rebaudioside-a and stevioside

Stevia rebaudiana Bertoni is a natural sweetener plant that is progressively used not only for its sweetening properties but also for its medicinal properties. The plant contains steviol glycoside (SG) which is reported to be up to 300 times sweeter than sucrose. The plant is said to have no side effects on human health and has been approved by FDA. On the basis of previous studies and available databases, this review discusses the extensive understanding of the different approaches for enhancements of SG in S. rebaudiana. To improve the SG biosynthesis, application of different stress, elicitors, induction of polyploidy, cell culture, genetic engineering, and transcriptomic approaches have been addressed. A brief discussion about the cloning and characterization of important genes of the metabolic pathway of SG biosynthesis is also discussed along with various metabolic engineering pathways viz. methylerythritol 4- phosphate (MEP) and mevalonate (MVA) pathways. This review paper also discusses the different aspects as well as the effects of various nanoparticles on S. rebaudiana growth and development, as well as SG biosynthesis.

Topics: Diterpenes, Kaurane; Gene Expression Regulation, Plant; Glucosides; Glycosides; Humans; Plant Leaves; Stevia

For health and safety concerns, traditional high-calorie sweeteners and artificial sweeteners are gradually replaced in food industries by natural and low-calorie sweeteners. As a natural and high-quality sugar substitute, steviol glycosides (SvGls) are continually scrutinized regarding their safety and application. Recently, the cultivation of organic stevia has been increasing in many parts of Europe and Asia, and it is obvious that there is a vast market for sugar substitutes in the future. Rebaudioside A, the main component of SvGls, is gradually accepted by consumers due to its safe, zero calories, clear, and sweet taste with no significant undesirable characteristics. Hence, it can be used in various foods or dietary supplements as a sweetener. In addition, rebaudioside A has been demonstrated to have many physiological functions, such as antihypertension, anti-diabetes, and anticaries. But so far, there are few comprehensive reviews of rebaudioside A. In this review article, we discuss the physicochemical properties, metabolic process, safety, regulatory, health benefits, and biosynthetic pathway of rebaudioside A and summarize the modification methods and state-of-the-art production and purification techniques of rebaudioside A. Furthermore, the current problems hindering the future production and application of rebaudioside A are analyzed, and suggestions are provided.

Topics: Dietary Sugars; Food Additives; Stevia; Sweetening Agents

This literature-based review synthesizes the available scientific information about steviol glycosides as natural sweeteners and molecules with therapeutic potential. In addition, it discusses the safety concerns regarding human consumption. Steviol glycosides exhibit a superior sweetener proficiency to that of sucrose and are noncaloric, noncariogenic, and nonfermentative. Scientific evidence encourages stevioside and rebaudioside A as sweetener alternatives to sucrose and supports their use based on their absences of harmful effects on human health. Moreover, these active compounds isolated from

Topics: Diterpenes, Kaurane; Glucosides; Glycosides; Humans; Stevia; Sucrose; Sweetening Agents

Nanotechnology has recently been emerged as a transformative technology that offers efficient and sustainable options for nano-bio interface. There has been a considerable interest in exploring the factors affecting elicitation mechanism and nanomaterials have been emerged as strong elicitors in medicinal plants. Stevia rebaudiana is well-known bio-sweetener and the presence of zero calorie, steviol glycosides (SGs) in the leaves of S. rebaudiana have made it a desirable crop to be cultivated on large scale to obtain its higher yield and maximal content of high quality natural sweeteners. Besides, phenolics, flavonoids, and antioxidants are abundant in stevia which contribute to its medicinal importance. Currently, scientists are trying to increase the market value of stevia by the enhancement in production of its bioactive compounds. As such, various in vitro and cell culture strategies have been adopted. In stevia agronanotechnology, nanoparticles behave as elicitors for the triggering of its secondary metabolites, specifically rebaudioside A. This review article discusses the importance of S. rebaudiana and SGs, conventional approaches that have failed to increase the desired yield and quality of stevia, modern approaches that are currently being applied to obtain utmost benefits of SGs, and future needs of advanced technologies for further exploitation of this wonder of nature.

Topics: Diterpenes, Kaurane; Flavonoids; Glucosides; Glycosides; Plant Leaves; Stevia; Sweetening Agents

Topics: Animals; Antineoplastic Agents, Phytogenic; Clinical Studies as Topic; Disease Models, Animal; Diterpenes, Kaurane; Drug Evaluation, Preclinical; Glucosides; Humans; Inhibitory Concentration 50; Metabolic Networks and Pathways; Molecular Structure; Stevia; Structure-Activity Relationship; Sweetening Agents

Stevia rebaudiana is one of the vastly acclaimed commercial plant in the world and belongs to Asteraceae family. The exclusive advantage of Stevia over artificial sweeteners is impeccable and targets its potentiality to the presence of diterpene glycosides. Moreover, the flaunting sweetness of steviol glycosides with associated medicinal benefits, turns the plant to be one of the most economic assets, globally. As compared to vegetative propagation through stem-cuttings, plant tissue culture is the most suitable approach in obtaining true-to-type plants of superior quality. During last few decades, significant in vitro propagation methods have been developed and still the research is ongoing. The present review discusses the tissue culture perspectives of S. rebaudiana, primarily focusing on the mineral nutrition, growth regulators and other accessory factors, motioning the optimum growth and development of the plant. Another crucial aspect is the generation of sweeter varieties in order to reduce the bitter-off taste, which is noticed after the consumption of the leaves. The in vitro cultures pose an efficient alternative system for production of steviol glycosides, with higher rebaudioside(s) content. Moreover, the review also covers the recent approaches pertaining to scale-up studies and genome editing perspectives.

Topics: Diterpenes, Kaurane; Glucosides; Glycosides; Plant Leaves; Stevia; Sweetening Agents

Steviol glycosides (SGs), as natural sweeteners from

Topics: Anthocyanins; Diterpenes, Kaurane; Glucosides; Glycosides; Humans; Plant Leaves; Stevia

Steviol glycosides (SvGls) are plant secondary metabolites belonging to a class of chemical compounds known as diterpenes. SvGls have been discovered only in a few plant species, including in the leaves of Stevia rebaudiana Bertoni. Over the last few decades, SvGls have been extensively researched for their extraordinary sweetness. As a result, the nutritional and pharmacological benefits of these secondary metabolites have grown increasingly apparent. In the near future, SvGls may become a basic, low-calorie, and potent sweetener in the growing natural foods market, and a natural anti-diabetic remedy, a highly competitive alternative to commercially available synthetic drugs. Commercial cultivation of stevia plants and the technologies of SvGls extraction and purification from plant material have already been introduced in many countries. However, new conventional and biotechnological solutions are still being sought to increase the level of SvGls in plants. Since many aspects related to the biochemistry and metabolism of SvGls in vivo, as well as their relationship to the overall physiology of S. rebaudiana are not yet understood, there is also a great need for in-depth scientific research on this topic. Such research may have positive impact on optimization of the profile and SvGls concentration in plants and thus lead to obtaining desired yield. This research summarizes the latest approaches and developments in SvGls production. KEY POINTS: • Steviol glycosides (SvGls) are found in nature in S. rebaudiana plants. • They exhibit nutraceutical properties. • This review provides an insight on different approaches to produce SvGls. • The areas of research that still need to be explored have been identified.

Topics: Diterpenes, Kaurane; Glucosides; Glycosides; Plant Leaves; Stevia; Sweetening Agents

The

Topics: Animals; Anti-Bacterial Agents; Antineoplastic Agents; Antioxidants; Cell Line, Tumor; Diterpenes, Kaurane; Drug Screening Assays, Antitumor; Ethnobotany; Ethnopharmacology; Flavonoids; Glucosides; HeLa Cells; Humans; Inhibitory Concentration 50; Medicine, Traditional; Mice; Phytochemicals; Plant Extracts; Plant Leaves; Rats; Stevia; Sweetening Agents

With the pursuit of natural non-calorie sweeteners, steviol glycosides (SGs) have become one of the most popular natural sweeteners in the market. The SGs in Stevia are a mixture of SGs synthesized from steviol (a terpenoid). SGs are diterpenoids. Different SGs depend on the number and position of sugar groups on the core steviol backbone. This diversity comes from the processing of glycoside steviol by various glycosyltransferases. Due to the differences in glycosylation, each SG has unique sensory properties. At present, it is more complicated to extract high-quality SGs from plants, so the excavation of the metabolic pathways of engineered microorganisms to synthesize SGs has been extensively studied. Specifically, the expression of different glycosyltransferases in microbes is key to the synthesis of various SGs by engineered microorganisms. To trigger more researches on the functional characterization of the enzymes encoded by these genes, this review describes the latest research progresses of the related enzymes involved in SG biosynthesis and metabolic engineering.Key points• Outlines the research progress of key enzymes in the biosynthetic pathway of SGs• Factors affecting the catalytic capacity of stevia glucosyltransferase• Provide guidance for the efficient synthesis of SGs in microbial cell factories.

Topics: Diterpenes, Kaurane; Glucosides; Glycosides; Glycosyltransferases; Metabolic Engineering; Plant Leaves; Stevia

Stevia rebaudiana Bertoni, a plant from South America and indigenous of Paraguay, has shown several biological effects and healthy properties, although it is especially used in South America and some Asiatic regions. In addition, it is a natural sweetener, almost 300 times sweeter than sucrose, being attributed to its phytoconstituents prominent antioxidant, antimicrobial, antidiabetic (antihyperglycemic, insulinotropic, and glucagonostatic), antiplatelet, anticariogenic, and antitumor effects. In this sense, this work aims to provide an extensive overview on the historical practices of stevia and its effects in human health based on its chemical composition and applications for both food and pharmaceutical industries.

Topics: Animals; Anti-Bacterial Agents; Antioxidants; Clinical Trials as Topic; Diterpenes, Kaurane; Drug Evaluation, Preclinical; Glucosides; Humans; Hypoglycemic Agents; Plant Extracts; Plant Leaves; Stevia; Sweetening Agents

Steviol glycoside sweeteners are extracted and purified from the Stevia rebaudiana Bertoni plant, a member of the Asteraceae (Compositae) family that is native to South America, where it has been used for its sweet properties for hundreds of years. With continued increasing rates of obesity, diabetes, and other related comorbidities, in conjunction with global public policies calling for reductions in sugar intake as a means to help curb these issues, low- and no-calorie sweeteners (LNCSs, also known as high-potency sweeteners) such as stevia are gaining interest among consumers and food manufacturers. This appeal is related to stevia being plant-based, zero calorie and with a sweet taste that is 50-350 times sweeter than sugar, making it an excellent choice for use in sugar- and calorie-reduced food and beverage products. Despite the fact that the safety of stevia has been affirmed by several food regulatory and safety authorities around the world, insufficient education about stevia's safety and benefits, including continuing concern with regard to the safety of LNCSs in general, deters health professionals and consumers from recommending or using stevia. Therefore, the aim of this review and the stevia symposium that preceded this review at the ASN's annual conference in 2017 was to examine, in a comprehensive manner, the state of the science for stevia, its safety and potential health benefits, and future research and application. Topics covered included metabolism, safety and acceptable intake, dietary exposure, impact on blood glucose and insulin concentrations, energy intake and weight management, blood pressure, dental caries, naturality and processing, taste and sensory properties, regulatory status, consumer insights, and market trends. Data for stevia are limited in the case of energy intake and weight management as well as for the gut microbiome; therefore, the broader literature on LNCSs was reviewed at the symposium and therefore is also included in this review.

Topics: Diterpenes, Kaurane; Glucosides; Humans; Plant Extracts; Plant Leaves; Stevia; Sweetening Agents

The isolation, structure elucidation, chemistry, biosynthesis and biological activity of the sweet steviol glycosides from Stevia rebaudiana, are reviewed.

Topics: Animals; Brazil; Chick Embryo; Diterpenes, Kaurane; Glucosides; Glycosides; Humans; Hydrolysis; Plant Extracts; Plant Leaves; Rats; Stevia; Sweetening Agents; Taste; Terpenes

Soaps are the oldest and perhaps most natural surfactants. However, they lost much of their importance since "technical surfactants", usually based on sulfates or sulfonates, have been developed over the last fifty years. Indeed, soaps are pH- and salt-sensitive and they are irritant, especially to the eyes. In food emulsions, although authorized, they have a bad taste, and long-chain saturated soaps have a high Krafft temperature. We believe that most or perhaps all of these problems can be solved with modern formulation approaches. We start this paper with a short overview of our present knowledge of soaps and soap formulations. Then we focus on the problem of the lacking soap solubility at neutral pH values. For example, it is well known that with the food emulsifier sodium oleate (NaOl), clear and stable aqueous solutions can only be obtained at pH values higher than 10. A decrease in the pH value leads to turbid and unstable solutions. This effect is not compatible with the formulation of aqueous stable and drinkable formulations with neutral or even acidic pH values. However, the pH value/phase behavior of aqueous soap solutions can be altered by the addition of other surfactants. Such a surfactant can be Rebaudioside A (RebA), a steviol glycoside from the plant Stevia rebaudiana which is used as a natural food sweetener. In a recent paper, we showed the influence of RebA on the apKa value of sodium oleate in a beverage microemulsion and on its clearing temperature. In the present paper, we report on the effect of the edible bio-surfactant RebA, on the macroscopic and microscopic phase behavior of simple aqueous sodium oleate solutions at varying pH values. The macroscopic phase behavior is investigated by visual observation and turbidity measurements. The microscopic phase behavior is analyzed by acid-base titration curves, phase-contrast and electron microscopy. It turned out that even at neutral pH, aqueous NaOl/RebA solutions can be completely clear and stable for more than 50days at room temperature. This is for the first time that a long chain soap could be really solubilized in water at neutral pH at room temperature. At last, these findings were applied to prepare stable, highly translucent and drinkable aqueous solutions of omega-3-fatty acids at a pH value of 7.5.

Topics: Diterpenes, Kaurane; Emulsifying Agents; Emulsions; Fatty Acids, Omega-3; Glucosides; Humans; Hydrogen-Ion Concentration; Micelles; Oleic Acid; Soaps; Solubility; Temperature; Water

Many different dietary supplements are currently marketed for the management of hypertension and diabetes, but the evidence for effectiveness is mixed. The objective of this systematic review was to critically appraise and evaluate the evidence for effectiveness of steviol glycosides (stevioside and rebaudioside A) on cardiovascular risk factors, using data from randomised clinical trials (RCTs).. Electronic searches were conducted in Medline, Embase, Amed, Cinahl and The Cochrane Library. We also searched Google Scholar, and hand searched the bibliography of retrieved full texts. The reporting quality of included studies was assessed using the Cochrane criteria. Two reviewers independently determined the eligibility, assessed the reporting quality, and extracted the data.. Nine studies with a total of 756 participants were included. There was a variation in the reporting quality of included studies. Meta-analysis revealed a non-significant difference in systolic blood pressure between steviol glycoside and placebo, mean difference (MD): -2.98 mm Hg (-6.23 to 0.27). Significant reductions in diastolic blood pressure and fasting blood glucose were observed. There was no significant effect on blood lipid profile. Heterogeneity was significant. Adverse events included abdominal fullness, epigastric pain, and dizziness.. The evidence from published RCTs suggests that stevioside may generate reductions in blood pressure and fasting blood glucose. The sizes of the effects are small, and the substantial heterogeneity limits the robustness of any conclusions. Rebaudioside A does not appear to have any significant effects on blood pressure or cardiovascular risk factors. Available clinical trials vary in design and reporting quality, and some are characterised by inadequate sample sizes. In addition, the participants in most of the trials have high cardiovascular risk. Further clinical trials and regulatory assessments are warranted.

Topics: Blood Glucose; Blood Pressure; Cardiovascular Diseases; Chi-Square Distribution; Diabetes Mellitus; Dietary Supplements; Diterpenes, Kaurane; Glucosides; Humans; Hypertension; Protective Factors; Randomized Controlled Trials as Topic; Risk Assessment; Risk Factors; Sweetening Agents

In addition to their widely recognized use as dietary supplement ingredients, plant-derived compounds are increasingly used as natural sweeteners. The search for nonnutritive sweeteners has been stimulated over the last 20-30 years by concern over demonstrated or suspected relationships between consumption of sucrose and high-fructose corn syrups and a variety of health-related conditions. In the USA, there is increased use of plant extracts known to contain highly sweet terpenoids. Purified extracts of Stevia rebaudiana (Bertoni) containing the diterpene glycosides stevioside and rebaudioside A are popular as sweeteners and are also used as dietary supplements, and soft drinks and nutritional and energy shakes incorporating extracts of Siraitia grosvenorii (Swingle) fruits containing sweet triterpene glycosides such as mogroside V are also on the market. Here, we review recent studies on these two important sources of noncaloric natural sweeteners, including analytical methods used to identify and quantify specific constituents and structural features relating to their sweetness. We also review the generally recognized as safe status of specific components and their status with respect to review by the Joint FAO/WHO Expert Committee on Food Additives.

Topics: Chromatography, High Pressure Liquid; Chromatography, Liquid; Cucurbitaceae; Diterpenes, Kaurane; Glucosides; Humans; Plant Extracts; Plant Leaves; Stevia; Sweetening Agents; Triterpenes

Stevia rebaudiana, a perennial herb from the Asteraceae family, is known to the scientific world for its sweetness and steviol glycosides (SGs). SGs are the secondary metabolites responsible for the sweetness of Stevia. They are synthesized by SG biosynthesis pathway operating in the leaves. Most of the genes encoding the enzymes of this pathway have been cloned and characterized from Stevia. Out of various SGs, stevioside and rebaudioside A are the major metabolites. SGs including stevioside have also been synthesized by enzymes and microbial agents. These are non-mutagenic, non-toxic, antimicrobial, and do not show any remarkable side-effects upon consumption. Stevioside has many medical applications and its role against diabetes is most important. SGs have made Stevia an important part of the medicinal world as well as the food and beverage industry. This article presents an overview on Stevia and the importance of SGs.

Topics: Cloning, Molecular; Diterpenes, Kaurane; Glucosides; Glycosides; Plant Leaves; Signal Transduction; Stevia

Stevioside, an ent-kaurene type of diterpenoid glycoside, is a natural sweetener extracted from leaves of Stevia rebaudiana (Bertoni) Bertoni. Stevioside and a few related compounds are regarded as the most common active principles of the plant. Such phytochemicals have not only been established as non-caloric sweeteners, but reported to exhibit some other pharmacological activities also. In this article, natural distribution of stevioside and related compounds, their structural features, plausible biosynthetic pathways along with an insight into the structure-sweetness relationship are presented. Besides, the pharmacokinetics, wide-range of pharmacological potentials, safety evaluation and clinical trials of these ent-kaurene glycosides are revisited.

Topics: Animals; Diterpenes, Kaurane; Glucosides; Humans; Plant Leaves; Stevia; Structure-Activity Relationship; Sweetening Agents

A review of the role of gut microbiota in the metabolism of the steviol glycosides, stevioside and rebaudioside A, indicates that they are not absorbed intact but undergo hydrolysis by the intestinal microflora to steviol. Steviol is not metabolized by the intestinal flora and is absorbed from the intestine. The rate of hydrolysis for stevioside is greater than for rebaudioside A. Recent studies using mass spectrometry have shown that steviol-16,17-epoxide is not a microbial metabolite of steviol glycosides. Bacteroides species are primarily responsible for hydrolysis via their beta-glucosidase activity. Fecal incubation studies with both human and animal mixed flora provide similar results, and this indicates that the rat is an appropriate model for studies on steviol glycosides. Given the similarity in the microbial metabolism of stevioside and rebaudioside A with the formation of steviol as the single hydrolysis product that is absorbed from the intestinal tract, the toxicological data on stevioside are relevant to the risk assessment of rebaudioside A.

Topics: Adaptation, Physiological; Animals; Bacteria; Cellulases; Diet; Diterpenes, Kaurane; Glucosides; Humans; Hydrolysis; Intestines

Stevioside is a natural sweetener extracted from leaves of Stevia rebaudiana (Bertoni) Bertoni. The literature about Stevia, the occurrence of its sweeteners, their biosynthetic pathway and toxicological aspects are discussed. Injection experiments or perfusion experiments of organs are considered as not relevant for the use of Stevia or stevioside as food, and therefore these studies are not included in this review. The metabolism of stevioside is discussed in relation with the possible formation of steviol. Different mutagenicity studies as well as studies on carcinogenicity are discussed. Acute and subacute toxicity studies revealed a very low toxicity of Stevia and stevioside. Fertility and teratogenicity studies are discussed as well as the effects on the bio-availability of other nutrients in the diet. The conclusion is that Stevia and stevioside are safe when used as a sweetener. It is suited for both diabetics, and PKU patients, as well as for obese persons intending to lose weight by avoiding sugar supplements in the diet. No allergic reactions to it seem to exist.

Topics: Animals; Diterpenes; Diterpenes, Kaurane; Fertility; Glucosides; Humans; Reproduction; Stevia; Sweetening Agents

Non-nutritive sweeteners have potential effects on brain function. We investigated neural correlates of responses to beverages differing in sweetness and calories. Healthy participants completed 4 randomised sessions: water vs. water with stevia, glucose, or maltodextrin. Blood-oxygenation level-dependent (BOLD) contrast was monitored for 30 min post-ingestion by functional Magnetic Resonance Imaging. A food visual probe task at baseline was repeated at 30 min. A significant interaction of taste-by-calories-by-time was demonstrated mainly in motor, frontal, and insula cortices. Consumption of the stevia-sweetened beverage resulted in greater BOLD decrease, especially in the 20-30 min period, compared to other beverages. There was a significant interaction of taste-by-time in BOLD response in gustatory and reward areas; sweet beverages induced greater reduction in BOLD compared to non-sweet. The interaction calories-by-time showed significantly greater incremental area under the curve in thalamic, visual, frontal, and parietal areas for glucose and maltodextrin 10-20 min post-consumption only, compared to water. In the visual cue task, the water demonstrated an increased response in the visual cortex to food images post-consumption; however, no difference was observed for the three sweet/caloric beverages. In conclusion, both sweet taste and calories exert modulatory effects, but stevia showed a more robust and prolonged effect.

Topics: Adult; Brain; Cross-Over Studies; Diterpenes, Kaurane; Glucose; Glucosides; Humans; Non-Nutritive Sweeteners; Stevia; Sweetening Agents; Water

Consumption of sugars in food and beverages has increased at an alarming rate. While excessive daily sugar intake has been well-associated as the onset of medical complications, additional sugars are still used in manufactured food products just to satisfy the consumers' needs. Hence, there is a need to develop sugar replacers that have low glycemic response without compromising the organoleptic characteristics of food products. This study aimed to determine if SUITENA™, a novel sweetener containing erythritol, xylitol, and Stevia, has low glycemic response upon consumption by human subjects.. Six human subjects were randomly chosen and were healthy at the point of experimentation. Capillary blood was collected via finger-prick method to monitor the glycemic response of every individual for 90 min after ingestion of sugar solution.. It was found that the mean area under the curve (AUC) of the dextrose standard was 11.8-fold higher (p < 0.05) than the AUC of SUITENA™. SUITENA™ was not able to increase blood glucose level for up to 90 min while a spike in blood glucose level was observed from 15 min post-consumption of dextrose solution. We found that SUITENA™ has elicited a glycemic response 8% relative to the standard. Such low glycemic response has been reported by studies on other novel sugars.. This preliminary finding suggested that SUITENA™ is a healthier alternative to fast sugars due to its low glycemic response. A larger sampling size is required to confirm the results.

