreased stevioside levels. Overall, this study offers new insights into the transcriptional response mechanisms in S. rebaudiana plants under the effect of the elicitors MeJa, SPD, SA, and PBZ.
Indra Prakash - One of the best experts on this subject based on the ideXlab platform.
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a novel diterpene Glycoside with nine glucose units from stevia rebaudiana bertoni
Biomolecules, 2017Co-Authors: Indra Prakash, Cynthia Bunders, Krishna Prasad Devkota, Romila D Charan, Catherine Ramirez, Tara M SnyderAbstract:Following our interest in new diterpene Glycosides with better taste profiles than that of Rebaudioside M, we have recently isolated and characterized Rebaudioside IX—a novel Steviol Glycoside—from a commercially‐supplied extract of Stevia rebaudiana Bertoni. This molecule contains a hexasaccharide group attached at C‐13 of the central diterpene core, and contains three additional glucose units when compared with Rebaudioside M. Here we report the complete structure elucidation—based on extensive Nuclear Magnetic Resonance (NMR) analysis (1H, 13C, Correlation Spectroscopy (COSY), Heteronuclear Single Quantum Coherence‐Distortionless Enhancement Polarization Transfer (HSQC‐DEPT), Heteronuclear Multiple Bond Correlation (HMBC), 1D Total Correlation Spectroscopy (TOCSY), Nuclear Overhauser Effect Spectroscopy (NOESY)) and mass spectral data—of this novel diterpene Glycoside with nine sugar moieties and containing a relatively rare 16 α‐linked Glycoside. A Steviol Glycoside bearing nine glucose units is unprecedented in the literature, and could have an impact on the natural sweetener catalog.
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Steviol Glycosides in purified stevia leaf extract sharing the same metabolic fate.
Regulatory toxicology and pharmacology : RTP, 2016Co-Authors: Sidd Purkayastha, Indra Prakash, Avetik Markosyan, Sachin Bhusari, George Pugh, Barry Lynch, Ashley RobertsAbstract:Abstract The safety of Steviol Glycosides is based on data available on several individual Steviol Glycosides and on the terminal absorbed metabolite, Steviol. Many more Steviol Glycosides have been identified, but are not yet included in regulatory assessments. Demonstration that these Glycosides share the same metabolic fate would indicate applicability of the same regulatory paradigm. In vitro incubation assays with pooled human fecal homogenates, using rebaudiosides A, B, C, D, E, F and M, as well as Steviolbioside and dulcoside A, at two concentrations over 24–48 h, were conducted to assess the metabolic fate of various Steviol Glycoside classes and to demonstrate that likely all Steviol Glycosides are metabolized to Steviol. The data show that glycosidic side chains containing glucose, rhamnose, xylose, fructose and deoxy-glucose, including combinations of α(1–2), β-1, β(1–2), β(1–3), and β(1–6) linkages, were degraded to Steviol mostly within 24 h. Given a common metabolite structure and a shared metabolic fate, safety data available for individual Steviol Glycosides can be used to support safety of purified Steviol Glycosides in general. Therefore, Steviol Glycosides specifications adopted by the regulatory authorities should include all Steviol Glycosides belonging to the five groups of Steviol Glycosides and a group acceptable daily intake established.
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a new diterpene Glycoside 15α hydroxy rebaudioside m isolated from stevia rebaudiana
Natural Product Communications, 2015Co-Authors: Indra Prakash, Cynthia Bunders, Tara M Snyder, Krishna Prasad Devkota, Romila D Charan, Catherine Ramirez, Christopher PriedemannAbstract:: In a continued search for novel diterpenoid Glycosides, we recently isolated and characterized a Rebaudioside M derivative with a hydroxyl group at position 15 in the central diterpene core from an extract of Stevia rebaudiana Bertoni. Here we report the complete structure elucidation of 15α-hydroxy-Rebaudioside M (2) on the basis of NMR (1H, 13C, COSY, HSQC-DEPT, HMBC, 1D TOCSY, NOESY) and mass spectral data. Steviol Glycoside with a hydroxyl group at C-15 in the central diterpene core has not been previously reported.
