Stevia is well-known for its unique non-caloric intense sweetness and recognising as one of the most acceptable sweeteners.Therefore,quality control and metabolite profiling perspectives,two isocratic reverse-phase UP...Stevia is well-known for its unique non-caloric intense sweetness and recognising as one of the most acceptable sweeteners.Therefore,quality control and metabolite profiling perspectives,two isocratic reverse-phase UPLC-PDA-ELSD-MS methods were developed to determine nine diterpene glycosides(method A)and isomeric compounds(steviol and isosteviol;method B).Both the methods were validated and found as per the ICH guidelines for accuracy(recovery),precision(Intraday,interday,and intermediate precision),linearity,and regression coefficient(r2),ruggedness(eluent concentration±2 mM,column oven temperature±3℃ and retention time),and system suitability(theoretical plate count,asymmetric factor,injection precision and resolution).LOD for steviol and its glycosides were found between 0.92 and 5.22μg/mL while LOQ were 2.78-18.29μg/mL,respectively.Validated methods had shown only the presence of steviol glycosides in Stevia rebaudina samples while LC-ESI-MS/MS revealed the identification of 46 different class of molecules.These methods will be helpful to monitor quality in Stevia production and its derived products.展开更多
The steviol glycosides(SGs)in stevia(Stevia rebaudiana Bertoni)leaves are becoming increasingly valuable due to its high sweetness but low calorific value,which is driving the development of stevia commercial cultivat...The steviol glycosides(SGs)in stevia(Stevia rebaudiana Bertoni)leaves are becoming increasingly valuable due to its high sweetness but low calorific value,which is driving the development of stevia commercial cultivation.Optimizing fertilization management can effectively increase SGs productivity,but knowledge on the relationship between potassium(K)fertilization and SGs production is still lacking.In this study,pot experiments were conducted in order to investigate the effect of K deficiency on SGs synthesis in stevia leaves,as well as the underlying mechanisms.Our results showed that when compared with standard K fertilization,K deficiency treatment has no significant effect on the biomass of stevia plant grown in a given soil with high K contents.However,K deficiency critically decreased leaf SGs contents as well as the expression of SGs synthesis-related genes.The contents of different sugar components decreased and the activities of sugar metabolism-related enzymes were inhibited under the K deficiency condition.Moreover,spraying sucrose on the leaves of stevia seedlings diminished the inhibitory effect caused by K deficiency.Our results also revealed the significant positive correlations between sucrose,glucose and SGs contents.Overall,our results suggest that K deficiency would suppress the synthesis of SGs in stevia leaves,and this effect may be mediated by the leaf sugar metabolism.Our findings provide new insights into the improvement of SGs production potential.展开更多
Stevia rebaudiana Bertoni is commonly called stevia and mostly found in the north east regions of South America.It is an herbaceous and shrubby plant belonging to the Asteraceae family.Stevia is considered as a natura...Stevia rebaudiana Bertoni is commonly called stevia and mostly found in the north east regions of South America.It is an herbaceous and shrubby plant belonging to the Asteraceae family.Stevia is considered as a natural sweetener and a commercially important plant worldwide.The leaves of S.rebaudiana contain steviol glycosides(SGs)which are highly potent and non-caloric sweeteners.The sweetening property of S.rebaudiana is contributed to the presence of these high potency,calorie free steviol glycosides.SGs are considerably suitable for replacing sucrose and other artificial sweetening agents which are used in different industries and pharmaceuticals.SGs amount in the plant mostly varies from 8%to 10%,and the enhancement of SGs is always in demand.These glycosides have the potential to become healthier alternatives to other table sugars for having desirable taste and zero calories.SGs are almost 300 times sweeter than sucrose.Being used as alternative sugar intensifier the commercial value of this plant in biopharmaceutical,food and beverages industries and in international market is increasing day by day.SGs have made stevia an important part of the medicinal world as well as the food and beverage industry,but the limited production of plant material is not fulfilling the higher global market demand.Therefore,researchers are working worldwide to increase the production of important SGs through the intercession of different biotechnological approaches in S.rebaudiana.This review aims to describe the emerging biotechnological strategies and approaches to understand,stimulate and enhance biosynthesis of secondary metabolites in stevia.Conventional and biotechnological methods for the production of steviol glycosides have been briefly reviewed and discussed.展开更多