Topics: Beverages; Blood Glucose; Diterpenes, Kaurane; Ethics; Food; Glucose; Glucosides; Glycemic Index; Humans; Malaysia; Plant Extracts; Polymers; Research Subjects; Single-Blind Method; Stevia; Sugars; Sweetening Agents

The effect of Stevia extracts on in vitro Streptococcus mutans biofilm formation and in vivo plaque pH was evaluated in this paper. Three 10% solutions containing stevioside, rebaudioside A or sucrose were prepared. MTT assay was used to evaluate microbiological counts in vitro. Twenty volunteers rinsed for 1 min with each solutions, and plaque pH was measured at 7 time points after each rinse. Higher in vitro S. mutans biofilm formation was observed in sucrose solution (p < 0.01). After 5, 10, 15 and 30 min, the sucrose in vivo rinse produced a statistically significantly lower pH value compared to the Stevia extracts (F = 99.45, p < 0.01).Stevia extracts can be considered nonacidogenic.

Topics: Adult; Bacterial Load; Bacteriological Techniques; Biofilms; Coloring Agents; Dental Caries; Dental Plaque; Diterpenes, Kaurane; Female; Follow-Up Studies; Glucosides; Humans; Hydrogen-Ion Concentration; Male; Mouthwashes; Pilot Projects; Plant Extracts; Spectrophotometry; Stevia; Streptococcus mutans; Sucrose; Sweetening Agents; Tetrazolium Salts; Thiazoles; Young Adult

This randomized, double-blind, cross-over study assessed the comparative pharmacokinetics of steviol and steviol glucuronide following single oral doses of rebaudioside A and stevioside in healthy adult male subjects. Steviol glucuronide appeared in the plasma of all subjects after administration of rebaudioside A or stevioside, with median tmax values of 12.0 and 8.00h post-dose, respectively. Steviol glucuronide was eliminated from the plasma, with similar t1/2 values of approximately 14h for both compounds. Administration of rebaudioside A resulted in a significantly (approximately 22%) lower steviol glucuronide geometric mean Cmax value (1472ng/mL) than administration of stevioside (1886ng/mL). The geometric mean AUC0-t value for steviol glucuronide after administration of rebaudioside A (30,788ngh/mL) was approximately 10% lower than after administration of stevioside (34,090ngh/mL). Steviol glucuronide was excreted primarily in the urine of the subjects during the 72h collection period, accounting for 59% and 62% of the rebaudioside A and stevioside doses, respectively. No steviol glucuronide was detected in feces. Pharmacokinetic analysis indicated that rebaudioside A and stevioside underwent similar metabolic and elimination pathways in humans with steviol glucuronide excreted primarily in the urine and steviol in the feces. No safety concerns were noted as determined by reporting of adverse events, laboratory assessments of safety or vital signs.

Topics: Administration, Oral; Adolescent; Adult; Cross-Over Studies; Diterpenes, Kaurane; Double-Blind Method; Glucosides; Humans; Male; Middle Aged; Sweetening Agents

The antihypertensive effect of crude stevioside obtained from the leaves of Stevia rebaudiana (Bertoni) Bertoni (Compositae) on previously untreated mild hypertensive patients was examined. Patients with essential hypertension were submitted to a placebo phase for 4 weeks. The volunteers selected in this phase were randomly assigned to receive either capsules containing placebo during 24 weeks or crude stevioside 3.75 mg/kg/day (7 weeks), 7.5 mg/kg/day (11 weeks) and 15.0 mg/kg/day (6 weeks). All capsules were prescribed twice a daily (b.i.d.), i.e. before lunch and before dinner. After the placebo phase and after each dose of crude stevioside, body mass index, electrocardiogram and laboratory tests were performed. During the investigation blood pressure (BP) was measured biweekly and the remaining data were collected at the end of each stevioside dose step. All adverse events were prospectively recorded but no major adverse clinical effects were observed during the trial. Systolic and diastolic BP decreased (p < 0.05) during the treatment with crude stevioside, but a similar effect was observed in the placebo group. Therefore, crude stevioside up to 15.0 mg/kg/day did not show an antihypertensive effect. Moreover, the results suggest that oral crude stevioside is safe and supports the well-established tolerability during long term use as a sweetener in Brazil.

Topics: Adult; Antihypertensive Agents; Diterpenes, Kaurane; Double-Blind Method; Female; Glucosides; Humans; Hypertension; Male; Middle Aged; Phytotherapy; Prospective Studies; Stevia

Steviol glycosides possess Bola-form amphiphilic structure, which can solubilize hydrophobic phytochemicals and exert physical modification to the hydrophilic matrix. However, the effect of steviol glycosides on the starch hydrogel is still unclear. Herein, the physicochemical properties, in vitro digestibility, and release behavior of starch hydrogel in the presence of steviol glycosides were investigated. The results showed that the addition of steviol glycosides promoted the gelatinization and gelation of starch, and endowed the starch hydrogel with softer texture, larger volume, and higher water holding capacity. The hydrophobic curcumin was well integrated into hydrogel by steviol glycosides, providing the gel with improved colour brilliance. The introduction of steviol glycosides hardly affected the digestibility of starch gel, but it promoted the release rate of curcumin. Notably, this release behavior was pH dependent, which tended to target the alkaline intestine. This work provided some theoretical supports for the development of sugar-free starchy foods.

Topics: Curcumin; Diterpenes, Kaurane; Glycosides; Hydrogels; Hydrogen-Ion Concentration; Starch; Stevia

Steviol glycosides have attracted great interest because of their high levels of sweetness and safety, and absence of calories. Improvement of their sensory qualities via glycosylation modification by glycosyltransferase is a research hotspot. In this study, YjiC, a uridine diphosphate-dependent glycosyltransferase from Bacillus subtilis 168, was found with the ability to glycosylate rebaudioside A (Reb A) to produce a novel mono β-1, 6-glycosylated Reb A derivative rebaudioside L2 (Reb L2). It has an improved sweetness compared with Reb A. Next, a cascade reaction was established by combining YjiC with sucrose synthase AtSuSy from Arabidopsis thaliana for scale-up preparation of Reb L2. It shows that Reb L2 (30.94 mg/mL) could be efficiently synthesized with an excellent yield of 91.34% within 12 h. Therefore, this study provides a potential approach for the production and application of new steviol glycoside Reb L2, expanding the scope of steviol glycosides.

Topics: Catalysis; Diterpenes, Kaurane; Glucosides; Glycosyltransferases; Stevia

Compared with steviol glycosides, the taste of glucosylated steviol glycosides is better and more similar to that of sucrose. At present, cyclodextrin glucanotransferase (CGTase) is primarily used to catalyze the conversion of steviol glycosides to glucosylated steviol glycosides, with soluble starch serving as a glycosyl donor. The main disadvantages of enzymatic transglycosylation are the limited number of enzymes available, the low conversion rates that result in low yields, and the lack of selectivity in the degree of glycosylation of the products. In order to fill these gaps, the proteome of Alkalihalobacillus oshimensis (also named Bacillus oshimensis) was used for mining novel CGTases.. Here, CGTase-15, a novel β-CGTase with a wide pH adaptation range, was identified and characterized. The catalyzed product of CGTase-15 tasted better than that of the commercial enzyme (Toruzyme® 3.0 L). In addition, two amino acid sites, Y199 and G265, which play important roles in the conversion of steviol glycosides to glucosylated steviol glycosides were identified by site-directed mutagenesis. Compared with CGTase-15, CGTase-15-Y199F mutant significantly increased the conversion rate of rebaudioside A (RA) to glucosylated steviol glycosides. Compared with CGTase-15, the content of short-chain glycosylated steviol glycosides catalyzed by CGTase-15-G265A mutant was significantly increased. Moreover, the function of Y199 and G265 was verified in other CGTases. The above mutation pattern has also been applied to CGTase-13 (a CGTase discovered by our laboratory with great potential in the production of glycosylated steviol glycosides), confirming that the catalytic product of CGTase-13-Y189F/G255A mutant has a better taste than that of CGTase-13.. This is the first report on the improvement of the sensory profiles of glycosylated steviol glycosides through site-directed mutagenesis of CGTase, which is significant for the production of glycosylated steviol glycosides.

Topics: Glucosides; Glycosylation

Stevia rebaudiana Bertoni is a valuable medicinal plant and an essential source of natural sweetener, steviol glycosides (SGs), with rebaudioside A (RA) being one of the main components of SGs. bHLH transcription factors play a crucial role in plant development and secondary metabolism. In this study, 159 SrbHLH genes were identified from the S. rebaudiana genome, and each gene was named based on its chromosome location. The SrbHLH proteins were then clustered into 18 subfamilies through phylogenetic analysis. The analysis of conserved motifs and gene structure further supported the classification of the SrbHLH family. Chromosomal location and gene duplication events of SrbHLH genes were also studied. Moreover, based on the RNA-Seq data of different tissues of S. rebaudiana, 28 SrbHLHs were co-expressed with structural genes involved in RA biosynthesis. The expression pattern of candidate SrbHLH genes were confirmed by qPCR. Finally, dual luciferase reporter assays (DLAs) and subcellular localization analysis verified SrbHLH22, SrbHLH111, SrbHLH126, SrbHLH142, and SrbHLH152 are critical regulators of RA biosynthesis. This study provides new insights into the function of SrbHLHs in regulating SGs biosynthesis and lays the foundation for future applications of SrbHLH genes in molecular breeding of S. rebaudiana.

Topics: Diterpenes, Kaurane; Glycosides; Phylogeny; Plant Leaves; Stevia; Transcription Factors

Stevia rebaudiana Bertoni possesses various medicinal and food industrial applications. This study is the first to explore the effect of the cytokinins meta-Topolin (mT; 6-(3-hydroxybenzylamino) purine), zeatin, kinetin, and BAP (6-benzylaminopurine) at concentrations of 0 (control), 5, 10, and 15 µM on shoot multiplication, as well as stevioside, rebaudioside A, phenolic acid, and flavonoid content in bioreactor cultures. The highest number of shoots (23.4 per explant) was obtained in the medium containing 5 μM of mT. However, 15 μM of mT was superior for fresh biomass production and dry biomass accumulation. Reversed-phase (RP)-HPLC analysis showed a beneficial effect of 5 μM mT on stevioside (11.43 mg/g dry weight [DW]) and rebaudioside A (10.74 mg/g DW) biosynthesis. In all conditions, the ratio of rebaudioside A/stevioside ranged from 0.75 to 1.12. The phenolic acids chlorogenic, neochlorogenic, isochlorogenic A, and rosmarinic were confirmed in the stevia extracts, as were the flavonoids isoquercetin, and quercitrin. The highest accumulations of chlorogenic and neochlorogenic acids and flavonoids were observed in shoot tissues derived from 5 µM mT, whereas 5 µM of BAP stimulated biosynthesis of chlorogenic, isochlorogenic A, and rosmarinic acids. This is the first report on the use of mT-cytokinin showing high potential in stevia cultures.

Topics: Bioreactors; Body Weight; Flavonoids; Stevia

In this study, the interaction between nanoparticles (0, 50, 100, and 150 mg L

Topics: DNA Damage; Glucosides; Nanotubes, Carbon; Plant Leaves; Secondary Metabolism; Stevia

Stevia rebaudiana is a medicinal herb that accumulates non-caloric sweeteners called steviol glycosides (SGs) which are approximately 300 times sweeter than sucrose. This study used alginate (ALG) as an elicitor to increase steviol glycosides accumulation and elucidate gene transcription in the steviol glycosides biosynthesis pathway.. To minimize the grassy taste associated with stevia sweeteners, plantlets were grown in complete darkness. ALG was applied to stevia plants grown in suspension culture with a Murashige and Skoog (MS) medium to determine its effect on SGs' content and the transcription profile of SG-related genes using the HPLC and RT-qPCR methods, respectively. Treatment with alginate did not significantly affect plantlet growth parameters such as shoot number, dry and fresh weight. Rebaudioside A (Reb A) content increased approximately sixfold in the presence of 1g L. The current study proposes that adding alginate to the MS suspension medium can increase Reb A levels by altering the SG biosynthesize pathway's transcription profile. The present experiment provides new insights into the biochemical and transcriptional response mechanisms of suspension-cultured stevia plants to alginate.

Topics: Alginates; Diterpenes, Kaurane; Glucosides; Glycosides; Plant Leaves; Stevia; Sweetening Agents

To improve the conversion efficiency of rebaudioside C, this study screened the Paenarthrobacter ilicis CR5301 from soil samples and identified it by 16S rRNA. The conversion experiment proved that P. ilicis CR5301 was capable of converting rebaudioside C. The effects of initial pH, temperature, inoculation amount, and substrate concentration on rebaudioside C conversion rate were investigated. The results showed that the conversion rate of rebaudioside C reached up to 100% when CR5301 was incubated in a conversion medium with an initial pH of 7.0 for 8 h at 28°C and 270 rpm. The conversion time was reduced by at least 16 h compared with previous studies. The conversion product was analyzed and identified as steviol by high performance liquid chromatography, ultra performance liquid chromatography-triple-time of flight mass spectrometer, and Fourier transform infrared spectroscopy methods. In addition, stevioside, rebaudioside A, dulcoside A, and some unknown components in steviol glycosides byproduct were all efficiently converted to steviol. These findings provide an efficient approach to the conversion of rebaudioside C and byproduct to steviol to simplify the subsequent industrial process and improve the reuse value of steviol glycosides.

Topics: Diterpenes, Kaurane; Glucosides; Glycosides; RNA, Ribosomal, 16S; Stevia

The relationship between excessive sugar consumption and many diseases such as dental caries, obesity, diabetes and coronary heart has been increasing in recent years. In this study, utilization of natural sugar replacer steviol glycosides and bifidogenic effect by

Topics: Dental Caries; Dietetics; Fermentation; Glucose; Glucosides; Humans; Inulin; Plant Leaves; Stevia; Sugars

Stevia rebaudiana (stevia) contains commercially important steviol glycosides, stevioside and rebaudioside A, these compounds have insulinotropic and anti-hyperglycemic effect. Steviol, stevioside and rebaudioside-A have taste modulation and insulin potentiation activity. Stevia leaves are composed of steviol (2-5%), stevioside (4-13%) and rebaudioside-A (1-6%). Stevioside has after-taste bitterness, rebaudioside-A is sweetest in taste among all the glycosides present. Therefore, lower ratio of rebaudioside-A to stevioside has bitter after-taste, which makes stevia plants unpalatable. By over-expressing the genes, SrUGT76G1 and SrKO, we propose to increase the ratio of RebA to stevioside in stevia. Various lines were generated and amongst them, seven lines had both the transgenes present. Co-overxpresion of SrUGT76G1 and SrKO led to the increased concentration of RebA in all the seven transgenic lines (KU1-KU7) than control plant and RebA to stevioside ratio also increased significantly. Steviol, stevioside and RebA showed a differential concentration in all the seven lines, but the pattern was the same in all of them and the ratio of RebA to stevioside increased dramatically. In transgenic line 2 (KU2), RebA showed a steep increase in concentration 52% the rebaudioside-A to stevioside ratio increased from 0.74 (control) to 2.83. In overall all the lines, RebA showed a positive correlation with steviol and stevioside. Overexpression of SrKO led to an increase in steviol which increased the stevioside, overexpression of SrUGT76G1 ultimately increased RebA concentration. In conclusion, concentration of RebA increased significantly with co- overexpression of SrUGT6G1 and SrKO genes. Lines with increased RebA are more palatable and commercially viable.

Topics: Diterpenes, Kaurane; Food Additives; Glycosides; Plant Leaves; Stevia

Nanoplastics (NPs) as environmental contaminants have received increased attention in recent years. Numerous studies have suggested possible negative effects of plants exposure to NPs, but more data are needed with various plants under different exposure conditions to clarify the underlying phytotoxicity mechanisms. In this study, we investigated the effect of polystyrene nanoplastics (PSNPs; 28.65 nm average diameter) exposure (10, 100 and 250 mg/L) on plant morphology and production of relevant metabolites (steviol glycosides, chlorophylls, carotenoids, and vitamins) of in vitro-grown Stevia rebaudiana plantlets. Additionally, we used dark field microscopy combined with fluorescence hyperspectral imaging for the visualization of internalized PSNPs inside plant tissues. At higher concentrations (>100 mg/L), PSNPs were shown to aggregate in roots and to be transported to leaves, having a significantly negative impact on plant growth (reduced size and biomass), while increasing the production of metabolites compared to controls, most probably because of response to stress. The production of steviol glycosides presented a biphasic dose-response suggestive of hormesis, with the highest values at 10 mg/L PSNPs (1.5-2.2-fold increase compared to controls), followed by a decline in production at higher concentrations (100 and 250 mg/L), but with values comparable to controls. These results are promising for future in vivo studies evaluating the effect of NP exposure on the production of steviol glycosides, the natural sweeteners from stevia.

Topics: Diterpenes, Kaurane; Glucosides; Glycosides; Microplastics; Plant Leaves; Polystyrenes; Stevia

Steviol glycosides obtained from

Topics: Diterpenes, Kaurane; Glycosides; Plant Leaves; Rhamnose; Stevia; Sweetening Agents; Xylose

Steviol glycosides (SGs) are a variety of important natural sweeteners. They are 200-350 times sweeter than sucrose without calories. Currently, their production is still mainly dependent on extraction from Stevia rebaudiana Bertoni (stevia). Oligosaccharides are environmentally friendly elicitors that promote plant growth and accumulation of secondary metabolites. In the present study, different concentrations of chitosan oligosaccharides (COS) and alginate oligosaccharides (AOS) were applied to stevia to explore their effect on growth and SGs biosynthesis. It was found that both COS and AOS promoted biomass production by increasing the leaf number and photosynthetic efficiency, which may be related to the decreased content of abscisic acid. The content of SGs was significantly increased after 50 mg/L AOS treatment, which not only increased the contents of stevioside (STV) and rebaudioside A (Reb A) significantly, but some important minority glucosides, like Reb E, Reb D, and Reb M. The increased SGs contents were the combined effect of the higher expression of SGs biosynthesis related genes, including KAH, UGT74G1, UGT85C2, and UGT91D2. The geometry changes of stem induced by COS and AOS may help to increase the lodging resistance of stevia. Thus, COS and AOS can be used in the field planting of stevia to increase the yield of SGs for industrial purposes.

Topics: Biomass; Diterpenes, Kaurane; Glucosides; Glycosides; Plant Leaves; Stevia; Sucrose

In diabetes, in parallel to hyperglycaemia, elevated serum lipids are also diagnosed, representing a high-risk factor for coronary heart disease and cardiovascular complications. The objective of this study was to unravel the mechanisms that underlie the potential of steviol glycosides (stevioside or rebaudioside A) administered at two doses (500 or 2500 mg/kg body weight for 5 weeks) to regulate lipid metabolism. In this paper, the expression of selected genes responsible for glucose and lipid metabolism (Glut4, Pparγ, Cebpa, Fasn, Lpl and Egr1) in the peripheral tissues (adipose, liver and muscle tissue) was determined using quantitative real-time PCR method. It was found that the supplementation of steviol glycosides affected the expression of Glut4, Cebpa and Fasn genes, depending on the type of the glycoside and its dose, as well as the type of tissue, whish in part may explain the lipid-regulatory potential of steviol glycosides in hyperglycaemic conditions. Nevertheless, more in-depth studies, including human trials, are needed to confirm these effects, before steviol glycosides can be used in the therapy of type 2 diabetes.

Topics: Diabetes Mellitus, Type 2; Gene Expression; Glycosides; Humans; Hyperglycemia; Lipid Metabolism; Stevia

Topics: Biological Products; Diterpenes, Kaurane; Food Additives; Glucosides; Glycosides; Metal-Organic Frameworks; Porosity

This study reports the increment in the secondary metabolites in Stevia rebaudiana plant after exposure to the elimination of Ca and Mg from Murashige and Skoog culture medium. The effect of nutrient stress on regenerants of S. rebaudiana is measured, which reveals significantly enhanced growth parameters, steviol glycosides (SGs) content, and nonenzymatic antioxidants; total phenolic content, total flavonoid content, total antioxidant capacity, total reducing power, and DPPH-free radical scavenging activity as compared with the control treatment. However, significantly highest amounts are obtained in a medium with only Ca deficiency. The amount of rebaudioside A (Reb A) and stevioside (ST) obtained in the case of Ca-deficient medium is 4.08 and 0.69%, respectively. It is followed by the results obtained from both Ca- and Mg-deprived medium [Reb A (3.23%) and ST (0.52%)] and the lowest values are obtained from medium lacking Mg only [Reb A (2.60%) and ST (0.40%)]. The most probable adaptation mechanism might be the production of reactive oxygen species by nutrients' stress, which results in secondary metabolites production as defensive moieties to overcome stress situation. This effective protocol needs to be refined to apply on an industrial scale in bioreactors for increasing quantities of commercially important pharmaceutical compounds.

Topics: Antioxidants; Biomass; Calcium; Magnesium; Pharmaceutical Preparations; Plant Leaves; Stevia

Natural sweeteners are in high demand as a part of a healthy lifestyle. Among them, sweeteners with decreased caloric value and suitability for diabetes patients are most requested. Extension in their consumption extends the need for their quality control. A fast gradient UHPLC coupled with charged aerosol detection enabling quantitation of stevioside, rebaudioside A-D, and steviolbioside in commercial sweeteners and Stevia rebaudiana plant extracts has been developed. The method was developed to achieve high efficiency, simplicity, versatility, and low solvent consumption. All steviol glycosides were baseline-separated in less than 4 min with a total run time of 7 min. Buffer-free eluents were used in the separations and only 2.45 mL solvent were needed per analysis. The Luna Omega Polar column featuring polar modification of the C18 stationary phase was employed with mobile phases composed of water and acetonitrile for the excellent separation of polar steviol glycosides. The flow rate of the mobile phase 0.35 mL/min, column temperature 50 °C and injection volume 2 µL were used. Critical pair of glycosides, stevioside and rebaudioside A, were baseline separated with a resolution of 2.41. The universal charged aerosol detector allowed quantitation of steviol glycosides with a limit of detection and quantitation 0.15 and 0.5 µg/mL, respectively. Method intra-day precision was less than 2% (RSD), and the recovery was 89.6-105.0% and 93.8-111.4% for plant material and sweetener tablets, respectively. The quantity of steviol glycosides in three out of four commercial sweeteners was 3.0-12.3% higher than declared. The content was about 12.4% less than declared in one sample. But the difference from the labeled content corresponded to trueness and precision of the developed method together with variability of sweeteners production. The most abundant glycoside detected in sweeteners was stevioside followed by rebaudioside A. A leaf-to-stem ratio describing the dominant accumulation of steviol glycosides in leaves affected the differences in the amount of steviol glycosides among plant samples.