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isolation and characterization of a novel rebaudioside m isomer from a bioconversion reaction of rebaudioside a and nmr comparison studies of rebaudioside m isolated from stevia rebaudiana bertoni and stevia rebaudiana morita
Biomolecules, 2014Co-Authors: Indra Prakash, Catherine Ramirez, Cynthia Unders, Krishna P Devkota, Romila D Chara, Christophe Priedema, Avetik MarkosyaAbstract:A minor product, rebaudioside M2 (2), from the bioconversion reaction of rebaudioside A (4) to rebaudioside D (3), was isolated and the complete structure of the novel Steviol Glycoside was determined. Rebaudioside M2 (2) is considered an isomer of rebaudioside M (1) and contains a relatively rare 1→6 sugar linkage. It was isolated and characterized with NMR (1H, 13C, COSY, HSQC-DEPT, HMBC, 1D-TOCSY, and NOESY) and mass spectral data. Additionally, we emphasize the importance of 1D and 2D NMR techniques when identifying complex Steviol Glycosides. Numerous NMR spectroscopy studies of rebaudioside M (1), rebaudioside D (3), and mixture of 1 and 3 led to the discovery that SG17 which was previously reported in literature, is a mixture of rebaudioside D (3), rebaudioside M (1), and possibly other related Steviol Glycosides.
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development of next generation stevia sweetener rebaudioside m
Foods, 2014Co-Authors: Indra Prakash, Avetik Markosya, Cynthia UndersAbstract:This work aims to review and showcase the unique properties of rebaudioside M as a natural non-caloric potential sweetener in food and beverage products. To determine the potential of rebaudioside M, isolated from Stevia rebaudiana Bertoni, as a high potency sweetener, we examined it with the Beidler Model. This model estimated that rebaudioside M is 200-350 times more potent than sucrose. Numerous sensory evaluations of rebaudioside M's taste attributes illustrated that this Steviol Glycoside possesses a clean, sweet taste with a slightly bitter or licorice aftertaste. The major reaction pathways in aqueous solutions (pH 2-8) for rebaudioside M are similar to rebaudioside A. Herein we demonstrate that rebaudioside M could be of great interest to the global food industry because it is well-suited for blending and is functional in a wide variety of food and beverage products.
Stijn Ceunen - One of the best experts on this subject based on the ideXlab platform.
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synthesis of an is and Steviol Glycoside analysis by a validated internal standard method
American Journal of Analytical Chemistry, 2018Co-Authors: Jan M C Geuns, Tom Struyf, Stijn Ceunen, Gert Steurs, Wim M De BorggraeveAbstract:The internal standard (IS) method is the best method for the analysis of samples, as it is independent of errors in injection volume, changes in sample volumes, and changes in sensitivity of the detector, etc. Use of an internal standard allows for the correction of losses due to sample clean-up of complex samples. An ideal IS is a compound that has properties very similar to, and that behaves as the compounds to be analysed. Ideally, only in the last step of analysis (HPLC), the IS should be well separated from the compounds of the mixture to be analysed. After testing several existing compounds with negative results, we decided to synthesise the 19-O-β-D-galactopyranosyl-13-O-β-D-glucopyranosyl-Steviol as IS. This is the 19-galactosyl ester of Steviolmonoside (13-O-β-D-glucopyranosyl-Steviol). The IS was made according to published methods. Steviolmonoside (SM) was made from purified commercial rubusoside (Rub) by refluxing it in 10% KOH for 2 h. SM was precipitated and crystallized from MeOH. The hydroxyls of the glucose unit of SM were protected by acetylation. The acetylated SM was crystallized from acetone and dissolved in 1,2-dichloroethane. Then Ag2CO3 on Celite and tetra-acetylated galactopyranosyl bromide were added and the mixture was refluxed for 2 h. After cooling, BaO in MeOH was added to remove the acetyl groups. The 1,2-dichloroethane fraction was then extracted three times with equal volumes of water and the water fraction containing the IS was further purified on a C18 flash chromatography column. Traces of unreacted SM were removed by preparative HPLC on an Alltima C18 column (250 mm × 22 mm, particle size 10 μm) with AcCN:water (35:65, 20 ml/min). Detection was at 210 nm (KNAUER, “Smartline” UV detector 2500). The collected IS fraction from the HPLC was completely dried. Mixtures of Steviol Glycosides (SVglys) containing IS could be purified over SPE cartridges without change of the SVgly over IS ratio. The calibration curves for rebaudioside A (RebA) and stevioside (ST) were linear between 0.012 and 0.95 and between 0.013 and 1.13 mM for RebA and ST, respectively. The accuracy was checked by the standard addition method. It was concluded that the IS method gives an excellent precision and accuracy.