Steviol glycosides are natural sweetener constituents found in the leaves of Stevia rebaudiana Bertoni (Asteraceae). The specifications for steviol glycosides were established by the Joint FAO/WHO Expert Committee on ...Steviol glycosides are natural sweetener constituents found in the leaves of Stevia rebaudiana Bertoni (Asteraceae). The specifications for steviol glycosides were established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2008, although there was a call in the following year for the modification of this assay method to enable the determination of nine steviol glycosides rather than just seven. In response, based on a proposed method by the Japan Stevia Association, we developed an improved method by changing the HPLC conditions and including the use of an octadecylsilyl column instead of an amino-bonded column to enable the rapid and reliable determination of the nine steviol glycosides by an isocratic HPLC-UV method. With the developed method, the nine steviol glycosides can be separately determined, and identified using individual reference chemicals as standards, unlike the previous identification method, which was based on the relative retention times. In addition, the single stevioside quantification standard was replaced with both stevioside and rebaudioside A quantification standards. Importantly, the validation of the developed method was successful. The limits of quantification for the nine steviol glycosides were between 0.2% and 0.6%. The developed assay method for the nine steviol glycosides was proposed to JECFA and adopted as the revised assay method for the steviol glycosides specifications at its 73rd meeting in 2010.展开更多
SrUGT76G1,the most well-studied diterpene glycosyltransferase in Stevia rebaudiana,is key to the biosynthesis of economically important steviol glycosides(SGs).However,the molecular regulatory mechanism of SrUGT76G1 h...SrUGT76G1,the most well-studied diterpene glycosyltransferase in Stevia rebaudiana,is key to the biosynthesis of economically important steviol glycosides(SGs).However,the molecular regulatory mechanism of SrUGT76G1 has rarely been explored.In this study,we identified a MYB transcription factor,SrMYB1,using a yeast one-hybrid screening assay.SrMYB1 belongs to the typical R2R3-type MYB protein and is specifically localized in the nucleus with strong transactivation activity.The transcript of SrMYB1 is predominantly accumulated in flowers,but is also present at a lower level in leaves.Yeast one-hybrid and electrophoretic mobility shift assays verified that SrMYB1 binds directly to the MYB binding sites in the F4-3 fragment(+50–(–141))of the SrUGT76G1 promoter.Furthermore,we found that SrMYB1 could significantly repress the expression of SrUGT76G1 in both epidermal cells of tobacco leaves and stevia callus.Taken together,our results demonstrate that SrMYB1 is an essential upstream regulator of SrUGT76G1 and provide novel insight into the regulatory network for the SGs metabolic pathway in S.rebaudiana.展开更多
In recent years there has been increasing demand for natural non-nutritive high intensity sweeteners with low-calorie value as an alternative to sucrose. Extracts of the leaves of Stevia rebaudiana (Bertoni), have bee...In recent years there has been increasing demand for natural non-nutritive high intensity sweeteners with low-calorie value as an alternative to sucrose. Extracts of the leaves of Stevia rebaudiana (Bertoni), have been known for their sweet taste. Steviosides and rebaudioside-A are the two major diterpenoid glucosides components present in the leaf extracts of the stevia, these glycosides are 300 times sweeter than sugar and also exhibits wide therapeutic activity. The conventional methods of isolation of steviosides involve long extraction and purification procedures;therefore optimization of product yields is a challenging problem. The present study, establishes a new improvised process of extraction of steviosides from the stevia leaves in which the dry treated leaves were grounded, defatted, and extracted through pressurized hot water extractor (PHWE), followed by purification and concentration of the sweet glycosides through ultra (UF) and nano (NF) membrane filtration in obtaining high (98.2%) purity steviosides. This process established “green” method for isolation of high quality steviol glycosides, with improved final yield is 10.1% from 11% of crude leaf extract and observed the improved organoleptic and biological activity (antioxidant). Thus the method confirms a simple, inexpensive and eco-friendly process in obtaining pure steviosides.展开更多
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 ...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.展开更多
To overcome the inconveniences related to its conventional obtainment of natural sweeteners and active compounds from Stevia rebaudiana Bertoni,methods involving application of technology,such as ultrasound assisted e...To overcome the inconveniences related to its conventional obtainment of natural sweeteners and active compounds from Stevia rebaudiana Bertoni,methods involving application of technology,such as ultrasound assisted extraction,microwave assisted extraction,extraction under pressurized conditions by means of pressurized liquid extraction,subcritical water extraction and supercritical fluid extraction have been investigated.In this review,these emerging techniques were analyzed and discussed,the process variables and operational strategies,their impacts on the extraction and their comparison against the conventional techniques were demonstrated.Simplicity and the possibility of operational automation,the use of less energy and solvents,in addition to the reduced complexity of the subsequent purification steps,make up viable alternatives suitable for a possible industrial application.These processes leverage the concept of green chemistry,but still suffer limitations related to acquisition and maintenance costs,in addition to the effects of the action of some of these technologies remaining unexplored.展开更多