Topics: Aerosols; Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Glucosides; Glycosides; Humans; Plant Extracts; Plant Leaves; Stevia; Sweetening Agents

Stevia rebaudiana leaf extracts contain stevioside and rebaudioside A, two steviol glycosides (SGs) used as natural sweeteners because of their non-toxic, thermally stable and non-caloric properties. Indeed, leaf extracts can be up to 300 times sweeter than sucrose. Stevioside and rebaudioside A have organoleptic differences, the first one having an undesirable bitterness and the second one a higher sweetener capacity. Selection of the S. rebaudiana varieties and the best environmental conditions that elicit higher SGs content and the appropriate composition is an important goal. In this study we quantified and compared the amount of stevioside and rebaudioside A in two of the most used S. rebaudiana cultivars, Morita II and Criolla. Our results show a strong differential ratio of stevioside and rebaudioside A accumulated in the leaf between these cultivars. The Criolla cultivar showed about 3 times more stevioside per mg of dry weight than Morita II, whereas the Morita II accumulated almost 10 times more rebaudioside A than that produced in Criolla. We observed an enhanced expression in Morita II of three genes (SrKA13H, SrUGT74G1 and SrUGT76G1) known to encode three enzymes that participate in SGs biosynthesis, likely contributing to the differences in the stevioside and rebaudioside A accumulation. Not only genetic variation can affect SGs composition, but also environmental factors and crop management. Numerous studies have shown that the light regime in which S. rebaudiana cultivars grow can affect SGs accumulation. However, the optimal light regime to increase total SGs content is currently controversial. By applying various light intensities, we detected an increase of expression of these three biosynthetic genes at higher light intensity, accompanied by higher levels of stevioside and rebaudioside A, demonstrating that light intensity influences the synthesis of SGs.

Topics: Diterpenes, Kaurane; Glucosides; Stevia

In this paper, a lutein-glucosyl stevioside (stevia-G)-hydroxypropyl methylcellulose (HPMC) complex was prepared via an antisolvent precipitation combined with dynamic high pressure microfluidization method. The solubility, microstructure, crystallinity and thermodynamic properties of the freeze-dried powder were investigated, as well as the formation mechanism and the storage stability of the produced complex. When the optimal mass ratio of lutein, stevia-G, and HPMC was 1: 40: 0.5, the apparent solubility of lutein reached 2805.47 ± 24.94 μg·mL

Topics: Diterpenes, Kaurane; Glucosides; Hypromellose Derivatives; Lutein; Methylcellulose; Solubility; Stevia

Topics: Flavonoids; Gene Expression; Stevia; Trichomes

Topics: Animals; Body Weight; Cognition; Male; Non-Nutritive Sweeteners; Rats; Rats, Sprague-Dawley; Stevia; Sweetening Agents

The current study aimed to scale up the favorable bio-stimulants for enhancing the growth and breeding strategies of Stevia rebaudiana to increase sugar productivity. Inoculation of 45-day-old S. rebaudiana plantlets with Bacillus cereus and Azospirillum brasilense alone or in combination for 30 days allowed comparisons among their effects on enhancement and improvement of plant growth, production of bioactive compounds and expression of steviol glycoside genes. B. cereus SrAM1 isolated from surface-sterilized Stevia rebaudiana leaves was molecularly identified using 16s rRNA and tested for its ability to promote plant growth. Beneficial endophytic B. cereus SrAM1 induced all plant growth-promoting traits, except solubilization of phosphate, therefore it showed high effectiveness in the promotion of growth and production of bioactive compounds. Treatment of plants with B. cereus SrAM1 alone revealed carbohydrates content of 278.99 mg/g, total soluble sugar of 114.17 mg/g, total phenolics content of 34.05 mg gallic acid equivalent (GAE)/g dry weight) and total antioxidants activity of 32.33 mg (A.A)/g dry weight). Thus, plantlets inoculated with B. cereus SrAM1 alone exhibited the greatest responses in physiological and morphological parameters, but plantlets inoculated with B. cereus SrAM1 + A. brasilense showed a maximal upregulation of genes responsible for the biosynthesis of steviol glycosides (Kaurene oxidase, ent-KO; UDP-dependent glycosyl transferases of UGT85C2, UGT74G1, UGT76G1). Taken together, the used bacterial strains, particularly B. cereus SrAM1 could significantly improve the growth of S. rebaudiana via dynamic interactions in plants.

Topics: Antioxidants; Azospirillum brasilense; Bacillus cereus; Diterpenes, Kaurane; Gallic Acid; Gene Expression Regulation, Plant; Glucosides; Glycosides; Molecular Biology; Phosphates; Plant Breeding; Plant Leaves; RNA, Ribosomal, 16S; Stevia; Sugars; Transferases; Uridine Diphosphate

Steviol glycosides have been widely applied as new sweeteners in food, beverages, health care, and daily chemical industry owing to the properties of high-intensity sweetness, low calorie, and good physiological characteristics. However, most of steviol glycosides have a bitter taste. Their organoleptic properties can be effectively improved by modifying the linked glycosyl units. In this study, UGT94D1, a uridine diphosphate-dependent glycosyltransferase from Sesamum indicum, was reported to selectively glycosylate rebaudioside A (Reb A) for the synthesis of rebaudioside D2 (Reb D2). Furthermore, a cascade reaction system was constructed to synthesize Reb D2 with 94.66% yield by coupling UGT94D1 with sucrose synthase AtSuSy from Arabidopsis thaliana. Thus, our study not only introduced a practical method for the synthesis of steviol glycosides but also provided the possibility for further exploration of Reb D2.

Topics: Catalysis; Diterpenes, Kaurane; Glucosides; Glycosylation; Stevia

Stevia rebaudiana is a high value crop due to the strong commercial demand for its metabolites (steviol glycosides) but has limited geographical cultivation range. In non-native environments with different daylength and light quality, Stevia has low germination rates and early flowering resulting in lower biomass and poor yield of the desired metabolites. In this study, artificial lighting with light-emitting diodes (LEDs) was used to determine if different light quality within and outside of the photosynthetically active radiation (PAR) range can be used to improve germination rates and yields for production of steviol glycosides for the herbal supplement and food industry.. Plants treated with red and blue light at an intensity of 130 μmol m. While red and blue light combinations are effective for plant growth, the use of supplemental non-PAR irradiation of UV-A wavelength significantly and desirably delayed flowering, enhanced germination, biomass, rebaudioside A and stevioside yields, while supplemental green light improved yield of biomass and rebaudioside A, but not stevioside. Overall, the combination of red, blue and UV-A light resulted in the best overall productivity for Stevia rebaudiana. © 2021 Society of Chemical Industry.

Topics: Biomass; Diterpenes, Kaurane; Flowers; Germination; Glucosides; Light; Photosynthesis; Seeds; Stevia

Obesity is a public health problem characterized by increased body weight due to abnormal adipose tissue expansion. Bioactive compound consumption from the diet or intake of dietary supplements is one of the possible ways to control obesity. Natural products with adipogenesis-regulating potential act as obesity treatments. We evaluated the synergistic antiangiogenesis, antiadipogenic and antilipogenic efficacy of standardized rebaudioside A, sativoside, and theasaponin E1 formulations (RASE1)

Topics: 3T3-L1 Cells; Adipocytes; Adipogenesis; Angiogenesis Inhibitors; Animals; Biological Products; Disease Models, Animal; Diterpenes, Kaurane; Drug Compounding; Drug Synergism; Female; Glucosides; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Lipid Metabolism; Lipogenesis; Lipolysis; Mice; Mice, Inbred ICR; Obesity; Oleanolic Acid; Phytotherapy; RNA, Messenger; Saponins; Signal Transduction; Stevia; Tea

Topics: Chromatography, High Pressure Liquid; Cluster Analysis; Diterpenes, Kaurane; Genetic Loci; Genetic Variation; Genotype; Glucosides; Glycosides; Microsatellite Repeats; Phylogeny; Principal Component Analysis; Seeds; Stevia

Artificial neural network (ANN), as one of the artificial intelligence methods, has been widely using in HPLC studies for modelling purposes. Stevia rebaudiana is an important industrial plant due to its bio-sweetener molecule, rebaudioside-a, in its leaves. Although rebaudioside-a is up to 300-fold sweeter than sucrose, its calorie is almost zero. In this study, HPLC optimization of rebaudioside-a was studied and the optimization data based on multilinear gradient retention times were modelled by ANN. The input parameters were selected as concentrations, column temperatures, initial acetonitrile percentage for the first step of gradient elution, initial acetonitrile percentage for the second step of gradient elution, slope of acetonitrile, wavelengths, flow rates. The retention time was the output. Also, dried S. rebaudiana leaves were extracted and the concentrations were evaluated by HPLC. According to the ANN results, the most effective parameters on the prediction of non-linear gradient retention time for rebaudioside-a were found as flow rate and initial acetonitrile percentage for the second step of gradient. The best back propagation was selected as Levenberg-Marquardt algorithm. The highest rebaudioside-a level was found as 96.53 ± 6.36 μg mL

Topics: Acetonitriles; Algorithms; Artificial Intelligence; Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Glucosides; Neural Networks, Computer; Plant Leaves; Stevia; Sweetening Agents

Steviol glycosides were subjected to bacteria present in a soil sample collected from a Stevia plantation in Paraguay. During the incubation experiments, next to the aglycon steviol, steviol degradation products were also formed. X-ray analysis and NMR methods in combination with chemical synthesis and GIAO NMR calculations were used to fully characterize the structure of these compounds as a tricyclic ketone and the corresponding reduced form. They were nicknamed

Topics: Diterpenes, Kaurane; Glucosides; Plant Leaves; Stevia

Steviol glycosides are the intensely sweet components of extracts from Stevia rebaudiana. These molecules comprise an invariant steviol aglycone decorated with variable glycans and could widely serve as a low-calorie sweetener. However, the most desirable steviol glycosides Reb D and Reb M, devoid of unpleasant aftertaste, are naturally produced only in trace amounts due to low levels of specific β (1-2) glucosylation in Stevia. Here, we report the biochemical and structural characterization of OsUGT91C1, a glycosyltransferase from Oryza sativa, which is efficient at catalyzing β (1-2) glucosylation. The enzyme's ability to bind steviol glycoside substrate in three modes underlies its flexibility to catalyze β (1-2) glucosylation in two distinct orientations as well as β (1-6) glucosylation. Guided by the structural insights, we engineer this enzyme to enhance the desirable β (1-2) glucosylation, eliminate β (1-6) glucosylation, and obtain a promising catalyst for the industrial production of naturally rare but palatable steviol glycosides.

Topics: Carbohydrate Sequence; Catalytic Domain; Diterpenes, Kaurane; Gene Expression; Glucose; Glucosides; Glycosylation; Glycosyltransferases; Humans; Kinetics; Models, Molecular; Oryza; Plant Proteins; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Engineering; Protein Interaction Domains and Motifs; Recombinant Proteins; Stevia; Substrate Specificity; Sweetening Agents; Taste; Uridine Diphosphate Glucose

Mother liquor sugar (MLS), as the by-product of stevia production, contained ~65% steviol glycosides (SGs). Recovery of the SGs from MLS was achieved by crystallization coupled with reversed-phase chromatography. Crystallization was done by dissolving MLS in methanol solution and SGs were crystallized due to the polarity difference from the medium. Composition of SGs crystals differed with various temperature, time, solid-to-liquid ratio and water content. SGs were 42.25% recovered with high purity of 96.89% under optimal conditions (solid-to-liquid ratio = 1:5 (w/v), T = 25 °C, t = 24 h) in absolute methanol. The liquid phase after crystallization was subsequently subjected to reversed-phase chromatography, whereby the impurities were firstly eluted with 35% (v/v) ethanol solution and the purified SGs were then desorbed by absolute ethanol, finally recovering 95.20% of SGs in the purity of 98.08%. The total SGs recovery of the whole procedure was 97.23%. The two-step purification was easy-to-operate and feasible to scale-up for industrial application.

Topics: Chromatography, Reverse-Phase; Crystallization; Diterpenes, Kaurane; Ethanol; Glucosides; Plant Leaves; Stevia

Stevia (

Topics: Diterpenes, Kaurane; Glucosides; Glycosides; Glycosyltransferases; Molecular Docking Simulation; Plant Leaves; Stevia; Uridine Diphosphate

Stevia leaves were subjected to convective hot-air, infrared and vacuum drying at 40, 60 and 80 °C, followed by an assessment of thermophysical properties and microstructure, along with drying kinetics modelling and evaluation of energy features for all drying operations.. Effective moisture diffusivity (D. The results of this study contribute to a better understanding of the drying behaviour and showed that thermophysical properties of dried Stevia leaves and energy features are affected by drying methods. © 2021 Society of Chemical Industry.

Topics: Desiccation; Diterpenes, Kaurane; Food Preservation; Glucosides; Hot Temperature; Kinetics; Plant Leaves; Stevia; Vacuum

Transcriptome analysis revealed the potential mechanism of nitrogen regulating steviol glycosides synthesis via shifting of leaf carbon metabolic flux or inducing certain transcription factors. Nitrogen (N) plays key regulatory roles in both stevia (Stevia rebaudiana) growth and the synthesis of its functional metabolite steviol glycosides (SGs), but the mechanism by which this nutrient regulates SGs synthesis remains to be elucidated. To address this question, a pot experiment was performed in a greenhouse where stevia plants fertilized with N (the control as CK plants) and compared with plants without the supply of N. Physiological and biochemical analyses were conducted to test the growth and metabolic responses of plants to N regimes. Our results showed that N deficiency significantly inhibited plant growth and leaf photosynthesis, while increased leaf SGs contents in stevia (49.97, 46.64 and 84.80% respectively for rebaudioside A, stevioside, and rebaudioside C), which may be partly due to "concentration effect". Then, transcriptome analysis was conducted to understand the underlying mechanisms. A total of 535 differentially expressed genes were identified, and carbon metabolism-related events were highlighted by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Many of these genes were significantly upregulated by N-deficiency, including those involved in "phenylpropanoid biosynthesis", "flavonoid biosynthesis" and "starch and sucrose metabolism". Our study also analyzed the expression patterns of SGs synthesis-related genes under two N regimes and the potential transcription factors linking N nutrition and SG metabolism. N-deficiency may promote SGs synthesis by changing the carbon metabolism flux or inducing certain transcription factors. Our results provide deeper insight into the relationship between N nutrition and SGs synthesis in stevia plants.

Topics: Carbon; Diterpenes, Kaurane; Gene Expression Profiling; Gene Expression Regulation, Plant; Glucosides; Glycosides; Nitrogen; Oligosaccharides; Plant Leaves; Plant Proteins; Reproducibility of Results; Stevia; Transcription Factors

Steviol glycosides are well-known food sweeteners; their consumption has steadily increased over time. A pretreatment method was developed and validated to better separate rebaudioside A and stevioside from various protein-rich and fatty foods for quantification. This method was applied to soy sauce in liquid type and fish cake and coffee in solid type. Parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision were calculated. Calibration curves were linear in the working range of 5-100 mg/l, with coefficients of determination ≥0.99. The LOD and LOQ were in the ranges of 0.16-0.39 and 0.52-1.28 mg/kg, respectively. The percentage recoveries of the fortified samples were in the 88.01%-103.09% range, and the relative standard deviation was <10%. Method validation predicted a desirable accuracy, linearity, and precision. Therefore, the developed method can be practically applied for the quantitation of steviol glycosides in various foods, including soy sauce in liquid type and fish cake and coffee in solid type.

Topics: Diterpenes, Kaurane; Food Analysis; Glucosides; Limit of Detection; Stevia; Sweetening Agents

Topics: Ammonia; Diterpenes, Kaurane; Gene Expression Profiling; Gene Expression Regulation, Plant; Glucosides; Nitrates; Stevia; Transcription, Genetic

Organic anion transporter 3 (OAT3) plays a critical role in the renal excretion of many xenobiotics. Because steviol acyl glucuronide (SVAG), an OAT3 substrate, is the major circulating metabolite after oral ingestion of steviol glycosides and is excreted into the urine, inhibition of OAT3 activity may alter pharmacokinetic profiles of SVAG. The present study showed that drugs such as probenecid and glimepiride displayed potent inhibition toward the OAT3-mediated SVAG transport, with IC

Topics: Animals; Biological Transport; Diterpenes, Kaurane; Glucosides; HEK293 Cells; Humans; Kidney; Male; Organic Anion Transporters, Sodium-Independent; Probenecid; Rats; Rats, Sprague-Dawley; Sulfonylurea Compounds

To explore the retention and separation of stevioside polar compounds on a sulfonic acid-functionalized cation exchange column, the effects of different organic solvent-water mobile phases on the retention behavior of polar rebaudioside A (RA) and its analogues on the column were investigated over a wide range of organic solvent contents. The obtained U-shape curves hinted that the retention of the compounds on the same column transitioned from a reversed-phase liquid chromatography (RPLC) mode to a hydrophilic interaction liquid chromatography (HILIC) mode when the water-rich state in the mobile phases changed to an organic solvent-rich state. Under the RPLC mode, no separation of RA from its analogues was observed. The HILIC mode was beneficial to the retention and separation of RA and its analogues. Compared with polar protic solvents, aprotic solvents were more conducive to the retention and separation of the polar compounds based on the HILIC mode in organic solvent-rich mobile phases. Three models were used to evaluate and discuss the HILIC retention and separation of the compounds on the column. In the aprotic solvent-rich mobile phase, the HILIC retention of RA and its analogues was effectively described by a mixed-mode model; in the polar proton solvent-rich mobile phase, the retention of analytes was best described by an linear solvation strength (LSS) model. The content and composition of the organic solvent in the mobile phase were determined to be important influencing factors that regulated the retention time for the RA and its analogues, and even the separation mechanism for HILIC. The present work provides a theoretical basis for guiding one to prepare high-purity RA from its analogues by predicting the retention time.

Topics: Chromatography, Reverse-Phase; Diterpenes, Kaurane; Glucosides; Hydrophobic and Hydrophilic Interactions; Ion Exchange; Organic Chemicals; Solvents; Sulfonic Acids; Water

Governments are creating regulations for consumers to reduce their sugar intake, prompting companies to increase the ratio of artificial sweeteners in their products. However, there is evidence of some deleterious effects ascribed to the aforementioned synthetic agents and therefore consumers and food manufacturers have turned their attention to natural dietary sweeteners, such as stevia, to meet their sweetening needs. Stevia is generally considered safe; however, emerging scientific evidence has implicated the agent in gut microbial imbalance. In general, regulation of microbial behavior is known to depend highly on signaling molecules via quorum sensing (QS) pathways. This is also true for the gut microbial community. We, therefore, evaluated the possible role of these stevia-based natural sweeteners on this bacterial communication pathway. The use of a commercial stevia herbal supplement resulted in an inhibitory effect on bacterial communication, with no observable bactericidal effect. Purified stevia extracts, including stevioside, rebaudioside A (Reb A), and steviol revealed a molecular interaction, and possible interruption of Gram-negative bacterial communication, via either the LasR or RhlR receptor. Our in-silico analyses suggest a competitive-type inhibitory role for steviol, while Reb A and stevioside are likely to inhibit LasR-mediated QS in a non-competitive manner. These results suggest the need for further safety studies on the agents.

Topics: Chromatography, Liquid; Dietary Supplements; Diterpenes, Kaurane; Food Additives; Glucosides; Mass Spectrometry; Models, Molecular; Molecular Structure; Plant Extracts; Quorum Sensing; Stevia; Sweetening Agents

Topics: Animals; Biomarkers; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Diet, High-Fat; Dietary Supplements; Diterpenes, Kaurane; Glucosides; Lipid Metabolism; Male; Rats; Rats, Wistar

Stevioside is a natural non-caloric sweetener obtained from Stevia rebaudiana Bertoni plant. The major challenge in the commercialization of stevioside as a natural sweetener is its bitter-off taste. In this study, we prepared molecular models of potential taste receptors of stevioside, both sweet and bitter. With appropriate modifications on the stevioside backbone, we performed molecular docking of prepared ligands with both sweet and bitter taste receptors. Based on binding energy, we found that one of the potential substituents, Kamiya-8, shows a good affinity towards sweet taste and a weak affinity for bitter receptors. Further, we selected Kamiya-8 for molecular dynamics simulations to improve the prediction of binding energy and to check the binding strength of Kamiya-8 with taste receptors. Moreover, we also performed MM-PBSA calculation for calculating the end state free energies of molecules in solvent and found that Kamiya-8 gives a 2-fold effect as it interacts with sweet receptors (T1R2, T1R3) with lowest binding energy conformation (-285.265 kcal/mol, -571.481 kcal/mol). Secondly, it gives high binding energy (-273.319 kcal/mol, -355.500 kcal/mol) with bitter taste receptors (T2R4, T2R14) as compared to stevioside. Based on this study, we found that Kamiya-8 can be the potential substituent that can improve the palatability of stevioside.

Topics: Diterpenes, Kaurane; Glucosides; Molecular Docking Simulation; Stevia; Taste

Leaf extracts of

Topics: Animals; Bacteria; Butyric Acid; Diterpenes, Kaurane; Erythritol; Gastrointestinal Microbiome; Glucosides; Humans; Pentanoic Acids; Plant Extracts; Sapajus apella; Stevia

Growing health concerns have increased interest in reducing the consumption of added sugars, which can be achieved by substituting or replacing sugar with sweeteners to maintain sensory intensity and quality. The growing availability of sweeteners has increased the complexity of the perceptual landscape as sweeteners differ in the qualitative, intensity, and temporal properties. A sweetener that can match the perceptual properties of sucrose in different food matrices is likely to have broad applications. In complex foods, sweetness is influenced by the taste interactions with the existing tastants and possible matrix effects that influence release and perception of sweetness. The current study compared the taste properties of three food matrices (black tea, chocolate milk, and natural yogurt) sweetened by sucrose to those sweetened using eight different sweeteners (acesulfame-K, aspartame, erythritol, luo han guo (Mogroside), palatinose (iso-maltulose), stevia (Reb-A), sucralose, and sucrose-allulose mixture) using Rate-All-That-Apply. The sensory properties of each sweetener differed across matrices, with sucrose-allulose mixture, aspartame, erythritol, palatinose, and sucralose having the most similar taste to sucrose across all foods. By contrast, acesulfame-K, stevia, and luo han guo had taste profiles that most varied from sucrose, characterized by side tastes such as bitterness, chemical taste, and a low sweetness. Sweeteners differed most from sucrose when presented in natural yogurt compared to tea and chocolate milk. A food's taste properties can suppress sweetness intensity and promote undesirable side tastes. Taken together, these findings highlight the importance of testing sweeteners in complex foods and help identify sweeteners and sweetener combinations that can replicate the sweetness of sucrose and support sugar reduction. PRACTICAL APPLICATION: Food manufacturers and researchers can refer to the results of the sensory profiles to identify suitable sweeteners substitutes for sucrose in foods with similar taste profiles to those tested. The current article highlights important changes to sweetener sensory properties when presented in different complex foods, and provides an indication of the potential for calorie reduction by substituting sucrose with a range of low or no calorie sweeteners.