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Validation of an HPLC method for direct measurement of Steviol equivalents in foods.
Food chemistry, 2015Co-Authors: Uria Bartholomees, Tom Struyf, Olivier Lauwers, Stijn Ceunen, Jan M C GeunsAbstract: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%.
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glucose sucrose and Steviol Glycoside accumulation in stevia rebaudiana grown under different photoperiods
Biologia Plantarum, 2013Co-Authors: Stijn Ceunen, Jan M C GeunsAbstract:The amount of free glucose and sucrose within the leaves of Stevia rebaudiana has been evaluated under different photoperiods and related to Steviol Glycoside (SVgly) accumulation. The diurnal dynamics of glucose and sucrose in leaves, internodes, apices, and roots revealed considerable fluctuations. Within leaves, glucose and sucrose content decreased up to three-fold during the night whereas SVgly amounts did not show any significant diurnal fluctuations. Ontogenetic variations in glucose content was markedly different under long-day (LD) and short-day (SD) regimes. Under LD, glucose content increased two-fold after the onset of flower bud formation, whereas under SD, a stagnation or small decrease was observed. This increase under LD was already noticed in the upper sink leaves at the end of the vegetative stage, possibly acting as a metabolic trigger for the phase transition to reproductive development. Under SD, glucose content appeared more controlled resulting in a better linear relationship with dry matter and SVgly content. The increased sink demands of the transitional apex led to a rapid decline in leaf sucrose which showed little or no correlation with SVgly amounts. From our results, it becomes clear that the correlation between glucose or sucrose as substrates and SVglys as products is very dynamic and is significantly influenced by day-length and ontogeny.
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Steviol Glycosides: chemical diversity, metabolism, and function.
Journal of natural products, 2013Co-Authors: Stijn Ceunen, Jan M C GeunsAbstract: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.
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influence of photoperiodism on the spatio temporal accumulation of Steviol Glycosides in stevia rebaudiana bertoni
Plant Science, 2013Co-Authors: Stijn Ceunen, Jan M C GeunsAbstract:Abstract The effect of photoperiodism on Steviol Glycoside (SVgly) accumulation was investigated in Stevia rebaudiana . Topped plants were cultivated to develop new branches under a 16 h or 8 h 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.
Simone Ribeiro Lucho - One of the best experts on this subject based on the ideXlab platform.
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Salt Stress-Induced Changes in In Vitro Cultured Stevia rebaudiana Bertoni: Effect on Metabolite Contents, Antioxidant Capacity and Expression of Steviol Glycosides-Related Biosynthetic Genes
Journal of Plant Growth Regulation, 2019Co-Authors: Simone Ribeiro Lucho, Marcelo Nogueira Amaral, Priscila Ariane Auler, Valmor João Bianchi, María Ángeles Ferrer, Antonio Asensio Calderón, Eugenia Jacira Bolacel BragaAbstract:Stevia rebaudiana is an industrially and medicinally important herb, mainly due to its Steviol Glycoside content, which is a calorie-free natural sweetener. Moreover, it contains other phytochemicals that make this species potentially beneficial for health. Salt stress affects plant growth, development and plant metabolism. The present study was executed with the objective of evaluating the effect of different NaCl concentrations (0.0, 0.5, 1.0 and 1.5 g L^−1) on growth parameters, total soluble sugars, Steviol Glycosides and phenolic compounds (total soluble phenols, flavonoids and hydroxycinnamic acids), as well as antioxidant capacity (with a FRAP and DPPH assay). We also analysed in this study the expression levels of genes involved in the three stages of the Steviol Glycosides biosynthesis pathway in S. rebaudiana . Treatment with NaCl decreased root numbers and weights (dry and fresh), while increasing antioxidant capacity (according to the FRAP assay), hydroxycinnamic acid and total soluble sugar content. Furthermore, the up-regulation of several genes ( CMS , CMK , HDR and UGT76G1 ) encoding key enzymes of the Steviol Glycoside biosynthetic pathways was observed after NaCl treatments. The results provide new insights into the physiological, biochemical and molecular response mechanisms of in vitro-grown stevia plants to NaCl.