文摘Stevia is well-known for its unique non-caloric intense sweetness and recognising as one of the most acceptable sweeteners.Therefore,quality control and metabolite profiling perspectives,two isocratic reverse-phase UPLC-PDA-ELSD-MS methods were developed to determine nine diterpene glycosides(method A)and isomeric compounds(steviol and isosteviol;method B).Both the methods were validated and found as per the ICH guidelines for accuracy(recovery),precision(Intraday,interday,and intermediate precision),linearity,and regression coefficient(r2),ruggedness(eluent concentration±2 mM,column oven temperature±3℃ and retention time),and system suitability(theoretical plate count,asymmetric factor,injection precision and resolution).LOD for steviol and its glycosides were found between 0.92 and 5.22μg/mL while LOQ were 2.78-18.29μg/mL,respectively.Validated methods had shown only the presence of steviol glycosides in Stevia rebaudina samples while LC-ESI-MS/MS revealed the identification of 46 different class of molecules.These methods will be helpful to monitor quality in Stevia production and its derived products.
基金supported by the Natural Science Foundation of Jiangsu Province,China(BK20180312)the Jiangsu Key Laboratory for the Research and Utilization of Plant Resources,China(JSPKLB201810)the Natural Science Foundation of Shanxi Province,China(201901D111230)。
文摘The steviol glycosides(SGs)in stevia(Stevia rebaudiana Bertoni)leaves are becoming increasingly valuable due to its high sweetness but low calorific value,which is driving the development of stevia commercial cultivation.Optimizing fertilization management can effectively increase SGs productivity,but knowledge on the relationship between potassium(K)fertilization and SGs production is still lacking.In this study,pot experiments were conducted in order to investigate the effect of K deficiency on SGs synthesis in stevia leaves,as well as the underlying mechanisms.Our results showed that when compared with standard K fertilization,K deficiency treatment has no significant effect on the biomass of stevia plant grown in a given soil with high K contents.However,K deficiency critically decreased leaf SGs contents as well as the expression of SGs synthesis-related genes.The contents of different sugar components decreased and the activities of sugar metabolism-related enzymes were inhibited under the K deficiency condition.Moreover,spraying sucrose on the leaves of stevia seedlings diminished the inhibitory effect caused by K deficiency.Our results also revealed the significant positive correlations between sucrose,glucose and SGs contents.Overall,our results suggest that K deficiency would suppress the synthesis of SGs in stevia leaves,and this effect may be mediated by the leaf sugar metabolism.Our findings provide new insights into the improvement of SGs production potential.
基金This work was financially supported by the National Natural Science Foundation of China(21676119,31671845,32021005)the Key Research and Development Program of China(2018YFA0900300,2018YFA0900504).
文摘Stevia rebaudiana Bertoni is commonly called stevia and mostly found in the north east regions of South America.It is an herbaceous and shrubby plant belonging to the Asteraceae family.Stevia is considered as a natural sweetener and a commercially important plant worldwide.The leaves of S.rebaudiana contain steviol glycosides(SGs)which are highly potent and non-caloric sweeteners.The sweetening property of S.rebaudiana is contributed to the presence of these high potency,calorie free steviol glycosides.SGs are considerably suitable for replacing sucrose and other artificial sweetening agents which are used in different industries and pharmaceuticals.SGs amount in the plant mostly varies from 8%to 10%,and the enhancement of SGs is always in demand.These glycosides have the potential to become healthier alternatives to other table sugars for having desirable taste and zero calories.SGs are almost 300 times sweeter than sucrose.Being used as alternative sugar intensifier the commercial value of this plant in biopharmaceutical,food and beverages industries and in international market is increasing day by day.SGs have made stevia an important part of the medicinal world as well as the food and beverage industry,but the limited production of plant material is not fulfilling the higher global market demand.Therefore,researchers are working worldwide to increase the production of important SGs through the intercession of different biotechnological approaches in S.rebaudiana.This review aims to describe the emerging biotechnological strategies and approaches to understand,stimulate and enhance biosynthesis of secondary metabolites in stevia.Conventional and biotechnological methods for the production of steviol glycosides have been briefly reviewed and discussed.