Topics: Animals; Aspartame; Camellia sinensis; Cattle; Chocolate; Diterpenes, Kaurane; Glucosides; Humans; Milk; Stevia; Sucrose; Sweetening Agents; Taste; Tea; Yogurt

Rebaudioside D is a sweetener from Stevia rebaudiana with superior sweetness and organoleptic properties, but its production is limited by its minute abundance in S. rebaudiana leaves. In this study, we established a multi-enzyme reaction system with S. rebaudiana UDP-glycosyltransferases UGT76G1, Solanum lycopersicum UGTSL2 and Solanum tuberosum sucrose synthase StSUS1, achieving a two-step glycosylation of stevioside to produce rebaudioside D. However, an increase in the accumulation of rebaudioside D required the optimization of UGTSL2 catalytic activity towards glucosylation of rebaudioside A and reducing the formation of the side-product rebaudioside M2. On the basis of homology modelling and structural analysis, Asn358 in UGTSL2 was subjected to saturating mutagenesis, and the Asn358Phe mutant was used instead of wild-type UGTSL2 for bioconversion. The established multi-enzyme reaction system employing the Asn358Phe mutant produced 14.4 g l

Topics: Diterpenes, Kaurane; Glucosides; Glycosides; Stevia

High content of steviol glycosides in stevia leaves is a cause of their high popularity as. a natural sweetener of various sugar-free food products. Stevioside (13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy]-ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester) is one of the main steviol glycosides in stevia leaves known for its hydrolytic instability responsible for the formation of simple steviol glucosides (steviolbioside, rubusoside, steviol monoside) and steviol. However, the formation of hydroxy and alkoxy adducts of stevioside and of its hydrolysis products has not yet been reported. The performed experiments prove that water and alkoxy adducts are formed not only during temperature processing of stevioside but also of stevia and stevia-containing food products. Their quantities depend on environment pH, water concentration and food composition. Although they are formed in small amounts their biological activity is unknown and should be recognized.

Topics: Diterpenes, Kaurane; Food Analysis; Glucosides; Hydrogen-Ion Concentration; Hydrolysis; Methanol; Plant Leaves; Stevia; Sweetening Agents; Temperature; Water

Topics: Antioxidants; Copper; Diterpenes, Kaurane; Glucosides; Nanoparticles; Nanotechnology; Regeneration; Static Electricity; Stevia; Tissue Culture Techniques; X-Ray Diffraction; Zinc Oxide

Melatonin (MEL) can act as a plant growth regulator and biostimulator in stressful situations. Using MEL in seed pretreatment also affects the future growth of plants. Therefore, this research investigated the effects of MEL on seed germination and seedling growth under NaCl in in vitro conditions. The additional effects of MEL on the accumulation of steviol glycosides (SGs) and on the expression of appropriate genes were also studied. Five μM of MEL was the best concentration for seed germination, while 20 μM exerted a positive impact on the biomass of stevia plantlets. NaCl significantly decreased seed germination, but MEL alleviated this effect when seeds were germinated in 50 mM of NaCl. Under salinity, the values of almost all morphological traits decreased as MEL concentration increased. The highest amounts of stevioside and rebaudioside A (Reb A) were observed as a result of treating seeds with 5 and 20 μM of MEL, respectively. When adding NaCl, positive impacts of MEL on the accumulation of both SGs were also observed. Expression analyses of the genes involved in SGs biosynthesis was explored in seeds and leaves, and the transcripts of key enzymes occurred in both the tissues. However, quantitative polymerase chain reaction (qPCR) analysis showed that all tested genes were upregulated in younger leaves, contrary to older ones. Also in younger, rather than older, leaves SG gene expression varied according to MEL concentration. This study, therefore, presents the promising potential of MEL for improving stevia seed germination under salinity conditions and for enhancing the production of SGs in stevia plants.

Topics: Diterpenes, Kaurane; Germination; Glucosides; Melatonin; Plant Leaves; Salinity; Seeds; Sodium Chloride; Stevia

Stevia has been proposed as a potential antidiabetic sweetener, mainly based on inconsistent results from stevioside or the plant extract, yet lacking relative experimental evidence from individual steviol glycosides (SGs) and their metabolites.. The results systematically revealed that the typical SGs and their final metabolite (steviol) presented an antidiabetic effect on streptozotocin (STZ) diabetic mice in all assayed antidiabetic aspects. In general, the performance strength of the samples followed the sequence steviol > steviol glucosyl ester > steviolbioside > rubusoside > stevioside > rebaudioside A, which is opposite to their sweetness strength order, and generally in accordance with the glucosyl group numbers in their molecules. This may imply that the antidiabetic effect of the SGs might be achieved through steviol, which presented antidiabetic performance similar to that of metformin with a dose of 1/20 that of metformin. Moreover, the. The SGs and steviol presented an antidiabetic effect on STZ diabetic mice in all assayed aspects, with an induction time to start the effect of the SGs. Stevioside and steviol could increase uptake of glucose in the myocardium and brain of the diabetic mice, and decrease accumulation of glucose in the liver and kidney. The performance strength of the SGs is generally in accordance with glucosyl group numbers in their molecules.

Topics: Animals; Diabetes Mellitus, Experimental; Diterpenes, Kaurane; Glucose; Glucosides; Humans; Hypoglycemic Agents; Kidney; Liver; Male; Mice; Mice, Inbred ICR; Plant Extracts; Plant Leaves; Stevia; Streptozocin

We herein report the preparation of a full-length raucaffricine-O-beta-D-glucosidase gene of stevia rebaudiana Bertoni (named SrRG1, GenBank accession number MK920450). Sequence analysis indicated SrRG1 consists of a 1650 bp open reading frame encoding a protein of 549 amino acids. Its deduced amino acid sequence showed a high identity of 82% with a raucaffricine-O-beta-D-glucosidase from H. annuus of glycoside hydrolase family 1. The expression pattern analyzed by real-time quantitative PCR showed no significant difference among different tissues, developmental stages, and cultivars under normal growth conditions. Furthermore, the gene function of SrRG1 was preliminarily studied by agrobacterium-mediated transformation on instantaneous expression. In the test of agrobacterium-mediated transformation on instantaneous expression, it was observed that overexpression of SrRG1 increased the accumulation of steviol content and decreased the major components and total SGs contents. Such results demonstrated that SrRG1 may participate in the steviol glycosides catabolic pathway. However, the effect of silencing construct infiltration on steviol and SGs content was not significant and its expression pattern was constitutive, which most probably, attributed the hydrolysis of SGs to the secondary activity of SrRG1. This study firstly identified the bate-glucosidase in stevia and advances our understanding of steviol glycosides hydrolyzation.

Topics: beta-Glucosidase; Diterpenes, Kaurane; Gene Expression Regulation, Plant; Glucosides; Glycosides; Plant Leaves; Stevia

Food products and botanicals on the global market need to be investigated in a more comprehensive way to detect effects, falsifications or adulterations. This is especially true for such ones containing Stevia leaves, Stevia extracts, or steviol glycosides. A multi-imaging profiling was developed exploiting hydrophilic interaction liquid chromatography (HILIC). A minimalistic sample preparation, different mixtures of acetonitrile and water/buffer on the silica gel phase as well as derivatization reagents and optional hyphenation with high-resolution mass spectrometry were exploited. The hydrophilic interaction high-performance thin-layer chromatography (HI-HPTLC) development took 10 min for 48 analyses. It was used to screen Stevia leaf extracts and 20 different food products. For the first time, the biological and biochemical profiling of Stevia leaf products by HI-HPTLC-UV/Vis/FLD-assay pointed to 19 different bioactive compound bands found in the more natural multicomponent Stevia leaf extracts, whereas most of these activities were not existent for the steviol glycosides. The chemically isolated, purified, and EU-regulated steviol glycosides ease risk assessment and food product development. However, multipotent botanicals may have subtle impact on homeostasis via several metabolic pathways, providing benefits for the consumer's health. Analyzed side by side by means of the effect-directed profiling, their individual activity profiles were visualized as image and individual substances of importance were pointed out. Multi-imaging (comprehensive detection) plus non-targeted bioprofiling (focus on known and unknown bioactivity) allows for a fast detection of questionable product changes that occur along the global food chain and are particularly related to food safety. Graphical abstract.

Topics: Chromatography, Thin Layer; Diterpenes, Kaurane; Food Analysis; Glucosides; Plant Leaves; Stevia

The ability to synthesize particular steviol glycosides (SvGls) was studied in Stevia rebaudiana Bertoni hairy roots (HR) grown in the light or in the dark under the influence of different osmotic active compounds. Manipulation of culture conditions led to changes in the morphology and growth rate of HR, as well as to an increase in oxidative stress manifested as an enhancement in endogenous hydrogen peroxide concentration in the cultured samples. The highest level of H

Topics: Agrobacterium; Biosynthetic Pathways; Culture Media; Diterpenes, Kaurane; Gene Expression Regulation, Plant; Glucosides; Glucosyltransferases; Light; Mixed Function Oxygenases; Osmotic Pressure; Oxidative Stress; Plant Proteins; Plant Roots; Plants, Genetically Modified; Stevia

Stevia is a plant containing many active compounds, but usually propagated by stem cuttings because of low seed-yield-germination ability. The aim of this study was to investigate the impact of plant-growth regulators on stevia callus induction and growth from somatic tissue, as well as to determine the effect α-naphthalene acetic acid (NAA) and proline (PRO) on the amount of stevioside, rebaudioside A, phenols, flavonoids, and antioxidant activity. Stem and leaf segments were inoculated on a Murashige and Skoog (MS) medium supplemented with different concentrations of NAA and 6-benzylaminopurine (BAP) for callus genesis. The amount of steviol glycosides (SGs) was evaluated using high-performance liquid chromatography (HPLC), and the amounts of total phenols, flavonoids, and antioxidant activity by spectrophotometric methods. The highest callus-induction frequency and callus-mass increase were obtained from the leaf explants in MS medium supplemented with 2.0 μM NAA. The highest amount of SGs, phenols, and flavonoids, and stronger antioxidant activity were determined in the cellular compounds of callus from leaf explant. PRO reduced the amount of SGs and flavonoids. The significantly highest amount of total phenolic compounds was obtained in the callus from leaf explants in the medium supplemented with 2.0 µM NAA and 2.0 µM PRO.

Topics: Antioxidants; Chromatography, High Pressure Liquid; Culture Media; Diterpenes, Kaurane; Flavonoids; Glucosides; Indoleacetic Acids; Phenols; Plant Growth Regulators; Plant Leaves; Proline; Stevia; Tissue Culture Techniques

The concentrations of steviol and its derivatives stimulating the growth of wheat plants were measured: 10

Topics: Diterpenes; Diterpenes, Kaurane; Glucosides; Plant Leaves; Stevia; Triticum

Ultrasound-assisted extraction is widely recognized as an eco-friendly technique due to low solvent consumption and time extraction as well as enhanced extraction efficiency with respect to conventional methods. Nevertheless, it would be convenient to avoid the usually used organic solvents to reduce the environment pollution. In this regard, Deep Eutectic Solvents (DES) represent nowadays a green and sustainable alternative for the extraction of bioactive compounds from natural sources. In this study, an efficient extraction of stevioside and rebaudioside A from Stevia rebaudiana coupling ultrasound with DES was developed. A solvent screening was performed using the predictive approach COnductor-like Screening MOdel for Real Solvent (COSMO-RS). The effect of three independent variables, namely % of water, temperature, and sonication amplitude, were investigated by the response surface methodology (RSM). Comparing ultrasound-assisted extraction (UAE) with conventional extraction, it has been demonstrated that the amount of steviol glycosides through UAE is almost three times higher than that obtained by the conventional method. Possible physicochemical factors involved in the UAE mechanism were discussed.

Topics: Chemical Fractionation; Diterpenes, Kaurane; Glucosides; Plant Leaves; Solvents; Stevia; Ultrasonics

Glycosylation is a key modification that contributes to determine bioactivity and bioavailability of plant natural products, including that of terpenoids and steviol glycosides (SVglys). It is mediated by uridine-diphosphate glycosyltransferases (UGTs), that achieve their activity by transferring sugars on small molecules. Thus, the diversity of SVglys is due to the number, the position and the nature of glycosylations on the hydroxyl groups in C-13 and C-19 of steviol. Despite the intense sweetener property of SVglys and the numerous studies conducted, the SVglys biosynthetic pathway remains largely unknown. More than 60 SVglys and 68 putative UGTs have been identified in Stevia rebaudiana. This study aims to provide methods to characterize UGTs putatively involved in SVglys biosynthesis. After agroinfiltration-based transient gene expression in Nicotiana benthamiana, functionality of the recombinant UGT can be tested simply and directly in plants expressing it or from a crude extract. The combined use of binary vectors from pGWBs series to produce expression vectors containing the stevia's UGT, enables functionality testing with many substrates as well as other applications for further analysis, including subcellular localization.

Topics: Biosynthetic Pathways; Diterpenes, Kaurane; Glucosides; Glycosylation; Glycosyltransferases; Plant Proteins; Recombinant Proteins; Stevia; Uridine Diphosphate

Stevia rebaudiana Bertoni is an important economic crop that is well known for its secondary metabolites, steviol glycosides (SGs), found in leaves. Because the enzymes of deglycosylation (glycoside hydrolases) play important roles in SGs biosynthetic processes, our study is focused on the functions of β-glucosidases in SGs catabolism in stevia. We cloned and characterized 19 stevia GH1 genes based on transcriptomic sequences. The 19 genes were divided into five putative subfamilies in Arabidopsis. Conserved motifs in the SrGH1 proteins were analysed using the online motif-based sequence analysis tool, MEME. Most of the identified proteins contain the conserved 'TFNEP' motif (contains the catalytic acid/base) and 'ITENG' motif (contains the catalytic nucleophile). Furthermore, the steviol glycoside content and expression of these 19 genes were characterized under constant darkness. The dark treatment lowered the steviol glycoside content significantly, while SrBGLU16 responded to darkness and was markedly upregulated. This study is the first transcriptome-wide analysis of the GH1 family in Stevia rebaudiana. The sequences of 19 SrGH1 members and their expression when grown in darkness were characterized. Among the 19 genes, SrBGLU16 was markedly upregulated by darkness. Thus, we identified SrBGLU16 for further investigation as a possible steviol glycoside beta-glucosidase.

Topics: Cellulases; Darkness; Diterpenes, Kaurane; Gene Expression Regulation, Plant; Genes, Plant; Glucosides; Stevia; Transcriptome

Steviol glycosides (SGs) and gibberellic acids share a part of their biosynthesis pathways. Despite the widespread studies on the effect of gibberellic acid 3 (GA

Topics: Benzyl Compounds; Cytokinins; Diterpenes, Kaurane; Gibberellins; Glucosides; Kinetin; Plant Leaves; Plant Shoots; Purines; Stevia

Stevia rebaudiana is an important medical plant for producing steviol glycosides (SGs) or stevioside. Autotetraploids (4x = 44) show an increasing level of morphology, physiology and tolerances comparing to diploids (2x = 22). However, little information regarded on the comparative transcriptome analysis between diploid and autotetraploid S. rebaudiana was found. In this study, synthetic autotetraploid was induced and morphological features were confirmed. A comprehensive transcriptome of stevia leaf, stem and root from the diploids and autotetraploids was constructed based on RNA-seq, yielded 1,000,892,422 raw reads and subsequently assembled into 251,455 transcripts, corresponded to 146,130 genes. Pairwise comparisons of the six leaf libraries between the diploids and autotetraploids revealed 4114 differentially expression genes (DEGs), in which 2105 (51.17%) were up-regulated in autotetraploids and associated with SGs biosynthesis, plant growth and secondary metabolism. Moreover, weighted gene co-expression network analysis showed co-expressed genes of fifteen genes of SG biosynthesis pathway were enriched in photosynthesis, flavonoid and secondary metabolic process, plant growth and morphogenesis. A hundred of DEGs related to plant resistance were identified by interviewing PlantPReS database. This study has highlighted molecular changes related to SGs metabolism of polyploidy, and advanced our understanding in plant resistance responsible for phenotypic change of autotetraploids.

Topics: Diploidy; Diterpenes, Kaurane; Gene Expression Regulation, Plant; Glucosides; Plant Leaves; Plant Roots; Stevia; Transcriptome

Stevia rebaudiana (Asteraceae), native from Paraguay, accumulates steviol glycosides (SGs) into its leaves. These compounds exhibit acaloric intense sweet taste which answers to consumer demands for reducing daily sugar intake. Despite the developpement of S. rebaudiana cultivation all over the world, the development of new cultivars is very recent, in particular due to a colossal lack of (1) germplasm collection and breeding, (2) studies on genetic diversity and its structuring, (3) genomic tools.. In this study, we developped 18 EST-SSR from 150,258 EST from The Compositae Genome Project of UC Davis ( http://compgenomics.ucdavis.edu/data/ ). We genotyped 145 S. rebaudiana individuals, issued from thirty-one cultivars and thirty-one landraces of various origins worldwide. Markers polymorphic information content (PIC) ranged between 0.60 and 0.84. An average of 12 alleles per locus and a high observed heterozygoty of 0.69 could be observed. The landraces revealed twice as many private alleles as cultivars. The genotypes could be clustered into 3 genetic populations. The landraces were grouped in the same cluster in which the oldest cultivars "Eirete" and "MoritaIII" type are also found. The other two clusters only include cultivated genotypes. One of them revealed an original genetic variability. SG phenotypes could not discriminate the three genetic clusters but phenotyping showed a wide range of composition in terms of bitter to sweet SGs.. This is the first study of genetic diversity in Stevia rebaudiana involving 145 genotypes, including known cultivars as well as landrace populations of different origin. This study pointed out the structuration of S. rebaudiana germplasm and the resource of the landrace populations for genetic improvement, even on the trait of SG's composition.

Topics: Alleles; Diterpenes, Kaurane; Genetic Variation; Genetics, Population; Genotype; Glucosides; Glycosides; Plant Breeding; Plant Leaves; Stevia; Taste

Efficient treatment of hypertension is vital. The inhibition of angiotensin-converting enzyme activity has been one of the major strategies for treating hypertension. The ethanol extract of stevia leaves, steviol glycosides (with 95% purity; natural sweeteners widely used in the food industry) isolated from the ethanol extract and stevia leaf protein hydrolysates inhibited 26.60%, 59.56% and 74.38% of angiotensin-converting enzyme activities, respectively. Their effect was dose-dependent, which can be beneficial for avoiding hypertension or hypotension just by the proper control of the amount of their intake, and it was found to be superior to that of pharmaceutical drugs. A sensory test indicated that the application of the mixtures of steviol glycosides and stevia protein hydrolysates to decaffeinated coffee or tea as well as a formulated peanut protein drink was found to be well accepted, and an animal test showed that they had a significantly antihypertensive effect in spontaneously hypertensive rats. Steviol glycosides and stevia leaf protein hydrolysates can be good ingredients for making functional or healthy food products or beverages targeted for the prevention or treatment of hypertension.

Topics: Animals; Antihypertensive Agents; Diterpenes, Kaurane; Enzyme Inhibitors; Female; Glucosides; Humans; Hypertension; Male; Peptidyl-Dipeptidase A; Plant Extracts; Plant Leaves; Plant Proteins; Protein Hydrolysates; Rats; Rats, Inbred SHR; Stevia; Taste

The application of chemical fertilizers to enhance crop production is a major concern due to associated environmental pollution and health hazards. Hence, there is an urgent need to develop an eco-friendly solution to improve crop production and promote sustainable agriculture simultaneously. Stevia rebaudiana is an important medicinal crop being substitute for sugar, superior flavor outline, extensive medicinal properties, and also of agronomic interest. In the present study, bacterium STJP isolated from the rhizospheric soil of S. rebaudiana and identified as Bacillus safensis on the basis of 16S rRNA gene sequencing, showed good amount of zinc (4.4 mg/L) and potassium (5.4 mg/L) solubilization. Paneer-whey (a dairy waste) based bioformulation (P-WBF) was developed utilizing isolate B. safensis STJP (accession number NAIMCC TB-2833) and inspected for the quality and ability to enhance the growth, nutrients uptake, and stevioside content in S. rebaudiana. The application of P-WBF displayed a significantly higher concentration (153.12%) of stevioside in S. rebaudiana as compared to control. P-WBF treated Stevia plants showed significantly higher fresh and dry weight as well (as compared to control). Further, enhancement of phosphorous, nitrogen, potassium, and zinc uptake in plant tissue was also recorded by application of P-WBF. This study suggests the use of P-WBF based biofertilizer using B. safensis STJP to increase stevioside content in Stevia plant by a nutrient(s) linked mechanism. This novel approach can also be beneficial for utilization of a dairy waste in preparation of bioformulation and, for enhancement of crop yield by an ecofriendly manner leading to sustainable agriculture.

Topics: Agriculture; Bacillus; Diterpenes, Kaurane; Fertilizers; Glucosides; Nitrogen; Nutrients; Phosphorus; Plant Development; RNA, Ribosomal, 16S; Stevia

A silica gel orthogonal method using acetonitrile: water was developed for the analyses of fractions rich in very polar steviol glycosides and resolve regions of co-elution of these compounds in reversed-phase. Additionally, we also used this normal phase analytical method to scale up the purification process of steviol glycosides. Using these approaches, one novel minor tetra-glucopyranosyl diterpene glycosides together with three known compounds were purified from a commercial

Topics: Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Glucosides; Glycosides; Magnetic Resonance Spectroscopy; Silica Gel; Stevia; Tandem Mass Spectrometry; Trisaccharides

The adverse health effects of sucrose overconsumption, typical for diets in developed countries, necessitate use of low-calorie sweeteners. Following approval by the European Commission (2011), steviol glycosides are increasingly used as high-intensity sweeteners in food. Stevioside is the most prevalent steviol glycoside in Stevia rebaudiana plant leaves, but it has found limited applications in food products due to its lingering bitterness. Enzymatic glucosylation is a strategy to reduce stevioside bitterness, but reported glucosylation reactions suffer from low productivities. Here we present the optimized and efficient α-glucosylation of stevioside using the mutant glucansucrase Gtf180-ΔN-Q1140E and sucrose as donor substrate. Structures of novel products were elucidated by NMR spectroscopy, mass spectrometry and methylation analysis; stevioside was mainly glucosylated at the steviol C-19 glucosyl moiety. Sensory analysis of the α-glucosylated stevioside products by a trained panel revealed a significant reduction in bitterness compared to stevioside, resulting in significant improvement of edulcorant/organoleptic properties.

Topics: Bacterial Proteins; Diterpenes, Kaurane; Glucosides; Glycosylation; Glycosyltransferases; Humans; Isomerism; Lactobacillus; Magnetic Resonance Spectroscopy; Mass Spectrometry; Mutagenesis; Plant Leaves; Stevia; Sucrose; Sweetening Agents; Taste

Kale is a vegetable that contains a high proportion of health-promoting compounds although its consumption as a beverage is very limited due to its bitter flavor. Nonetheless, the bitter flavor of Brassica may be masked by sweetening. The effects were studied of different stevia extracts (CTRL, S0.5 (g L. The addition of the stevia extracts did not affect the physicochemical quality of spheres. In particular, S2.5 spheres showed the least color changes after 7 days. All spheres showed good microbiological quality throughout storage, with loads < 7 log CFU g. The addition of stevia to the kale juice spheres led to a better flavor without altering product quality during refrigerated storage. © 2018 Society of Chemical Industry.