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Validation of reference genes for RT-qPCR studies in Stevia rebaudiana in response to elicitor agents
Physiology and Molecular Biology of Plants, 2018Co-Authors: Simone Ribeiro Lucho, Marcelo Nogueira Amaral, Valmor João Bianchi, Cristini Milech, Letícia Carvalho Benitez, Alitcia Moraes Kleinowski, Eugenia Jacira Bolacel BragaAbstract:Stevia rebaudiana is an important source of natural Steviol Glycosides and is of increasing interest in various fields of study. Therefore, understanding the molecular processes regulating its metabolism is of great importance. In this study, the stability of seven reference genes ( 18S ribosomal RNA, Actin, Aquaporin, Calmodulin, Eukaryote elongation factor 1 - α, Malate dehydrogenase, and Ubiquitin ) under the effect of three stress-related elicitors (methyl jasmonate, salicylic acid, and spermidine) was evaluated in stevia plants. We used RefFinder software, which makes use of the four main currently available algorithms for reference gene selection: geNorm, NormFinder, BestKeeper, and the Comparative ∆Ct method. The results indicated that Ubiquitin and Actin can be used as reference genes under all tested experimental conditions. The genes, 18S ribosomal RNA, traditionally used as a reference gene, along with Calmodulin , showed the lowest stability. The expression of Deoxyxylulose - 5 - phosphate synthase and Kaurenoic acid hydroxylase genes was used to confirm the validated reference genes, showing that inadequacy of the reference gene may lead to erroneous results. This is the first study on the stability of reference genes in Stevia rebaudiana plants, and is of great relevance for further analysis of the gene expression of the Steviol Glycoside biosynthetic pathway.
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elicitor induced transcriptional changes of genes of the Steviol Glycoside biosynthesis pathway in stevia rebaudiana bertoni
Journal of Plant Growth Regulation, 2018Co-Authors: Simone Ribeiro Lucho, Valmor João Bianchi, Marcelo Nogueira Do Amaral, Cristini Milech, M A Ferrer, A A Calderon, Eugenia Jacira Bolacel BragaAbstract:Stevia rebaudiana is a medicinal plant widely used as a sweetener, and the sweetening power comes from the diterpenoid Glycosides known as Steviol Glycosides (SGs). The studies of gene expression patterns in response to elicitors treatment are of extreme importance for biotechnological purposes. Methyl jasmonate (MeJa), spermidine (SPD), salicylic acid (SA), and paclobutrazol (PBZ) (100 µM) were applied to stevia plants growing in a hydroponic system to examine the effect of these elicitors along the time (24, 48, 72, and 96 h). SGs’ contents and transcription levels of fifteen genes involved in the three stages comprising their biosynthesis pathway were evaluated. The results provide direct evidence that HDR, GGDPS, CDPS, KS, KO, and KAH are elicitor-responsive genes and they can be effectively regulated at least at the transcriptional level. MeJa and SPD treatments resulted in positive effects on the transcription of SGs biosynthetic genes, with SPD exhibiting the highest number of regulated genes at 48 h of exposure. PBZ treatment down-regulated genes encoding kaurenoid enzymes. SA treatment did not affect transcription of UGT85C2, UGT74G1, and UGT76G1 and decreased stevioside levels. Overall, this study offers new insights into the transcriptional response mechanisms in S. rebaudiana plants under the effect of the elicitors MeJa, SPD, SA, and PBZ.