文摘Steviol glycosides are natural sweetener constituents found in the leaves of Stevia rebaudiana Bertoni (Asteraceae). The specifications for steviol glycosides were established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2008, although there was a call in the following year for the modification of this assay method to enable the determination of nine steviol glycosides rather than just seven. In response, based on a proposed method by the Japan Stevia Association, we developed an improved method by changing the HPLC conditions and including the use of an octadecylsilyl column instead of an amino-bonded column to enable the rapid and reliable determination of the nine steviol glycosides by an isocratic HPLC-UV method. With the developed method, the nine steviol glycosides can be separately determined, and identified using individual reference chemicals as standards, unlike the previous identification method, which was based on the relative retention times. In addition, the single stevioside quantification standard was replaced with both stevioside and rebaudioside A quantification standards. Importantly, the validation of the developed method was successful. The limits of quantification for the nine steviol glycosides were between 0.2% and 0.6%. The developed assay method for the nine steviol glycosides was proposed to JECFA and adopted as the revised assay method for the steviol glycosides specifications at its 73rd meeting in 2010.
基金supported by the National Natural Science Foundation of China(31901597)the Natural Science Foundation of Jiangsu Province,China(BK20201243)。
文摘SrUGT76G1,the most well-studied diterpene glycosyltransferase in Stevia rebaudiana,is key to the biosynthesis of economically important steviol glycosides(SGs).However,the molecular regulatory mechanism of SrUGT76G1 has rarely been explored.In this study,we identified a MYB transcription factor,SrMYB1,using a yeast one-hybrid screening assay.SrMYB1 belongs to the typical R2R3-type MYB protein and is specifically localized in the nucleus with strong transactivation activity.The transcript of SrMYB1 is predominantly accumulated in flowers,but is also present at a lower level in leaves.Yeast one-hybrid and electrophoretic mobility shift assays verified that SrMYB1 binds directly to the MYB binding sites in the F4-3 fragment(+50–(–141))of the SrUGT76G1 promoter.Furthermore,we found that SrMYB1 could significantly repress the expression of SrUGT76G1 in both epidermal cells of tobacco leaves and stevia callus.Taken together,our results demonstrate that SrMYB1 is an essential upstream regulator of SrUGT76G1 and provide novel insight into the regulatory network for the SGs metabolic pathway in S.rebaudiana.
文摘In recent years there has been increasing demand for natural non-nutritive high intensity sweeteners with low-calorie value as an alternative to sucrose. Extracts of the leaves of Stevia rebaudiana (Bertoni), have been known for their sweet taste. Steviosides and rebaudioside-A are the two major diterpenoid glucosides components present in the leaf extracts of the stevia, these glycosides are 300 times sweeter than sugar and also exhibits wide therapeutic activity. The conventional methods of isolation of steviosides involve long extraction and purification procedures;therefore optimization of product yields is a challenging problem. The present study, establishes a new improvised process of extraction of steviosides from the stevia leaves in which the dry treated leaves were grounded, defatted, and extracted through pressurized hot water extractor (PHWE), followed by purification and concentration of the sweet glycosides through ultra (UF) and nano (NF) membrane filtration in obtaining high (98.2%) purity steviosides. This process established “green” method for isolation of high quality steviol glycosides, with improved final yield is 10.1% from 11% of crude leaf extract and observed the improved organoleptic and biological activity (antioxidant). Thus the method confirms a simple, inexpensive and eco-friendly process in obtaining pure steviosides.
文摘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.
基金The authors would like to thank the Coordenaç˜ao de Aperfeiçoamento de Pessoal de Nível Superior(CAPES,process number 88882.449245/2019-01),for the financial support.
文摘To overcome the inconveniences related to its conventional obtainment of natural sweeteners and active compounds from Stevia rebaudiana Bertoni,methods involving application of technology,such as ultrasound assisted extraction,microwave assisted extraction,extraction under pressurized conditions by means of pressurized liquid extraction,subcritical water extraction and supercritical fluid extraction have been investigated.In this review,these emerging techniques were analyzed and discussed,the process variables and operational strategies,their impacts on the extraction and their comparison against the conventional techniques were demonstrated.Simplicity and the possibility of operational automation,the use of less energy and solvents,in addition to the reduced complexity of the subsequent purification steps,make up viable alternatives suitable for a possible industrial application.These processes leverage the concept of green chemistry,but still suffer limitations related to acquisition and maintenance costs,in addition to the effects of the action of some of these technologies remaining unexplored.