Topics: Brassica; Diterpenes, Kaurane; Food Storage; Fruit and Vegetable Juices; Glucosides; Humans; Plant Leaves; Quality Control; Refrigeration; Stevia; Sweetening Agents; Taste

Stevia rebaudiana produces sweet steviol glycosides that are 300 times sweeter than sugar and have the beneficial effects on human health including anti-hyperglycaemic. Tissue culture is the best method with high efficacy to propagate stevia. Abiotic stress has an impact on steviol glycoside contents in stevia. Therefore, we investigated the effect of mannitol on the expression of four genes involved in the biosynthesis of stevia including UGT74G1, UGT76G1, kaurene oxidase and kaurene synthase genes and steviol glycosides accumulation in stevia under in vitro conditions. The highest expression of UGT76G1 gene occurred in the plants grown under 20 g/l mannitol. While for the kaurene synthase gene, the highest amount of gene expression was observed at 40 g/l mannitol. The results were different about kaurene oxidase gene. As the highest and lowest gene expression were seen in 50 and 30 g/l mannitol conditions respectively. There were the same results for UGT74G1 that means the most appropriate and also the most inopportune treatment for the gene expression were same as the condition for the kaurene oxidase gene. Compared with control, adding mannitol to media in all concentrations increases the expression of UGT76G1 gene. Estimation of steviol glycosides contents under different treatments of mannitol carried out by HPLC. According to the results, the highest amount of stevioside was produced under 20 g/l mannitol treatment. However, rebaudioside A was accumulated in its maximum amounts under 30 g/l mannitol. It can be concluded that adding mannitol to media in the certain concentration increases steviol glycoside contents in the stevia.

Topics: Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Gene Expression Regulation, Plant; Genes, Plant; Glucosides; Glycosides; Mannitol; Plant Leaves; Stevia; Tissue Culture Techniques

The reduction of sugar consumption is one of the major challenges for nutritionists and food industry. Therefore, it is significant to replace sucrose with other types of sweeteners, especially, natural ones. The aim of the present study is to produce low-calorie, sucrose-free mango nectar and to optimize the formulation by employing response surface methodology. The two independent variables were stevia, as a low-calorie sugar replacer (0, 1.5, and 3% w/w) and inulin as a prebiotic texturizer (0, 3, and 6% w/w) in order to compensate sugar elimination defect on viscosity and °Brix. The fitted models indicated a high coefficient of determination. The results revealed that stevia and inulin are as the independent variables which had significant effects on °Brix, viscosity, and sensory scores (p < 0.05). Also, pH was affected by stevia concentration. The rheological behavior of the sucrose-free mango nectar was non-Newtonian, shear thinning as Herschel-Bulkley model which was not different from the reported behavior for normal mango nectar-containing sucrose. The optimization of the variables, based on the response surface three-dimensional plots, demonstrated that utilizing 6% w/w inulin and 3% w/w stevia produced the optimum mango nectar with the desirability of 0.85 without undesirable changes in the physicochemical and organoleptic properties. The optimum sample was produced in triplicate to validate the optimum model as well.

Topics: Caloric Restriction; Chemical Phenomena; Diterpenes, Kaurane; Fruit; Fruit and Vegetable Juices; Glucosides; Humans; Inulin; Mangifera; Prebiotics; Rheology; Sensation; Stevia; Sucrose; Sweetening Agents

Steviol glycosides (SGs) are extracted from Stevia leaves for use as a natural sweetener. Among SGs, stevioside is most abundant in leaf extracts followed by rebaudioside A (Reb A). However, Reb A is of particular interest because of its sweeter and more pleasant taste compared to stevioside. Therefore, the development of new Stevia varieties with a higher Reb A to stevioside ratio would be desirable for the production of higher quality natural sweeteners. Here, we generated transgenic Stevia plants overexpressing Stevia UDP-glycosyltransferase 76G1 (SrUGT76G1) that is known to convert stevioside to Reb A through 1,3-β-d-glucosylation in vitro. Interestingly, by overexpressing SrUGT76G1, the Reb A to stevioside ratio was drastically increased from 0.30 in wild-type (WT) plants up to 1.55 in transgenic lines without any significant changes in total SGs content. This was contributed by a concurrent increase in Reb A content and a decrease in stevioside content. Additionally, we were able to find an increase in the Reb C to dulcoside A ratio in transgenic lines. Using the glutathione S-transferase-tagged SrUGT76G1 recombinant protein for an in vitro glucosyltransferase assay, we further demonstrated that Reb C can be produced from the glucosylation of dulcoside A by SrUGT76G1. Transgenic Stevia plants having higher Reb A to stevioside ratio were visually indistinguishable from WT plants. Taken together, our results demonstrate that the overexpression of SrUGT76G1 in Stevia is an effective way to generate new Stevia varieties with higher proportion of the more preferred Reb A without compromising on plant development.

Topics: Diterpenes, Kaurane; Food Technology; Gene Expression; Glucosides; Glycosyltransferases; Plant Leaves; Stevia; Uridine Diphosphate

Stevia rebaudiana produces sweet-tasting steviol glycosides (SGs) in its leaves which can be used as natural sweeteners. Metabolic engineering of Stevia would offer an alternative approach to conventional breeding for enhanced production of SGs. However, an effective protocol for Stevia transformation is lacking.. Here, we present an efficient and reproducible method for Agrobacterium-mediated transformation of Stevia. In our attempts to produce transgenic Stevia plants, we found that prolonged dark incubation is critical for increasing shoot regeneration. Etiolated shoots regenerated in the dark also facilitated subsequent visual selection of transformants by green fluorescent protein during Stevia transformation. Using this newly established transformation method, we overexpressed the Stevia 1-deoxy-d-xylulose-5-phosphate synthase 1 (SrDXS1) and kaurenoic acid hydroxylase (SrKAH), both of which are required for SGs biosynthesis. Compared to control plants, the total SGs content in SrDXS1- and SrKAH-overexpressing transgenic lines were enhanced by up to 42-54% and 67-88%, respectively, showing a positive correlation with the expression levels of SrDXS1 and SrKAH. Furthermore, their overexpression did not stunt the growth and development of the transgenic Stevia plants.. This study represents a successful case of genetic manipulation of SGs biosynthetic pathway in Stevia and also demonstrates the potential of metabolic engineering towards producing Stevia with improved SGs yield.

Topics: Diterpenes, Kaurane; Genetic Engineering; Glucosides; Mixed Function Oxygenases; Plant Leaves; Plant Proteins; Plant Shoots; Plants, Genetically Modified; Stevia; Transferases

The present work reports the preparation of CO

Topics: Adsorption; Carbon; Carbon Dioxide; Charcoal; Diterpenes, Kaurane; Glucosides; Hydrogen-Ion Concentration; Kinetics; Stevia; Surface Properties; Temperature; Thermodynamics; Triclosan

In the metabolic glycosylation grid of steviol glycosides, UGT76G1 was shown to catalyze at least eight different glucosylation steps, including the formation of rebaudioside B (Reb B) and rebaudioside A (Reb A) (Olsson et al., 2016). In this study, the accumulation of steviolbioside, Reb B, stevioside (ST) and Reb A in more than 140 samples of stevia leaves collected from different regions in China were analyzed by high-performance liquid chromatography (HPLC), and five genotypes, 'N01-N05', with significantly different levels of the abovementioned glycosides were discovered. Mutations in the UGT76G1 gene cloned from cDNAs from these five genotypes were identified, and the functions of the recombinant UGT76G1 variants were ascertained by adding steviolbioside and ST substrates. In addition, homology modeling and molecular docking were used to elucidate the functional differences between variants and UGT76G1. Comparing the sequences of the five variants 'N01-N05' with UGT76G1 (AY345974.1) revealed that base substitutions were not observed in 'N01'. By contrast, 'N02' exhibited 9 single nucleotide polymorphisms (SNPs) and 9 associated amino acid substitutions or insertions with notable variations in the protein structure; however, an enzyme assay showed similar functionalities between the variant and UGT76G1. In 'N03', 49 SNPs and 29 associated amino acid substitutions or insertions were identified and shown to induce significant variations in the protein structure, especially in the binding pocket, resulting in the lack of functionality of this variant in the enzyme assay. These results were in agreement with the docking profiles. Moreover, a nonsense mutation of p.1090T > G in 'N04' and an insertion of a 68 base fragment in 'N05' were found, and both produced a premature protein without any catalytic activity. Therefore, UGT76G1, which is vital to the content of main steviol glycosides, should be a key gene marker for the molecular breeding of Stevia rebaudiana. Our investigations also revealed the location and orientation of active groups of the receptors and donors in the UGT76G1 enzyme, which play key roles in determining whether the enzyme has any enzymatic activity.

Topics: Biocatalysis; Cloning, Molecular; Diterpenes, Kaurane; Glucosides; Glycosyltransferases; Models, Molecular; Mutation; Protein Conformation; Stevia; Uridine Diphosphate

Stevia rebaudiana Bertoni has been promoted for having sweet leaves as well as pharmaceutical and industrial properties. The sweet taste of Stevia leaves is due to the presence of steviol glycosides (a group of diterpene glycosides) found in a small number of plants. In the biosynthetic pathway of steviol glycosides (SGs), 15 enzymes that express the genes are associated with these enzymes under the influence of the elicitors. Due to the individuality of the stevia and few studies on the biosynthesis pathway of SGs, this paper attempted to investigate the effects of some of the elicitors, including methyl jasmonate (MeJA), salicylic acid (SA), auxins (Aux), cytokinins (CKs), gibberellins (GAs) and its inhibitors including paclobutrazol (BPZ) and chloroquate (CCC)), on the responsible genes for the biosynthesis of SGs. Some of these elicitors, including MeJA, SA and GA have great potential in increasing secondary metabolites. Moreover, the biosynthetic pathway of GAs and SGs are shared till ent-kaurenoic acid (ent-KA) biosynthesis, which raises the question of whether this hormone and its inhibitors are effective in the SGs biosynthesis.

Topics: Biosynthetic Pathways; Diterpenes, Kaurane; Gene Expression Regulation, Plant; Glucosides; Stevia

miRNAs are major regulators of gene expression and have proven their role in understanding the genetic regulation of biosynthetic pathways. Stevioside and rebaudioside-A, the two most abundant and sweetest compounds found in leaf extract of Stevia rebaudiana, have been used for many years in treatment of diabetes. It has been found that the crude extract is more potent than the purified extract. Stevioside, being accumulated in higher concentration, imparts licorice like aftertaste. Thus, in order to make the sweetener more potent and palatable, there is a need to increase the intrinsic concentration of steviol glycosides and to alter the ratio of rebaudioside-A to stevioside. Doing so would significantly increase the quality of the sweeteners, and the potential to be used on a wider scale. To do so, in previous report, miRNAs associated with genes of steviol glycosides biosynthetic pathway were identified in S. rebaudiana. In continuation to that in this study, the two miRNAs (miR319g and miRStv_11) targeting key genes of steviol glycosides biosynthetic pathway were modulated and their impact was evaluated on steviol glycosides contents.. The over-expression results showed that miRStv_11 induced, while miR319g had repressive action on its target genes. The knock-down constructs for miR319g and miRStv_11 were then prepared and it was demonstrated that the expression of anti-miR319g produced inhibitory effect on its target miRNA, resulting in enhanced expression of its target genes. On the other hand, anti-miRStv_11 resulted in down-regulation of miRStv_11 and its target gene. Further miRStv_11 and anti-miR319gwere co-expressed which resulted in significant increase in stevioside (24.5%) and rebaudioside-A (51%) contents.. In conclusion, the role of miR319g and miRStv_11 was successfully validated in steviol gycosides biosynthetic pathway gene regulation and their effect on steviol gycosides contents. In this study, we found the positively correlated miRNA-mRNA interaction network in plants, where miRStv_11 enhanced the expression of KAH gene. miRNAs knock-down was also successfully achieved using antisense precursors. Overall, this study thus reveals more complex nature and fundamental importance of miRNAs in biosynthetic pathway related gene networks and hence, these miRNAs can be successfully employed to enhance the ratio of rebaudioside-A to stevioside, thus enhancing the sweetening indices of this plant and making it more palatable.

Topics: Diterpenes, Kaurane; Gene Expression Regulation, Plant; Gene Knockout Techniques; Gene Silencing; Glucosides; MicroRNAs; Plant Leaves; Promoter Regions, Genetic; RNA, Plant; Stevia; Sweetening Agents

Diets high in sugar are recognized as a serious health problem, and there is a drive to reduce their consumption. Steviol glycosides are natural zero-calorie sweeteners, but the most desirable ones are biosynthesized with low yields. UGT76G1 catalyzes the β (1-3) addition of glucose to steviol glycosides, which gives them the preferred taste. UGT76G1 is able to transfer glucose to multiple steviol substrates yet remains highly specific in the glycosidic linkage it creates. Here, we report multiple complex structures of the enzyme combined with biochemical data, which reveal that the enzyme utilizes hydrophobic interactions for substrate recognition. The lack of a strict three-dimensional recognition arrangement, typical of hydrogen bonds, permits two different orientations for β (1-3) sugar addition. The use of hydrophobic recognition is unusual in a regio- and stereo-specific catalysis. Harnessing such non-specific hydrophobic interactions could have wide applications in the synthesis of complex glycoconjugates.

Topics: Arabidopsis Proteins; Catalysis; Catalytic Domain; Crystallography, X-Ray; Diterpenes, Kaurane; Glucose; Glucosides; Glycosides; Glycosyltransferases; Hydrophobic and Hydrophilic Interactions; Plant Proteins; Protein Conformation; Stevia; Substrate Specificity; Sweetening Agents

The use of steviol glycosides as non-caloric sweeteners has proven to be beneficial for patients with type 2 diabetes mellitus (T2D), obesity, and metabolic syndrome. However, recent data demonstrate that steviol and stevioside might act as glucocorticoid receptor (GR) agonists and thus correlate with adverse effects on metabolism. Herein, we evaluated the impact of steviol, steviol glycosides, and a Greek-derived stevia extract on a number of key steps of GR signaling cascade in peripheral blood mononuclear cells (PBMCs) and in Jurkat leukemia cells. Our results revealed that none of the tested compounds altered the expression of primary GR-target genes (GILZ, FKPB5), GR protein levels or GR subcellular localization in PBMCs; those compounds increased GILZ and FKPB5 mRNA levels as well as GRE-mediated luciferase activity, inducing in parallel GR nuclear translocation in Jurkat cells. The GR-modulatory activity demonstrated by stevia-compounds in Jurkat cells but not in PBMCs may be due to a cell-type specific effect.

Topics: Adrenocorticotropic Hormone; Cell Nucleus; Cell Survival; Dexamethasone; Diterpenes, Kaurane; Gene Expression Regulation; Glucosides; Humans; Hydrocortisone; Jurkat Cells; Leukocytes, Mononuclear; Luciferases; Neoplasms; Plant Extracts; Receptors, Glucocorticoid; Response Elements; RNA, Messenger; Signal Transduction; Stevia; Tacrolimus Binding Proteins; Transcription Factors

Stevia rebaudiana Bertoni is one of the most important biologically sourced and low-calorie sweeteners that contains a lots of Steviol glycosides. Tissue culture is the best method for propagation of stevia and micro nutrients can affect both morphological traits and steviol glycosides production. In the present study, we investigated the effect of different concentrations of glutamine (10, 20, 30 and 40 g/l) on expression of UGT74G1 and UGT76G1 genes and stevioside and rebaudioside A accumulation in the leaves of stevia under in vitro conditions. The highest level of expression for UGT74G1 (1.000 Total lab unit) was seen at plants grown in MS media without glutamine and the highest gene expression level for UGT76G1 (1.321 Total lab unit) was observed at plants grown in 2% glutamine. Based on HPLC results, the highest amount of stevioside (22.74) was accumulated in plants which were under 3% glutamine treatment and the lowest production level of stevioside (16.19) was resulted under MS (0 glutamine) medium. The highest rebaudioside A (12.19) accumulation was observed under 2% glutamine treatment and the lowest accumulation of rebaudioside A (8.41) was seen at plants grown in MS medium.

Topics: Culture Media; Diterpenes, Kaurane; Gene Expression; Genes, Plant; Glucosides; Glutamine; Plant Leaves; Stevia; Tissue Culture Techniques

Stevia rebaudiana Bertoni is One of the most important biologically sourced and low-calorie sweeteners that known as "Sweet Weed". It contains steviol glycosides that they are about 200-300 times sweeter than sucrose. Tissue culture is the best method with high efficiency that can overcome to problems of traditional methods, and it is the most useful tools for studying stress tolerance mechanisms under in vitro conditions to obtain drought tolerance. In the present research, we investigated the impact of life cycle, leaves location and the harvesting time on expression of UGT74G1 and UGT76G1 as well as steviol glycosides accumulation. The highest gene expression of both UGT74G1 and UGT76G1 (207.677 and 208.396 Total Lab unit, respectively) was observed in young leaves in the second vegetative year. Also, the highest amount of stevioside accumulation (13.04) was due to the old leaves in vegetative stage which had significant differences with other effects whereas the lowest accumulation (7.47) was seen at young leaves at vegetative stage. Interestingly, the highest level of rebaudioside a production (15.74) was occurred at the young leaves at vegetative stage. There was significant differences between life cycle and leaves location on steviol glycoside production in stevia.

Topics: Diterpenes, Kaurane; Gene Expression Regulation, Plant; Genes, Plant; Glucosides; Life Cycle Stages; Plant Leaves; Stevia; Time Factors

Stevia rebaudiana (Bertoni) is a non-caloric sweetener belonging to Asteraceae family. Stevia compounds such as steviol glycosides (SGs) are 200 times sweeter than sugar. Stevioside and rebaudioside A are the two major steviol glycosides. Nitrogen is an essential element for plant growth and development. In this study the effects of nitrogen influenced by different concentrations of NH4NO3 (0, 825 and 1650 mg/l) and KNO3 (0, 950 and 1900 mg/l) is examined in MS medium. To analysis the UGT74G1 and UGT76G1 genes expression, involved in the synthesis of SGs, RT-qPCR technique was performed. Data showed that there were significant differences between all media. The shoot length, seedlings dry weight and leaf fresh weight of stevia increased with applying NH4NO3 along with KNO3. The highest expression of UGT74G1 gene, was observed in plantlets grown on MS medium with 0 mg/l NH4NO3 and 950 mg/l KNO3 (1.291 total lab unit) but the highest expression of UGT76G1 gene, was observed in plantlets grown on MS medium added by 1650 mg/l NH4NO3 +950 mg/l KNO3 (1.08 total lab unit). Moreover, the lowest value of UGT74G1 gene expression were revealed in MS medium added by 1650 mg/l NH4NO3 +0 mg/l KNO3 (0.80 total lab unit) and the lowest values of UGT76G1 gene expression seen in MS medium with 0 mg/l NH4NO3 +950 mg/l KNO3 (0.85 total lab unit) concentrations. The results of this study could be valuable in stevia breeding programs through glycosides biosynthesis pathways.

Topics: Culture Media; Diterpenes, Kaurane; Gene Expression Regulation, Plant; Genes, Plant; Glucosides; Nitrates; Plant Breeding; Plant Leaves; Plant Shoots; Potassium Compounds; Seedlings; Stevia; Tissue Culture Techniques

Stevia rebaudiana Bertoni is one of two species that contains steviol glycosides. Among steviol glycosides that extracted from leaves, stevioside and rebaudioside A are the two major and the sweetest glycosides that are about 200-300 times sweeter than sucrose with zero calories. The best method for stevia propagation is tissue culture. So, for investigation of nutrients in medium, we studied the effect of different concentrations of MS media (MS, 0.5 MS, 0.25 MS, 0 MS) on morphological traits, UGT74G1 and UGT76G1 genes expression and accumulation of steviol glycosides in stevia leaves. The best growth rate (0.472 mm/d) has occurred in plants grown in MS media. Also, the highest gene expression of UGT74G1 gene (1.000 Total lab unit) was seen under MS treatment. However, the highest expression level of UGT76G1 gene (1.701 Total lab unit) was observed at plants grown in 0 MS. The highest amount of both Stevioside and Rebaudioside A (14.23 and 8.12, respectively) were accumulated in plants under MS treatment. Obviously, dilution of MS media associated with decreasing in both expression of the intended genes and accumulation of steviol glycosides.

Topics: Culture Media; Diterpenes, Kaurane; Gene Expression; Gene Expression Regulation, Plant; Genes, Plant; Glucosides; Plant Extracts; Plant Leaves; Stevia; Tissue Culture Techniques

Stevia rebaudiana Bertoni is a kind of perennial medicinal plant with sweetening properties which belongs to Asteraceae family. Its leaves with fundamental glycoside compounds consist of both a sugar part and a non-sugar sector. One of the glycoside compounds is Rebaudioside- A which has a greater importance in business. This experiment was conducted to evaluate the effects of Ag2O, CrO3, PbO, Fe2O3, BaO and TiO2 on the expression pattern of these genes in the Stevia rebaudiana. Rebaudioside- A biosynthesis was repeated 3 times with concentrations of 50, 100 and 200µM. Also, the results of the study pertaining to the expression pattern of these genes showed that metal oxides have led to an increase in the expression of the regulatory genes involved in biosynthesis of Rebaudioside- A. According to the expression profile, it was found that its effect on DXR, HDS, HDR, IDI and CPPS genes is more than other genes. The peak HPLC indicated for stevioside and Rebaudioside- A represents an increase in the production of this active ingredient under the influence of all treatments. In general, the expression profile of these genes and the results of HPLC show that whatever going to the end of the pathway of production of Rebaudioside- A, the activity of the enzymes increases under the influence of these treatments, and eventually a greater amount of Rebaudioside- A will be produced. This process shows that metal oxides will have a significant effect on the biosynthesis of Rebaudioside- A.

Topics: Diterpenes, Kaurane; Gene Expression Regulation, Plant; Genes, Plant; Glucosides; Metals; Oxides; Plant Leaves; Stevia; Transcriptome; Up-Regulation

The physiological effects of the Stevia-derived compounds, rebaudioside A, stevioside and steviol have been the focus of several studies due to their use as sweeteners in food. Despite that, little is known about their potential food-drug interactions. In the present study, IPEC-J2 cells and primary hepatocytes were used to investigate the effect of rebaudioside A, stevioside and steviol on cytochrome p450 (CYP) mRNA expression. Moreover, hepatic microsomes were used to investigate direct interactions between the compounds and specific CYP activity. In IPEC-J2 no changes in mRNA expression of CYP1A1 or CYP3A29 were observed with the Stevia-derived compounds. In primary hepatocytes all three tested compounds induced a significant increase in CYP3A29 expression. The tested compounds had no direct effect on specific CYP activity. In conclusion, rebaudioside A, stevioside and steviol induce only minor or no changes to the CYP expression and activity, and are not likely to cause food-drug interactions.

Topics: Animals; Cell Survival; Cells, Cultured; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP1A1; Cytochrome P-450 Enzyme System; Diterpenes, Kaurane; Glucosides; Hepatocytes; Occludin; Swine; Transcriptome

A quantitative analysis by HPLC of α-glucosyltransferase-treated stevia in foods was considered. This analysis is the way which hydrolyzed α-glucosyltransferase-treated stevia in the stevioside (SS) and the rebaudioside A (RS) using a glucoamylase. Recovery (%) of α-glucosyltransferase-treated stevia, spiked at 200 mg/kg in various foods, were more than 80% and the relative standard deviations were less than 5.0% as SS and RS for the rate of collection. A qualitative analysis by LC-MS/MS was performed 36 products of commercial foods containing stevia. We quantified of 11 products in which α-glucosyltransferase-treated stevia was detected. Quantitative value was at most 180 mg/kg as SS, at most 70 mg/kg as RS.

Topics: Chromatography, High Pressure Liquid; Chromatography, Liquid; Diterpenes, Kaurane; Food Analysis; Glucan 1,4-alpha-Glucosidase; Glucosides; Glucosyltransferases; Hydrolysis; Stevia; Sweetening Agents; Tandem Mass Spectrometry

This study provides a comprehensive investigation on the impact of increasing NaCl concentrations on hydroponically grown Stevia rebaudiana cultivars (Shoutian-2 and Fengtian). Growth parameters including plant height, biomass and physiological responses including osmotic potential were measured. In addition, the levels of steviol glycosides, elements and primary metabolites were measured and statistically evaluated. The cultivar Fengtian grew faster, accumulated less Na

Topics: Chlorophyll; Diterpenes, Kaurane; Gas Chromatography-Mass Spectrometry; Gluconates; Glucosides; Osmotic Pressure; Potassium; Proline; Salt Stress; Sodium; Spectrometry, Mass, Electrospray Ionization; Stevia

Stevia extracts are obtained from Stevia rebaudiana commonly used as natural sweeteners. It is ∼250-300 times sweeter than sucrose. Common use of stevia prompted us to investigate its genotoxicity in human peripheral blood lymphocytes. Stevia (active ingredient steviol glycoside) was dissolved in pure water. Dose selection was done using ADI (acceptable daily intake) value. Negative control (pure water), 1, 2, 4, 8 and 16 μg/ml concentrations which were equivalent to ADI/4, ADI/2, ADI, ADI × 2 and ADI × 4 of Stevia were added to whole-blood culture. Two repetitive experiments were conducted. Our results showed that there was no significant difference in the induction of chromosomal aberrations and micronuclei between the groups treated with the concentrations of Stevia and the negative control at 24 and 48 h treatment periods. The data showed that stevia (active ingredient steviol glycosides) has no genotoxic activity in both test systems. Our results clearly supports previous findings.

Topics: Adult; Diterpenes, Kaurane; Female; Glucosides; Humans; Lymphocytes; Male; Micronuclei, Chromosome-Defective; Micronucleus Tests; Plant Extracts; Risk Assessment; Stevia; Sweetening Agents

The present study aims to assess the effect of different nitrogen (N) rates on the chemical composition and antioxidant properties of stevia frozen fresh and dried leaves, and to define the best growing conditions to maximize the levels of bioactive compounds. In general, processing affects more significantly the tocopherol and sugar contents than N fertilization. The most abundant sugars were xylose, arabinose + fructose and sucrose, presenting dried samples with higher contents than frozen fresh ones, while the latter better retained tocopherols than dry samples. Regarding phenolic compounds, greater levels were found in dried samples and in those fertilized with 25 kg N ha

Topics: Antioxidants; Desiccation; Diterpenes, Kaurane; Fertilizers; Freezing; Glucosides; Nitrogen; Phenols; Plant Leaves; Stevia

Stevia rebaudiana Bertoni leaves are a natural source of diterpenic glycosides, and various bioactive compounds. The objectives were to characterize antioxidants and steviol glycosides in the extracts obtained from Stevia after "green" pressurized hot water extraction (PHWE). PHWE extracts were obtained at different temperatures (100, 130, 160 °C); static extraction times (5 and 10 min), and cycle numbers (1, 2, 3) using a constant pressure of 10.34 MPa. Temperature was the most important parameter for extraction, where the highest recoveries of all bioactive compounds (except for carotenoids) were at 160 °C. Extracts obtained at longer static times had more steviol glycosides, condensed tannins, and chlorophyll A. Higher amounts of total phenols, condensed tannins, and steviol glycosides were obtained under higher cycle numbers. This study indicated that PHWE is useful for recovering polar and nonpolar antioxidants and steviol glycosides. PHWE may be a suitable technique for scale-up to industrial applications.

Topics: Antioxidants; Chemical Fractionation; Diterpenes, Kaurane; Glucosides; Green Chemistry Technology; Hot Temperature; Plant Leaves; Pressure; Stevia; Water

Pure stevioside was extracted from leaves of Stevia rebaudiana (Bertoni) to be used as a reference material in the instrument calibration or method validation process. The mass fraction was determined by comparison between the mass balance method and quantitative nuclear magnetic resonance (qNMR) spectroscopy. The impurities in the sample were analyzed by Karl Fischer titration for moisture content and thermogravimetric analysis for inorganic residue. Homogeneity, together with short term and long term stability, were also studied and the uncertainty was reported. The certified value of this mass fraction is 0.986 ± 0.0019 (k = 2) with 1 year stability.

Topics: Calibration; Diterpenes, Kaurane; Drug Contamination; Drug Stability; Glucosides; Magnetic Resonance Spectroscopy; Plant Leaves; Quality Control; Reference Standards; Reproducibility of Results; Stevia

The objective of this study was to evaluate the sensory profile and the influence of the information on the acceptance of the symbiotic chocolate ice cream made with sucrose and different sweeteners (aspartame, sucralose, neotame, Stevia with 60%, 85%, 95%, and 97% of rebaudioside A) through analysis of variance (ANOVA), Tukey's test, and partial least of square (PLS) regression. Quantitative descriptive analysis (QDA) was carried out by 18 assessors, who evaluated the samples in relation to the raised descriptors. Additionally, two acceptance tests (blind/informed) were performed with 120 consumers. The samples sweetened with sucralose and rebaudioside 97% presented similar profile to the control sample, thus having a better potential to replace sucrose in chocolate ice cream. The acceptance test carried out with information had higher scores for the attributes appearance, aroma, flavor, texture, and overall impression. The correlation between data from the acceptance tests and QDA showed that the descriptors "low-energy" and "natural sweetener" claims interfered negatively in the drivers of liking of chocolate ice cream. Therefore, we can conclude that some characteristics unnoticed by consumers were highlighted after providing the information about the product's characteristics.. This research is important and contributes to the manufacture and development of low-calorie chocolate ice cream with functional properties, guiding, through suitable sensory and statistical tools, the application of stevia and other artificial sweeteners in products with reduction or total absence of sucrose and highlighting the impact of the labeling of these products on consumer perception.

Topics: Adolescent; Adult; Aspartame; Cacao; Chocolate; Consumer Behavior; Diterpenes, Kaurane; Energy Intake; Female; Food Additives; Food Handling; Functional Food; Glucosides; Humans; Ice Cream; Male; Middle Aged; Probiotics; Stevia; Sucrose; Sweetening Agents; Synbiotics; Taste; Young Adult

Jelly and confectionery products are high in sugar and calories. Xylitol and stevioside are natural low-calorie sweeteners and they can be used as an alternative; however, their effects on fish gelatin are unknown. The gelatin was extracted from cod skins and added to xylitol or stevioside (0, 2, 6, 10, 14, and 20% (w/v)) to form gel products. This paper investigated how xylitol and stevioside affected the physical and rheological behaviors of fish gelatin, such as color, gel strength, texture profile analysis, storage modulus (G'), loss modulus (G″), and viscosity. Results showed that the change of color and viscosity in gel products were similar when various concentrations of xylitol or stevioside were added to the fish gelatin. But the effects of xylitol/stevioside on texture profile analysis and G', G″ were different, which might due to the structure variation in xylitol and stevioside. The linear structure of xylitol resulted in ionic interaction, hydrogen bonding, van der Waals forces, and hydrophobic association between xylitol and fish gelatin. Therefore, xylitol is a promising sweetener substitute, which was probably related to its greater solubility and number of -OH groups.

Topics: Animals; Color; Diterpenes, Kaurane; Energy Intake; Food Analysis; Food Handling; Food Quality; Gadiformes; Gelatin; Glucosides; Humans; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Plant Extracts; Rheology; Skin; Stevia; Sweetening Agents; Viscosity; Xylitol

Stevia rebaudiana extracts are used as sweeteners in several countries worldwide. Several extracts of diverse composition are available on the market, and their taste depends on the contents of the various steviol glycosides. This study presents an accurate method for the qualitative and quantitative determination of steviol glycosides in 40 Stevia extracts, 7 sweeteners and 3 Stevia-sweetened beverages by a UHPLC coupled to an Orbitrap mass spectrometer. The sub-2 μm amide column provided the separation of all the target analytes in a run time of 30 min with high resolution. The effect of different eluent compositions on the ionisation efficiency of the steviol glycosides was studied. The optimal ionisation conditions were achieved in negative mode using 0.05% formic acid. Under this condition, adducts were not found, [M-H]

Topics: Beverages; Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Food Additives; Food Analysis; Glucosides; Glycosides; Mass Spectrometry; Plant Extracts; Stevia; Sweetening Agents

Topics: Carbon; Carbon Dioxide; Diterpenes, Kaurane; Glucosides; Light; Osmoregulation; Plant Leaves; Stevia; Water

Biosynthesis of steviol glycosides in planta proceeds via two cytochrome P450 enzymes (CYPs): kaurene oxidase (KO) and kaurenoic acid hydroxylase (KAH). KO and KAH function in succession with the support of a NADPH-dependent cytochrome P450 reductase (CPR) to convert kaurene to steviol. This work describes a platform for recombinant production of steviol glucosides (SGs) in Saccharomyces cerevisiae, demonstrating the full reconstituted pathway from the simple sugar glucose to the SG precursor steviol. With a focus on optimization of the KO-KAH activities, combinations of functional homologues were tested in batch growth. Among the CYPs, novel KO75 (CYP701) and novel KAH82 (CYP72) outperformed their respective functional homologues from Stevia rebaudiana, SrKO (CYP701A5) and SrKAH (CYP81), in assays where substrate was supplemented to culture broth. With kaurene produced from glucose in the cell, SrCPR1 from S. rebaudiana supported highest turnover for KO-KAH combinations, besting two other CPRs isolated from S. rebaudiana, the Arabidopsis thaliana ATR2, and a new class I CPR12. Some coexpressions of ATR2 with a second CPR were found to diminish KAH activity, showing that coexpression of CPRs can lead to competition for CYPs with possibly adverse effects on catalysis.

Topics: Arabidopsis; Arabidopsis Proteins; Basic Helix-Loop-Helix Transcription Factors; Cloning, Molecular; Cytochrome P-450 Enzyme System; Diterpenes, Kaurane; Glucosides; Plant Proteins; Plasmids; Saccharomyces cerevisiae; Stevia; Substrate Specificity

Steviol glycosides (SGs), such as stevioside and rebaudioside A, are natural, non-caloric sweet-tasting organic molecules, present in extracts of the scrub plant Stevia rebaudiana, which are widely used as sweeteners in consumer foods and beverages. TRPM5 is a Ca

Topics: Animals; Blood Glucose; Diabetes Mellitus, Type 2; Diet, High-Fat; Diterpenes, Kaurane; Female; Glucosides; HEK293 Cells; Humans; Insulin; Insulin Secretion; Insulin-Secreting Cells; Male; Mice; Mice, Knockout; Patch-Clamp Techniques; Sweetening Agents; Taste; TRPM Cation Channels

Stevia rebaudiana Bertoni is a famous medicinal plant for its low calorific value compounds which are named steviol glycosides (SGs) and they are 150-300 times sweeter than sugar. Among various SGs, stevioside and rebaudioside A considered to be the main sweetening compounds.  Soil salinity is one of the most essential stress in the world. Salinity affects the survival and yield of crops. In current study the effects of salinity and osmotic stress caused by different concentration of NaCl (0, 20, 40, 60 and 80 mM) on morphological traits, genes expressionand amount of both stevioside and rebaudioside Aunder in vitro conditions has been investigated. The morphological traits such as bud numbers, root numbers, shoot length (after 15 and 30 days) were evaluated. With increasing salinity, the values of all studied morphological traits decreased. To investigation of UGT74G1 and UGT76G1 genes expression that are involved in the synthesis of SGs, RT-PCR was done and there were significant differences between all media. The highest expression of both genes was observed in plantlets grown on MS media (with NaCl-free). Also, the lowest amounts of gene expression of the both genes were seen in MS+ 60 mM NaCl. Based on HPLC results, the highest amount of both stevioside and rebaudioside A were observed in plantlets grown in MS media (with NaCl-free). Finally, it can be concluded that stevia can survive under salt stress, but it has the best performance in the lower salinity.

Topics: Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Gene Expression Regulation, Plant; Genes, Plant; Glucosides; Plant Leaves; Plant Proteins; Plant Shoots; Salinity; Sodium Chloride; Stevia

Stevia rebaudiana is one of the most important biologically sourced and low-calorie sweeteners Bertoni that has a lot of steviol glycosides. Tissue culture is the best for propagation of stevia and micro nutrients can affect both morphological traits and steviol glycosides production. Therefore, the effect of different concentrations of KH2PO4on stevia growth factors and gene expression had been studied by tissue culture methods, RT-PCR and HPLC. According the results, bud numbers had increased significantly in MS + 0.034 mMKH2PO4 media and the highest measured length was seen in plants grown under MS + 0.034 mM KH2PO4 treatment. Also, the highest growth rate (1.396 mm/d) was observed in MS + 0.034 mMKH2PO4.The best concentration of KH2PO4 for expression of UGT74G1 was 0.00425mMand the best one for UGT76G1 expression was 0.017mM. Interestingly, the best media for both stevioside and rebaudioside A accumulation was 0.017mM KH2PO4containing media. There was positive correlation between the best media for gene expression and the best one for steviol glycosides production.

Topics: Analysis of Variance; Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Gene Expression Regulation, Plant; Genes, Plant; Glucosides; Phosphates; Plant Proteins; Plant Shoots; Potassium Compounds; Stevia

Topics: Beverages; Diterpenes, Kaurane; Electrophoresis, Capillary; Food Analysis; Glucosides; Stevia; Sweetening Agents

Stevioside and rebaudioside A are the main diterpene glycosides present in the leaves of the Stevia rebaudiana plant, which is used in the production of foods and low-calorie beverages. The difficulties associated with their extraction and purification are currently a problem for the food processing industries. The objective of this study was to develop an effective and economically viable method to obtain a high-quality product while trying to overcome the disadvantages derived from the conventional transformation processes. For this reason, extractions were carried out using a conventional maceration (CM) and a cyclically pressurized extraction known as rapid solid-liquid dynamic extraction (RSLDE) by the Naviglio extractor (NE). After only 20 min of extraction using the NE, a quantity of rebaudioside A and stevioside equal to 1197.8 and 413.6 mg/L was obtained, respectively, while for the CM, the optimum time was 90 min. From the results, it can be stated that the extraction process by NE and its subsequent purification developed in this study is a simple, economical, environmentally friendly method for producing steviol glycosides. Therefore, this method constitutes a valid alternative to conventional extraction by reducing the extraction time and the consumption of toxic solvents and favouring the use of the extracted metabolites as food additives and/or nutraceuticals. As an added value and of local interest, the experiment was carried out on stevia leaves from the Benevento area (Italy), where a high content of rebaudioside A was observed, which exhibits a sweet taste compared to stevioside, which has a significant bitter aftertaste.

Topics: Dietary Supplements; Diterpenes, Kaurane; Food Additives; Glucosides; Glycosides; Plant Leaves; Stevia; Sweetening Agents

An enzymatic method for specific determination of stevioside content was established. Recombinant β-glucosidase BT_3567 (rBT_3567) from Bacteroides thetaiotaomicron HB-13 exhibited selective hydrolysis of stevioside at β-1,2-glycosidic bond to yield rubusoside and glucose. Coupling of this enzyme with glucose oxidase and peroxidase allowed for quantitation of stevioside content in Stevia samples by using a colorimetric-based approach. The series of reactions for stevioside determination can be completed within 1 h at 37 °C. Stevioside determination using the enzymatic assay strongly correlated with results obtained from HPLC quantitation (r

Topics: Bacterial Proteins; Bacteroides thetaiotaomicron; beta-Glucosidase; Biocatalysis; Colorimetry; Diterpenes, Kaurane; Enzyme Assays; Glucose Oxidase; Glucosides; Plant Extracts; Stevia; Sweetening Agents

The effect of Stevia rebaudiana Bertoni on the hemolytic potential of Listeria monocytogenes was studied by means of the assessment of the Listeriolysin O (LLO) production. The three factors under study, stevia concentration in the range [0-2.5] % (w/v), incubation temperature (10 and 37°C), and exposure time (0-65h) significantly affected (p≤0.05) the hemolytic activity of L. monocytogenes. Results showed that at the lower incubation temperature the hemolytic potential of the bacterium was significantly reduced, from 100% at 37°C to 8% at 10°C (after 65h of incubation) in unsupplemented substrate (0% stevia). Irrespective of the temperature, 10 or 37°C, supplementation of the medium with stevia at 2.5 % (w/v) reduced the bacterium's hemolytic activity by a maximum of 100%. Furthermore, the time of exposure to 2.5 % (w/v) stevia concentration was also a significant factor reducing the hemolytic capability of L. monocytogenes. The possibility of reducing the pathogenic potential of L. monocytogenes (hemolysis) by exposure to stevia should be confirmed in real food matrices, opening a research niche with a valuable future impact on food safety.

Topics: Bacterial Toxins; Cytotoxins; Diterpenes, Kaurane; Glucosides; Heat-Shock Proteins; Hemolysin Proteins; Listeria monocytogenes; Plant Extracts; Stevia; Temperature

Diabetes mellitus is a chronic disease characterized by abnormal carbohydrate, lipid and protein metabolism due to a lack of insulin or reduced target cell sensitivity to insulin. Stevia rebaudiana is an important source of biochemically active substances with proven anti-diabetic effect. The aim of this study was to determine anti-diabetic effects of the low dose of stevioside in NMRI Haan mice. Aqueous stevioside solution (20mg/kg body weight) was administered by oral route of administration. Anti-diabetic effect of stevioside was estimated by oral glucose tolerance test, adrenaline test after a 10day stevioside treatment, and alloxan induced hyperglycaemia in mice (two experimental groups, 10day stevioside treatment before and after alloxan administration). Aqueous stevioside solution prevented significant increase in glycaemia in oral glucose tolerance test (9.22±1.13 to 9.85±1.32mmol/l, P<0.05), and not in adrenaline test. Significant difference in glycaemia was detected in mice pre-treated with saline and stevioside in alloxan induced hyperglycaemia (saline 23.32±2.14, stevioside 14.70±4.95mmol/l, P<0.05). In mice pre-treated with stevioside, smallest β cells loss was found compared to other alloxan treated groups. Preserved normal cytoarchitectonic arrangement in islets was detected. Based on the given results we presume there exist a potential therapeutic use of low dose stevioside in diabetes.

Topics: Alloxan; Animals; Blood Glucose; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Diterpenes, Kaurane; Glucose Tolerance Test; Glucosides; Hyperglycemia; Hypoglycemic Agents; Insulin; Male; Mice; Phytotherapy; Plant Extracts; Stevia

Topics: Antioxidants; Diterpenes, Kaurane; Glucosides; Minerals; Oxidative Stress; Plant Leaves; Reactive Oxygen Species; Salt Tolerance; Sodium Chloride; Stevia

We investigated the effect of natural sweetener Stevia rebaudiana and its constituent stevioside in cisplatin (CP)-induced kidney injury. Male BALB/cN mice were orally administered 10, 20, and 50 mg/kg body weight of Stevia rebaudiana ethanol extract (SE) or stevioside 50 mg/kg, 48 h after intraperitoneal administration of CP (13 mg/kg). Two days later, CP treatment resulted in histopathological changes showing kidney injury. Increased expression of 4-hydroxynonenal (4-HNE), 3-nitrotyrosine (3-NT), and heme oxygenase-1 (HO-1) in mice kidneys suggested oxidative stress. CP treatment also increased renal expression of nuclear factor-kappaB (NF-κB) p65 subunit and phosphorylated inhibitor of NF-κB (IκBα), as well as expression of pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α). Induction of apoptosis and inhibition of the cell cycle in kidneys was evidenced by increased expression of p53, Bax, caspase-9, and p21, proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), with concomitant suppression of Bcl-2 and cyclin D1 expression. The number of apoptotic cells in kidneys was also assessed. CP administration resulted in activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3). Both SE and stevioside attenuated CP nephrotoxicity by suppressing oxidative stress, inflammation, and apoptosis through mechanism involving ERK1/2, STAT3, and NF-κB suppression.

Topics: Animals; Antineoplastic Agents; Apoptosis; Caspase 9; Chemical and Drug Induced Liver Injury; Cisplatin; Diterpenes, Kaurane; Glucosides; Heme Oxygenase-1; Humans; Kidney; Male; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinase 3; NF-kappa B; Oxidative Stress; Poly(ADP-ribose) Polymerases; Protective Agents; Stevia; Tumor Necrosis Factor-alpha

Stevia rebaudiana (Bert.) Bertoni is known as sweet plant which it contains a high level of steviol glycosides in the leaves.  This plant has been used from centuries ago as a sweetener for tea. One of the most important steviol glycosides is stevioside that is attractive for diabetic persons. Tissue culture is the only rapid process for the mass propagation of stevia. One of the most important factors in the medium is sucrose that is a necessary for plant growth. In the present study, we use nodal segments of the stem as explants in mediums with different sucrose concentration (50 mM, 100mM and 150mM). Several morphological traits were measured in a 28 day period. Results analysis showed a significant variation between treatments. The highest growth rate, rooting and leaf production was obtained in medium with 100mM sucrose. The correlation between measured traits was significant at the 0.01 level. To investigation of UGT74G1, UGT76G1, UGT85C2 and KS genes expression that are involved in the synthesis of SGs, RT- PCR was done with the housekeeping gene of as internal control. There were significant differences between all media. The results showed thatsucrose 100 mM containing media was more desirable than others for expression of UGT76G1 and UGT85C2 genes. Whereas, the best medium for expression of UGT74G1 was sucrose 150 mM and sucrose 50 mM for KS gene. Totally, it seems that sucrose at a concentration of 100 mMprovides the best condition for stevia growth and steviol glycosides production.

Topics: Culture Media; Diterpenes, Kaurane; Gene Expression Regulation, Plant; Genes, Plant; Glucosides; Plant Leaves; Plant Roots; Stevia; Sucrose; Sweetening Agents; Tissue Culture Techniques

Oxidative stress and hepatic inflammatory response is primarily implicated in the pathogenesis of LPS induced acute liver injury. Stevioside, a diterpenoidal glycoside isolated from the Stevia rebaudiana leaves, exerts potent anti-oxidant, anti-inflammatory and immunomodulatory activities. The present study was aimed to investigate the hepatoprotective effect of hydroalcoholic extract of Stevia rebaudiana leaves (STE EXT) and its major phytochemical constituent, stevioside (STE) in LPS induced acute liver injury. The hepatoprotective activity of STE EXT (500mg/kg p.o) and STE (250mg/kg p.o) was investigated in lipopolysaccharide (LPS 5mg/kg i.p.) induced acute liver injury in male wistar rats. Our results revealed that both STE EXT and STE treatment ameliorated LPS induced hepatic oxidative stress, evident from altered levels of reduced SOD, Catalase, GSH, MDA, NO. Histopathological observations revealed that both STE EXT and STE attenuated LPS induced structural changes and hepatocellular apoptosis providing additional evidence for its hepatoprotective effect. Further, STE EXT and STE significantly restored the elevated serum and tissue levels of AST and ALT in LPS treated rats. Furthermore, both STE EXT and STE rescued hepatocellular dysfunctions to normal by altering the level of proinflammatory cytokines such as TNF-α, IL-1β and IL-6 exhibiting its anti-inflammatory potential. In conclusion, both STE EXT and STE demonstrated excellent hepatoprotective effects against endotoxemia induced acute liver injury possibly through suppression of hepatic inflammatory response and oxidative stress, attributing to its medicinal importance in treating various liver ailments.

Topics: Acute Disease; Animals; Biphenyl Compounds; Chromatography, Thin Layer; Cytokines; Diterpenes, Kaurane; Ethanol; Flavonoids; Free Radical Scavengers; Glucosides; Inflammation Mediators; Lipid Peroxidation; Lipopolysaccharides; Liver; Liver Function Tests; Male; Nitric Oxide; Oxidative Stress; Phenols; Phytochemicals; Phytotherapy; Picrates; Plant Extracts; Rats, Wistar; Stevia; Water

Excess dietary fructose intake associated with metabolic syndrome and insulin resistance and increased risk of developing type 2 diabetes. Previous animal studies have reported that diabetic animals have significantly impaired behavioural and cognitive functions, pathological synaptic function and impaired expression of glutamate receptors. Correction of the antioxidant status of laboratory rodents largely prevents the development of fructose-induced plurimetabolic changes in the nervous system. We suggest a novel concept of efficiency of Stevia leaves for treatment of central diabetic neuropathy.. By in vivo extracellular studies induced spike activity of hippocampal neurons during high frequency stimulation of entorhinal cortex, as well as neurons of basolateral amygdala to high-frequency stimulation of the hippocampus effects of Stevia rebaudiana Bertoni plant evaluated in synaptic activity in the brain of fructose-enriched diet rats. In the conditions of metabolic disorders caused by fructose, antioxidant activity of Stevia rebaudiana was assessed by measuring the NOX activity of the hippocampus, amygdala and spinal cord.. In this study, the characteristic features of the metabolic effects of dietary fructose on synaptic plasticity in hippocampal neurons and basolateral amygdala and the state of the NADPH oxidase (NOX) oxidative system of these brain formations are revealed, as well as the prospects for development of multitarget and polyfunctional phytopreparations (with adaptogenic, antioxidant, antidiabetic, nootropic activity) from native raw material of Stevia rebaudiana. Stevia modulates degree of expressiveness of potentiation/depression (approaches but fails to achieve the norm) by shifting the percentage balance in favor of depressor type of responses during high-frequency stimulation, indicating its adaptogenic role in plasticity of neural networks. Under the action of fructose an increase (3-5 times) in specific quantity of total fraction of NOX isoforms isolated from the central nervous system tissue (amygdala, hippocampus, spinal cord) was revealed. Stevia exhibits an antistress, membrane-stabilizing role reducing the level of total fractions of NOX isoforms from central nervous system tissues and regulates NADPH-dependent O. Generally, in condition of metabolic disorders caused by intensive consumption of dietary fructose Stevia leaves contributes to the control of neuronal synaptic plasticity possibly influencing the conjugated NOX-specific targets.

Topics: Action Potentials; Animals; Brain; Brain Chemistry; Dietary Sugars; Diterpenes, Kaurane; Fructose; Glucosides; Male; Metabolic Diseases; NADPH Oxidases; Neuronal Plasticity; Rats; Stevia

The demand for zero and reduced-sugar food products containing cocoa is expanding continuously. The present study was designed to evaluate the feasibility of producing high-quality chocolate sweetened with a crude extract of Stevia rebaudiana (Bertoni) prepared by a green microwave-assisted water-steam extraction procedure. Seven approximately isosweet chocolate formulations were developed, mixing cocoa paste, sucrose, commercial stevioside, crude green extract and maltitol in different proportions. All samples were analyzed for the determination of polyphenol and flavonoid content, antioxidant activity, and sensory acceptability.. The use of a crude stevia extract allowed low-sugar, high-quality chocolates to be obtained that were also acceptable by consumers and had a significant increased antioxidant activity. Moreover, consumers' segmentation revealed a cluster of consumers showing the same overall liking for the sample with 50% sucrose replaced by the stevia crude extract as that obtained with the commercial stevioside and the control sample (without sucrose replacement).. The results provide information that can contribute to promoting the development of sweet food products, with advantages in terms of an improved nutritional value (reduced sugar content and increased antioxidant activity) and a reduced impact of the production process on the environment. © 2016 Society of Chemical Industry.

Topics: Adolescent; Adult; Aged; Antioxidants; Chocolate; Diterpenes, Kaurane; Female; Flavonoids; Food Handling; Glucosides; Humans; Male; Middle Aged; Plant Extracts; Plant Leaves; Stevia; Sweetening Agents; Taste

Stevia is currently a well-known plant thanks to its sweeting power. Numerous studies that elucidate its composition were exclusively focused on determination of steviol and its glycosides. Untargeted analysis was applied to obtain a profile of main compounds present in extracts from Stevia (Stevia rebaudiana Bertoni) leaves using LC-MS in high resolution mode with a quadrupole-time of flight analyzer. Eighty-nine compounds were tentatively identified and classified into different families: flavonoids; quinic and caffeic acids and derivatives; diterpenoids (including steviol and glycosides); sesquiterpenoids; amino acids and derivatives; fatty amides and derivatives; fatty acids and derivatives; oligosaccharides; glycerolipids; purines; and retinoids. New steviol glycosides were tentatively identified and their possible structures proposed. Other compounds were tentatively identified in Stevia for the first time, such as fatty acid amides. These results reveal the wide range of compounds present in Stevia, which could be responsible for the nutraceutical effects ascribed to their leaves.

Topics: Chromatography, Liquid; Diterpenes; Diterpenes, Kaurane; Flavonoids; Glucosides; Glycosides; Plant Extracts; Plant Leaves; Stevia; Tandem Mass Spectrometry

Substantial evidence suggests influence of color, physical state, and other extrinsic features on consumer perception and acceptability of food products. In this study, 560 subjects evaluated liking and emotional responses associated with 5 sweeteners (sucralose, stevia, saccharin, aspartame, and sucrose) under 2 eliciting conditions: control (brand name only) and informed (brand name/packet image), to assess impact of the packet color. For a given condition, 5 identical tea samples each labeled with a sweetener type were rated for sweetness and overall liking (9-point) and emotions (5-point). Nonsignificant interactions between eliciting condition and sweetener type were found for liking attributes and emotions (except peaceful), indicating their independent effects. However, overall differences existed among sweetener types and eliciting conditions based on both hedonic and emotional responses (MANOVA, P < 0.05), suggesting modulating effects of packet color on sweetener type in the sensory-emotion space. The sensory-emotion profile for sucrose was separate from that of nonnutritive sweeteners, with statistically significant Mahalanobis distances among sample centroids. Increases in positive emotion intensities contrasted with a decrease in negative emotion intensities were observed for some sweeteners moving from the control to informed condition. Sweetness liking was strongly correlated with the emotion satisfied (sucralose, saccharin) only in the control condition, whereas it was strongly correlated with the emotions pleased and satisfied (stevia), disgusted (aspartame), and satisfied (sucrose) only in the informed condition. Overall, results suggested that sensory liking and emotions during the consumption experience are related not entirely to the type of sweetener, but also the color of the packet.

Topics: Adolescent; Adult; Aspartame; Color; Consumer Behavior; Cues; Diterpenes, Kaurane; Emotions; Female; Food Labeling; Food Packaging; Glucosides; Humans; Male; Non-Nutritive Sweeteners; Perception; Personal Satisfaction; Saccharin; Stevia; Sucrose; Sweetening Agents; Taste; Tea; Young Adult

Sugar is commonly substituted with stevia-based products in food industry and in our daily-life. This substitution results in a change in food product characteristic formula and properties that may affect the growth dynamics of food pathogenic and spoilage bacteria. This work studies the effect of table sugar (TS), laboratory sucrose (LS), commercial stevia (St) and steviol glycosides (SG) on the growth dynamics of Salmonella Typhimurium and Listeria monocytogenes. Experiments were carried out in general and minimal culture media at 3 equivalent concentration levels in terms of sweetness intensity (TS and LS at 3, 9 and 15% (w/v); St at 0.3, 0.9 and 1.5% (w/v); and SG at 0.01, 0.03 and 0.05% (w/v)). Incubation temperatures were: 4, 8 and 20°C for general media, and for minimal media 20°C. To decipher the role of these sweeteners, their concentration evolution in minimal media was determined via HPLC analysis. The results revealed slow maximum specific growth rates (μ

Topics: Chromatography, High Pressure Liquid; Colony Count, Microbial; Culture Media; Dietary Sucrose; Diterpenes, Kaurane; Food Microbiology; Glucosides; Hydrogen-Ion Concentration; Listeria monocytogenes; Models, Theoretical; Non-Nutritive Sweeteners; Salmonella typhimurium; Stevia; Temperature

The use of sweeteners extracted from leaves of the plant species Stevia rebaudiana is increasing worldwide. They are recognized as generally recognized as safe by the US-FDA and approved by EU-European Food Safety Authority, with some recommendation on the daily dosage that should not interfere with glucose metabolism. The results presented here introduce an easy analytical approach for the identification and assay of Stevia sweeteners in commercially available soft drink, based on liquid chromatography coupled to tandem mass spectrometry, using a natural statin-like molecule, Brutieridin, as internal standard. Copyright © 2017 John Wiley & Sons, Ltd.

Topics: Beverages; Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Glucosides; Plant Extracts; Plant Leaves; Stevia; Sweetening Agents; Tandem Mass Spectrometry

Stevioside and rebaudioside A are the chief diterpene glycosides present in the leaves of Stevia rebaudiana. Rebaudioside A imparts a desirable sweet taste, while stevioside produces a residual bitter aftertaste. Enzymatic synthesis of rebaudioside A from stevioside can increase the ratio of rebaudioside A to stevioside in steviol glycoside products, providing a conceivable strategy to improve the organoleptic properties of steviol glycoside products. Here, we demonstrate the efficient conversion of stevioside to rebaudioside A by coupling the activities of recombinant UDP-glucosyltransferase UGT76G1 from S. rebaudiana and sucrose synthase AtSUS1 from Arabidopsis thaliana. The conversion occurred via regeneration of UDP-glucose by AtSUS1. UDP was applicable as the initial material instead of UDP-glucose for UDP-glucose recycling. The amount of UDP could be greatly reduced in the reaction mixture. Rebaudioside A yield in 30 h with 2.4 mM stevioside, 7.2 mM sucrose, and 0.006 mM UDP was 78%.

Topics: Cloning, Molecular; Diterpenes, Kaurane; Escherichia coli; Food Technology; Gene Expression; Glucosides; Glucosyltransferases; Humans; Plant Leaves; Plasmids; Recombinant Proteins; Saccharomyces cerevisiae; Stevia; Sucrose; Sweetening Agents; Taste Perception; Uridine Diphosphate; Uridine Diphosphate Glucose

Rebaudioside A has superior taste quality among the steviol glycosides extracted from Stevia rebaudiana leaves. Given its high purity as a general-purpose sweetener, rebaudioside A has received significant attention and has been widely applied in food and beverages in recent decades. Stevioside is one of the major steviol glycosides and can be converted to rebaudioside A by the uridine-diphosphate dependent glucosyltransferase UGT76G1 in S. rebaudiana. To explore the applicability of and limits in producing rebaudioside A from stevioside through whole-cell biocatalysis, the engineered Saccharomyces cerevisiae expressing UGT76G1, using a newly constructed constitutive expression vector, was used as the whole-cell biocatalyst. Citrate was added to the reaction mixture to allow metabolic regulation when glucose was fed to provide the activated sugar donor UDP-glucose for glycosylation of stevioside in vivo. In an evaluation of the whole-cell reaction parameters involving cell permeability, temperature, pH, citrate and Mg(2+) concentrations, and glucose feeding, production of 1160.5 mg/L rebaudioside A from 2 g/L stevioside was achieved after 48 h without supplementation of extracellular UDP-glucose.

Topics: Catalysis; Diterpenes, Kaurane; Food Additives; Glucosides; Glucosyltransferases; Metabolic Engineering; Saccharomyces cerevisiae; Stevia; Sweetening Agents; Uridine Diphosphate; Uridine Diphosphate Glucose

Rebaudioside-A is the second most abundant sweet diterpene glycoside (1-3%) present in the leaves of Stevia rebaudiana Bertoni, and is now being considered as a possible sucrose substitute due to its pleasant organoleptic properties and associated health benefits. In the present study, a novel in situ enzymatic transglycosylation of stevioside has been developed by pre-treating the stevia leaves with cellulase and adding soluble starch as the glucosyl donor. The results confirm that the transglycosylation of stevioside led to an enrichment in the rebaudioside-A content from 4% to 66%. This was further purified by multiple column chromatography to obtain 95% pure rebaudioside-A. The isolated rebaudioside-A showed concentration-dependent α-glucosidase inhibitory activity with IC50=35.01 μg/ml. Thus the study highlights the biotransformation of stevioside present in stevia leaves to rebaudioside-A by a simple, inexpensive and eco-friendly process that has commercial potential.

Topics: Cellulase; Diterpenes, Kaurane; Food Additives; Glucosides; Plant Leaves; Stevia; Sweetening Agents

Steviol glycosides (SG's) from Stevia rebaudiana (Bertoni) have been used as a natural low-calorie sweeteners. Its aftertaste bitterness restricts its use for human consumption and limits its application in food and pharmaceutical products. In present study, we have performed computational analysis in order to investigate the interaction of two major constituents of SG's against homology model of the hTAS2R4 receptor. Molecular simulation study was performed using stevioside and rebaudioside A revealed that, sugar moiety at the C-3'' position in rebaudioside A causes restriction of its entry into the receptor site thereby unable to trigger the bitter reception signaling cascade. Encouraged by the current finding, we have also developed a greener route using β-1,3-glucanase from Irpex lacteus for the synthesis of de-bittered rebaudioside A from stevioside. The rebaudioside A obtained was of high quality with percent conversion of 62.5%. The results here reported could be used for the synthesis of rebaudioside A which have large application in food and pharmaceutical industry.

Topics: Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Glucan 1,3-beta-Glucosidase; Glucosides; Humans; Receptors, G-Protein-Coupled; Stevia; Sweetening Agents; Taste

The surface and foaming properties of native soy glycinin (11S) and its heat-induced fibrillar aggregates, in the presence of natural surfactant steviol glycoside (STE), were investigated and compared at pH 7.0 to determine the impact of protein structure modification on protein-surfactant interfacial interactions. The adsorption at, and nonlinear dilatational rheological behavior of, the air-water interface were studied by combining drop shape analysis tensiometry, ellipsometry, and large-amplitude oscillatory dilatational rheology. Lissajous plots of surface pressure versus deformation were used to analyze the surface rheological response in terms of interfacial microstructure. The heat treatment generates a mixture of long fibrils and unconverted peptides. The presence of small peptides in 11S fibril samples resulted in a faster adsorption kinetics than that of native 11S. The addition of STE affected the adsorption of 11S significantly, whereas no apparent effect on the adsorption of the 11S fibril-peptide system was observed. The rheological response of interfaces stabilized by 11S-STE mixtures also differed significantly from the response for 11S fibril-peptide-STE mixtures. For 11S, the STE reduces the degree of strain hardening in extension and increases strain hardening in compression, suggesting the interfacial structure may change from a surface gel to a mixed phase of protein patches and STE domains. The foams generated from the mixtures displayed comparable foam stability to that of pure 11S. For 11S fibril-peptide mixtures STE only significantly affects the response in extension, where the degree of strain softening is decreased compared to the pure fibril-peptide system. The foam stability of the fibril-peptide system was significantly reduced by STE. These findings indicate that fibrillization of globular proteins could be a potential strategy to modify the complex surface and foaming behaviors of protein-surfactant mixtures.

Topics: Diterpenes, Kaurane; Elasticity; Globulins; Glucosides; Glycine max; Protein Aggregates; Rheology; Soybean Proteins; Surface Tension; Surface-Active Agents; Viscosity

A method for purifying and preparing rebaudioside A (RA) from steviol glycosides at preparative scale was developed with resin based on hydrophilic interaction liquid chromatography (HILIC). In pure water and acetone-water system, the adsorption capacity and selectivity of five anion resins for RA, rebaudioside C (RC) and stevioside (ST) were examined and discussed. Strongly basic IRA458 with the quaternary amine group was chosen as the resin used for separating and preparing RA. The hydroxide form of IRA458 (IRA458-OH) showed the best results in terms of the adsorption behaviors for RA, RC and ST. The retentions of RA, RC and ST on IRA458-OH resin at high concentration of acetone solution followed HILIC mode, in which retention is probably based on surface adsorption of the resin. Under optimized chromatographic conditions, the pooled purity and yield of RA were up to 98.18 and 98.62%, the relative standard deviations (n = 3) for these two parameters were 1.2 and 5.7%, respectively. The present method has the characteristics of simple, low cost, high purity and high yield. The study will be a promising tool for RA industrialized production and also provides a possible mode for purifying and preparing polar components from their analogs.

Topics: Chemistry Techniques, Analytical; Chromatography; Diterpenes, Kaurane; Glucosides; Hydrophobic and Hydrophilic Interactions

A rapid dialysis method for the analysis of stevioside (SS) and rebaudioside A (RS) in foods was developed. Minced samples (10 g) were packed into 30 cm net length dialysis tubing with 30% methanol to increase the dialysis efficiency. The dialysis tubing was put in a 100 mL centrifuge tube, and the total fluid volume was made up to 100 mL with 30% methanol. Dialysis was done with shaking while heating at 50℃. The dialysis times were reduced from 48-72 hr in the conventional method to 2 hr under these conditions. The dialysate was loaded on a C18 solid- phase extraction cartridge, and the cartridge was washed with 40% methanol. SS and RS were eluted from the cartridge with 80% methanol, and separated by reversed-phase HPLC. Recovery yields (%)of SS and RS, spiked at 0.02 g/kg in various foods, were 83.0-105.1% and the relative standard deviations were mostly less than 5%.

Topics: Chromatography, High Pressure Liquid; Dialysis; Diterpenes, Kaurane; Food Additives; Food Analysis; Glucosides; Solid Phase Extraction; Temperature; Time Factors

Steviol glycosides are currently being used as natural sweeteners by the food industry and Stevia rebaudiana has long been used as a sweet plant in South America for patients suffering from diabetes. In this study, a Stevia rebaudiana ethanolic extract (SREE) was prepared, analysed and tested for antioxidant activity in terms of free radical scavenging properties and antiproliferative effects in cervix (HeLa), pancreatic (MiaPaCa-2) and colonic (HCT116) cancer cells. The antiproliferative mechanism was confirmed by testing the effects on cyclin D1-CDK4. Bioassays were also performed for the diterpene glycoside stevioside. Our results demonstrate that the extract acts as an antioxidant being able to scavenge free radicals, but this activity was not due to stevioside. The extract also induced cell death in the three cell lines, being more active against cervix cancer cells (HeLa); however, the concentration of stevioside needed to produce antiproliferative effects was higher than the amount of steviol glycosides found in a lower dose of extract inducing cell death. In addition, the extract clearly inhibited CDK4 whereas stevioside did not, concluding that the antiproliferative activity of stevia may be due to inhibition of cyclin-dependent kinases performed by other compounds of the extract.

Topics: Antioxidants; Cell Line, Tumor; Cell Proliferation; Diterpenes, Kaurane; Glucosides; Humans; Neoplasms; Plant Extracts; Plant Leaves; Stevia

Stevia (Stevia rebaudiana Bertoni) is a medicinal plant having sweet, diterpenoid glycosides known as steviol glycosides which are 200-300 times sweeter than sucrose (0.4 % solution). They are synthesized mainly in the leaves via plastid localized 2-C-methyl-D-erythrose-4-phosphate pathway (MEP pathway). Fifteen genes are involved in the formation of these glycosides. In the present protocol, a method for the quantification of transcripts of these genes is shown. The work involves RNA extraction and cDNA preparation, and therefore, procedures for the confirmation of DNA-free cDNA preparation have also been illustrated. Moreover, details of plant treatments are not mentioned as this protocol may apply to relative gene expression profile in any medicinal plant with any treatment. The treatments are numbered as T0 (Control), T1, T2, T3, and T4.

Topics: Biosynthetic Pathways; Diterpenes, Kaurane; DNA, Complementary; DNA, Plant; Gene Expression; Genes, Plant; Glucosides; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Plant; Secondary Metabolism; Stevia; Transcriptome

Stevioside is one of the most important food additives that has become well known for its sweetness. The aim of this study was to evaluate the impact of Stevioside on the heart.. 4-day-old embryonated chickens eggs were inoculated with Stevioside and kept until hatching. Shortly after, the heart tissue samples were taken to examine organ Oxidative stresses by measuring Malondialdehyde (MDA) and glutathione (GSH) levels, ferric reducing /antioxidant power (FRAP) and cupric ion reducing assay (CUPRIC).. There was no significant difference in glutathione level, lipid peroxidation, FRAP, and cupric assay.. It was suggested that stevioside did not cause marked damages to heart tissues in chicken embryo model.

Topics: Animals; Chick Embryo; Chickens; Diterpenes, Kaurane; Glucosides; Glutathione; Heart; Lipid Peroxidation; Malondialdehyde; Oxidative Stress; Stevia; Sweetening Agents

Stevia is one of the sweeteners with the greatest consumer demand because of its natural origin and minimal calorie content. Steviol glycosides (SG) are the main active compounds present in the leaves of Stevia rebaudiana and are responsible for its sweetness. However, recent in vitro studies in HEK 293 cells revealed that SG specifically activate the hT2R4 and hT2R14 bitter taste receptors, triggering this mouth feel. The objective of this study was to characterize the interaction of SG with these two receptors at the molecular level. The results showed that SG have only one site for orthosteric binding to these receptors. The binding free energy (ΔG

Topics: Binding Sites; Diterpenes, Kaurane; Glucosides; Glycosides; Humans; Molecular Dynamics Simulation; Receptors, G-Protein-Coupled; Stevia; Sweetening Agents

The Stevia rebaudiana plant is likely to become a major source of high-potency sweetener for the growing natural-food market. S. rebaudiana is the source of a number of sweet diterpenoid glycosides, but the major sweet constituents are rebaudioside A and stevioside. These two constituents have similar pharmacokinetic and metabolic profiles in rats and humans, and thus, studies carried out with either steviol glycoside are relevant to both. Other studies illustrate the diversity of voluntary sweet intake in mammals.. This study was done using a series of two-bottle tests that compared a wide range of sweetener concentrations versus saccharin concentrations and versus water.. Wistar rats displayed preferences for stevia extract and pure rebaudioside A solutions over water at a range of concentrations (0.001% to 0.3%), and their intake peak occurred at 0.1% concentration. They also preferred solutions prepared with a commercial rebaudioside A plus erythritol mixture to water, and their peak was at 2% concentration.. The present study provides new information about the responses of Wistar rats to stevia compounds and commercial stevia products such as Truvia. These results could help with the appropriate dosage selection for focused behavioral and physiological studies on stevia.

Topics: Animals; Diterpenes, Kaurane; Female; Food Preferences; Glucosides; Plant Extracts; Rats; Rats, Wistar; Stevia; Sweetening Agents

The glucosyltransferase UGT76G1 from Stevia rebaudiana is a chameleon enzyme in the targeted biosynthesis of the next-generation premium stevia sweeteners, rebaudioside D (Reb D) and rebaudioside M (Reb M). These steviol glucosides carry five and six glucose units, respectively, and have low sweetness thresholds, high maximum sweet intensities and exhibit a greatly reduced lingering bitter taste compared to stevioside and rebaudioside A, the most abundant steviol glucosides in the leaves of Stevia rebaudiana.. In the metabolic glycosylation grid leading to production of Reb D and Reb M, UGT76G1 was found to catalyze eight different reactions all involving 1,3-glucosylation of steviol C. Screening of the mutant library identified mutations with positive impact on the accumulation of Reb D and Reb M. The effect of the introduced mutations on other reactions in the metabolic grid was characterized. This screen made it possible to identify variants, such as UGT76G1

Topics: Amino Acid Sequence; Diterpenes, Kaurane; Glucosides; Glycosyltransferases; Stevia; Sweetening Agents

There is a close interaction between Type 2 Diabetes, obesity and liver disease. We have studied the effects of the two most abundant Stevia-derived steviol glycosides, stevioside and rebaudioside A, and their aglycol derivative steviol on liver steatosis and the hepatic effects of lipotoxicity using a mouse model of obesity and insulin resistance. We treated ob/ob and LDLR-double deficient mice with stevioside (10 mg⋅kg(-1)⋅day-1 p.o., n = 8), rebaudioside A (12 mg⋅kg(-1)⋅day-1 p.o., n = 8), or steviol (5 mg⋅kg(-1)⋅day(-1) p.o., n = 8). We determined their effects on liver steatosis and on the metabolic effects of lipotoxicity by histological analysis, and by combined gene-expression and metabolomic analyses. All compounds attenuated hepatic steatosis. This could be explained by improved glucose metabolism, fat catabolism, bile acid metabolism, and lipid storage and transport. We identified PPARs as important regulators and observed differences in effects on insulin resistance, inflammation and oxidative stress between Stevia-derived compounds. We conclude that Stevia-derived compounds reduce hepatic steatosis to a similar extent, despite differences in effects on glucose and lipid metabolism, and inflammation and oxidative stress. Thus our data show that liver toxicity can be reduced through several pathophysiological changes. Further identification of active metabolites and underlying mechanisms are warranted.

Topics: Amino Acids; Animals; Bile Acids and Salts; Disease Models, Animal; Diterpenes, Kaurane; Fatty Liver; Glucose; Glucosides; Glutathione; Insulin Resistance; Lipid Metabolism; Liver; Male; Metabolomics; Mice; Mice, Obese; Obesity; Oxidative Stress; Peroxisome Proliferator-Activated Receptors; Plant Preparations; Stevia; Transcriptome

Stevia rebaudiana (Bertoni) produces steviol glycosides (SGs)--stevioside (stev) and rebaudioside-A (reb-A) that are valued as low calorie sweeteners. Inoculation with arbuscular mycorrhizal fungi (AMF) augments SGs production, though the effect of this interaction on SGs biosynthesis has not been studied at molecular level. In this study transcription profiles of eleven key genes grouped under three stages of the SGs biosynthesis pathway were compared. The transcript analysis showed upregulation of genes encoding 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway enzymes viz.,1-deoxy-D-xylulose 5-phospate synthase (DXS), 1-deoxy-D-xylulose 5-phospate reductoisomerase (DXR) and 2-C-methyl-D-erytrithol 2,4-cyclodiphosphate synthase (MDS) in mycorrhizal (M) plants. Zn and Mn are imperative for the expression of MDS and their enhanced uptake in M plants could be responsible for the increased transcription of MDS. Furthermore, in the second stage of SGs biosynthesis pathway, mycorrhization enhanced the transcription of copalyl diphosphate synthase (CPPS) and kaurenoic acid hydroxylase (KAH). Their expression is decisive for SGs biosynthesis as CPPS regulates flow of metabolites towards synthesis of kaurenoid precursors and KAH directs these towards steviol synthesis instead of gibberellins. In the third stage glucosylation of steviol to reb-A by four specific uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) occurs. While higher transcription of all the three characterized UGTs in M plants explains augmented production of SGs; higher transcript levels of UGT76G1, specifically improved reb-A to stev ratio implying increased sweetness. The work signifies that AM symbiosis upregulates the transcription of all eleven SGs biosynthesis genes as a result of improved nutrition and enhanced sugar concentration due to increased photosynthesis in M plants.

Topics: Diterpenes, Kaurane; Erythritol; Gene Expression Regulation, Plant; Genes, Plant; Glucosides; Glycosides; Glycosyltransferases; Manganese; Mycorrhizae; Photosynthesis; Plant Proteins; Stevia; Sugar Phosphates; Sweetening Agents; Symbiosis; Transcription, Genetic; Uridine Diphosphate; Zinc

Stevia products are advertised as a zero-calorie sweetener. Glucose should not be an intrinsic component of this product, but it has been identified from some of stevia products in a preliminary study. An UHPLC-UV method was developed for the quantitative determination of glucose from stevia products. After stevia products reacted with 1-phenyl-3-methyl-5-pyrazolone (PMP), PMP derivatives were analysed and glucose was found in seven out of 35 products in the range 0.3-91.5% (w/w). Two products, SPR-12 and SPR-27, showed remarkable amounts of glucose at 61.6% and 91.5%, respectively. In addition, an UHPLC-UV-evaporative light-scattering detector (ELSD) method was developed for the quantitative determination of rebaudioside A, stevioside, rebaudioside D, dulcoside A and steviolbioside from Stevia rebaudiana and related products. In a 12 min run, five steviol glycosides were baseline-separated. ELSD and ultraviolet (UV) detections showed comparable results. The LC methods were validated for linearity, repeatability, accuracy, limits of detection (LOD) and limits of quantification (LOQ). For steviol glycosides, the LODs and LOQs were found to be less than 10 and 30 μg ml(-1), respectively. The RSD for intra- and inter-day analyses was less than 2.5%, and the recovery was 90-94%. For PMP derivative of glucose, the LOD and LOQ were 0.01 and 0.05 μg ml(-1), respectively. Repeatability (RSD) was less than 2.6%; recovery was 98.6-101.7%. The methods are useful for the identification, quality assurance, and adulterant assessment of S. rebaudiana and steviol glycosides sweeteners (stevia products).

Topics: Antipyrine; Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Edaravone; Food Analysis; Food Contamination; Glucose; Glucosides; Glycosides; Limit of Detection; Non-Nutritive Sweeteners; Reproducibility of Results; Sensitivity and Specificity; Stevia

Stevioside is a non-caloric, natural, high-intensity sweetener. However, the bitter aftertaste of stevioside restricts its utilization for human consumption and limits its application in the food industry. In this study, a high efficiency enzymatic modification system was investigated to improve stevioside taste quality. A cyclodextrin glucanotransferase (CGTase) producing strain Paenibacillus sp. CGMCC 5316 was isolated from Stevia planting soil. With starch as glycosyl donor, this CGTase can transform stevioside into a single specific product which is an isomer of rebaudioside A and identified as mono-glycosylated stevioside. The taste of stevioside is improved noticeably by generating mono-glycosylated stevioside, which possesses a sucrose-like taste and has sweetness increased significantly by 35.4%. Next, the parameters influencing CGTase production were optimized. Compared to initial conditions, CGTase activity increased by 214.7% under optimum conditions of 3.9 g/L starch, 17.9 g/L tryptone, and 67.6 h of culture time, and the transglycosylation rate of stevioside was remarkably increased by 284.8%, reaching 85.6%. This CGTase modification system provides a promising solution for improving the sweetness and taste quality of stevioside. The efficiency of CGTase transformation can be greatly increased by optimizing the culture conditions of Paenibacillus sp. CGMCC 5316.

Topics: Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Glucosides; Glucosyltransferases; Glycosylation; Mass Spectrometry; Paenibacillus; Starch; Sweetening Agents

Following the approval of steviol glycosides as a food additive in Europe in December 2011, large-scale stevia cultivation will have to be developed within the EU. Thus there is a need to increase the efficiency of stevia evaluation through germplasm enhancement and agronomic improvement programs. To address the need for faster and reproducible sample throughput, conditions for automated extraction of dried stevia leaves using Accelerated Solvent Extraction were optimised. A response surface methodology was used to investigate the influence of three factors: extraction temperature, static time and cycle number on the stevioside and rebaudioside A extraction yields. The model showed that all the factors had an individual influence on the yield. Optimum extraction conditions were set at 100 °C, 4 min and 1 cycle, which yielded 91.8% ± 3.4% of total extractable steviol glycosides analysed. An additional optimisation was achieved by reducing the grind size of the leaves giving a final yield of 100.8% ± 3.3%.

Topics: Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Europe; Glucosides; Models, Theoretical; Plant Extracts; Plant Leaves; Reproducibility of Results; Solvents; Stevia

Steviol glycosides (SVglys) and gibberellins are originated from the shared biosynthesis pathway in Stevia (Stevia rebaudiana Bertoni). In this research, two experiments were conducted to study the opposing effects of external gibberellin (GA3) and Daminozide (a gibberellin inhibitor) on Stevia growth and metabolites. Results showed that GA3 significantly increased the stem length and stem dry weight in Stevia. Total soluble sugar content increased while the SVglys biosynthesis was decreased by external GA3 applying in Stevia leaves. In another experiment, the stem length was reduced by Daminozide spraying on Stevia shoots. The Daminozide did not affect the total SVglys content, while in 30 ppm concentration, significantly increased the soluble sugar production in Stevia leaves. Although the gibberellins biosynthesis pathway has previously invigorated in Stevia leaf, the Stevia response to external gibberellins implying on high precision regulation of gibberellins biosynthesis in Stevia and announces that Stevia is able to kept endogenous gibberellins in a low quantity away from SVglys production. Moreover, the assumption that the internal gibberellins were destroyed by Daminozide, lack of Daminozide effects on SVglys production suggests that gibberellins biosynthesis could not act as a competitive factor for SVglys production in Stevia leaves.

Topics: Diterpenes, Kaurane; Gibberellins; Glucosides; Plant Growth Regulators; Plant Leaves; Plant Stems; Stevia; Succinates; Sweetening Agents

New stevia amino acid sweeteners, stevia glycine ethyl ester (ST-GL) and stevia l-alanine methyl ester (ST-GL), were synthesised and characterised by IR, NMR ((1)H NMR and (13)C NMR) and elemental analysis. The purity of the new sweeteners was determined by HPLC and their sensory properties were evaluated relative to sucrose in an aqueous system. Furthermore, the stevia derivatives (ST-GL and ST-AL) were evaluated for their acute toxicity, melting point, solubility and heat stability. The novel sweeteners were stable in acidic, neutral or basic aqueous solutions maintained at 100 °C for 2 h. The sweetness intensity rate of the novel sweeteners was higher than sucrose. Stevia amino acid (ST-GL and ST-AL) solutions had a clean sweetness taste without bitterness when compared to stevioside. The novel sweeteners can be utilised as non-caloric sweeteners in the production of low-calorie food.

Topics: Amino Acids; Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Glucosides; Glycine; Spectrum Analysis; Stevia

This report describes a fragment-based approach to the examination of congeneric organic compounds by NMR spectroscopy. The method combines the classic interpretation of 1D- and 2D-NMR data sets with contemporary computer-assisted NMR analysis. Characteristic NMR profiles of key structural motifs were generated by (1)H iterative full spin analysis and then joined together as building blocks to recreate the (1)H NMR spectra of increasingly complex molecules. To illustrate the methodology described, a comprehensive analysis of steviol (1), seven steviol glycosides (2-8) and two structurally related isosteviol compounds (9, 10) was carried out. The study also assessed the potential impact of this method on relevant aspects of natural product research including structural verification, chemical dereplication, and mixture analysis.

Topics: Diterpenes, Kaurane; Glucosides; Glycosides; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Stevia

In order to determine the impact of Stevia rebaudiana (SR) addition on bioactive compounds bioaccessibility of a new developed functional beverage based on exotic fruits (mango juice, papaya juice and açaí) mixed with orange juice and oat, an in vitro gastrointestinal digestion was performed. Ascorbic acid, total carotenoids, total phenolics, total anthocyanins, total antioxidant capacity and steviol glycosides were evaluated before and after a simulated gastrointestinal digestion. Salivary and gastric digestion had no substantial effect on any of the major phenolic compounds, ascorbic acid, total antioxidant capacity and steviol glycosides, whereas carotenoids and anthocyanins diminished significantly during the gastric step. All analysed compounds were significantly altered during the pancreatic-bile digestion and this effect was more marked for carotenoids and total anthocyanins. However, phenolic compounds, anthocyanins, total antioxidant capacity and steviol glycosides bioaccessibility increased as did SR concentration. Ascorbic acid bioaccessibility was negatively affected by the SR addition.

Topics: Anthocyanins; Antioxidants; Ascorbic Acid; Avena; Beverages; Carotenoids; Digestion; Diterpenes, Kaurane; Fruit; Glucosides; Humans; Phenols; Stevia

The aim of our study was to determine the antioxidant activities, cytotoxicity and proliferative properties in Stevia rebaudiana leaves and stems. Leaves extracts exhibited a higher antioxidant activity than stems extract, through oxygen radical absorbance capacity (ORAC) and cellular antioxidant activity (CAA) assays. Stevioside and rebaudioside A, the main sweetening metabolites in stevia leaves, exhibited a low ORAC value in comparison with plant extracts, while did not elicit any CAA. Stevia rebaudiana did not exhibit toxicity against HepG2 (hepatocellular carcinoma) human cells. No proliferative nor catalase modulations were observed in cells treated with such extracts. Our findings support the promising role of stevia that, apart from its sweetness, can act as a source of antioxidants, even at the intracellular level. This activity makes S. rebaudiana crude extract an interesting resource of natural sweetness with antioxidant properties which may find numerous applications in foods and nutritional supplements industries.

Topics: Antioxidants; Diterpenes, Kaurane; Glucosides; Hep G2 Cells; Humans; Plant Extracts; Plant Leaves; Plant Stems; Stevia; Sweetening Agents

Docking studies were performed on natural sweeteners from Stevia rebaudiana by constructing homology models of T1R2 and T1R3 subunits of human sweet taste receptors. Ramachandran plot, PROCHECK results and ERRAT overall quality factor were used to validate the quality of models. Furthermore, docking results of steviol glycosides (SG's) were correlated significantly with data available in the literature which enabled to predict the exact sweetness rank order of SG's. The binding pattern indicated that Asn 44, Ans 52, Ala 345, Pro 343, Ile 352, Gly 346, Gly 47, Ala 354, Ser 336, Thr 326 and Ser 329 are the main interacting amino acid residues in case of T1R2 and Arg 56, Glu 105, Asp 215, Asp 216, Glu 148, Asp 258, Lys 255, Ser 104, Glu 217, Leu 51, Arg 52 for T1R3, respectively. Amino acids interact with SG's mainly by forming hydrogen bonds with the hydroxyl group of glucose moieties. Significant variation in docked poses of all the SG's were found. In this study, we have proposed the mechanism of the sweetness of the SG's in the form of multiple point stimulation model by considering the diverse binding patterns of various SG's, as well as their structural features. It will give further insight in understanding the differences in the quality of taste and will be used to improve the taste of SG's using semi-synthetic approaches.

Topics: Amino Acids; Diterpenes, Kaurane; Glucosides; Humans; Models, Biological; Receptors, G-Protein-Coupled; Stevia; Sweetening Agents; Taste

The defective responsiveness of body tissues to insulin involves the insulin receptors of cell membranes. The binding of insulin to its receptor induce an increase of high affinity glucose transporter molecules in target cell surface that enhance the uptake of glucose in to these cells. The WHO expert committee recommended the importance to investigate the hypoglycemic agents from plant origin, which are used in traditional medicine for the treatment of diabetes. Stevioside, a natural sweetener and a diterpene glycoside extracted from Stevia rebaudiana (Bertoni) has been used as an anti-hyperglycemic agent for the treatment of diabetes for decades.. To reveal the molecular mechanism underlying the insulinomimetic activity of stevioside and its aglycone metabolite, steviol using cell line models.. Efficacy of stevioside and steviol in inducing glucose absorption was studied at transcript level, protein level and by measuring glucose absorption in the cell using in-vitro cell line studies.. Quantification of glucose transporter (GLUT4) transcript was done in 3T3-L1 adipocytes and L6 myotubes by qPCR using RPL23 as the internal control. GLUT4 protein was quantified using anti GLUT4 antibody by ELISA and radioactive glucose uptake studies were done to measure the rate of glucose absorption.. The absolute and relative quantitation of GLUT4 gene by qPCR showed the activation of GLUT4 transcript at lower concentration of steviol (1 µM) and higher concentration of stevioside (100 µM) in both L6 myotubes and 3T3-L1 adipocytes. The increased level of glut4 protein and the glucose uptake in both the cell lines using the same concentration of steviol and stevioside further supports the qPCR data. The copy number and the expression level of GLUT4 gene, the amount of GLUT4 protein and the glucose uptake efficacy support the insulinomimetic effect of steviol and stevioside.. The results of the study clearly demonstrate the functional similarity of steviol and stevioside with that of insulin in controlling the level of glucose in both the cell lines. In other words, the insulinomimetic property of stevioside and steviol was evident from the data.

Topics: 3T3-L1 Cells; Adipocytes; Animals; Cell Differentiation; Diterpenes, Kaurane; Glucose; Glucose Transporter Type 4; Glucosides; Hypoglycemic Agents; Insulin; Mice; Myoblasts; Rats; Stevia; Sweetening Agents

Stevia (Stevia rebaudiana) produces not only a group of diterpenoid glycosides known as steviol glycosides (SGs), but also other labdane-type diterpenoids that may be spatially separated from SGs. However, their biosynthetic routes and spatial distribution in leaf tissues have not yet been elucidated. Here, we integrate metabolome and transcriptome analyses of Stevia to explore the biosynthetic capacity of leaf tissues for diterpenoid metabolism. Tissue-specific chemical analyses confirmed that SGs were accumulated in leaf cells but not in trichomes. On the other hand, Stevia leaf trichomes stored other labdane-type diterpenoids such as oxomanoyl oxide and agatholic acid. RNA sequencing analyses from two different tissues of Stevia provided a comprehensive overview of dynamic metabolic activities in trichomes and leaf without trichomes. These metabolite-guided transcriptomics and phylogenetic and gene expression analyses clearly identified specific gene members encoding enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate pathway and the biosynthesis of steviol or other labdane-type diterpenoids. Additionally, our RNA sequencing analysis uncovered copalyl diphosphate synthase (SrCPS) and kaurene synthase1 (SrKS1) homologs, SrCPS2 and KS-like (SrKSL), which were specifically expressed in trichomes. In vitro and in planta assays showed that unlike SrCPS and SrKS1, SrCPS2 synthesized labda-13-en-8-ol diphosphate and successively catalyzed the formation of manoyl oxide and epi-manoyl oxide in combination with SrKSL. Our findings suggest that Stevia may have evolved to use distinct metabolic pathways to avoid metabolic interferences in leaf tissues for efficient production of diverse secondary metabolites.

Topics: Alkyl and Aryl Transferases; Base Sequence; Diterpenes; Diterpenes, Kaurane; Erythritol; Glucosides; Metabolome; Molecular Sequence Data; Mutation; Organ Specificity; Phylogeny; Plant Leaves; Plant Proteins; Sequence Analysis, RNA; Stevia; Sugar Phosphates; Transcriptome; Trichomes

The ideal sucrose concentration and equivalent concentrations of the stevia, sucralose, aspartame, and neotame in chocolate milk with chia oil as well as the dynamic behavior of certain sensory attributes were investigated using a time-intensity methodology. The use of just-about-right (JAR) identified an ideal sucrose concentration of 9% (w/w). In addition, the magnitude estimation method showed that stevia had the lowest sweetness power whereas neotame presented the highest. Furthermore, the time-intensity analysis indicated that there was no significant change between the maximum intensities of the sweetness for any evaluated sweeteners. In general, the desired sensory profile and some economic considerations are decisive on the choice of which sweetener is better to be used in chocolate milk formulation added with chia oil.

Topics: Animals; Aspartame; Cacao; Dietary Sucrose; Dipeptides; Diterpenes, Kaurane; Flavoring Agents; Food Handling; Glucosides; Humans; Milk; Plant Oils; Salvia; Stevia; Sucrose; Sweetening Agents; Taste

Use of stevia-derived sweeteners was recently officially approved by the European Commission, and their application in the food industry has increased, especially in functional foods. However, there are scarce data about the influence of stevia on probiotic bacteria, which are important both as an inhabitant of the human gut and as a functional food additive. Taking into consideration the broad application of Lactobacillus reuteri in functional foods, the aim of the research was to evaluate the influence of stevia glycosides on its growth. Six Lact. reuteri strains were tested for their ability to grow in the presence of stevioside and rebaudioside A (0·2-2·6 g l(-1) ). The effect of stevia glycosides on biomass concentration, cell count, pH and lactic and acetic acid synthesis was analysed. Both glycosides impaired the growth of analysed strains. However, the inhibitory effect was strain specific, and the concentration-dependent effect was not observed for all parameters. The most pronounced concentration-dependent effect was on lactic and acetic acid production. Taking into account the observed strain-specific inhibitory effect of stevia glycosides, it could be suggested to evaluate the influence of them on each strain employed before their simultaneous application in functional foods.. The study showed that the growth of Lactobacillus reuteri strains was inhibited in the presence of stevia sweeteners stevioside and rebaudioside A. Probiotics, for example Lact. reuteri strains, are often used as functional additives in health foods and are an important natural inhabitant of the human gastrointestinal tract. Stevia glycosides application in food is increasing; yet, there are no data about the influence of stevia glycosides on Lact. reuteri growth and very few data on growth of other lactobacilli, either in probiotic foods or in the gastrointestinal tract. This research shows that it is necessary to evaluate the influence of stevia glycosides on other groups of micro-organisms in further research.

Topics: Diterpenes, Kaurane; Glucosides; Limosilactobacillus reuteri; Stevia; Sweetening Agents

The adsorptive separation of each steviol glycoside from aqueous solutions by polymeric adsorbents has attracted a lot of interest in recent years. The adsorption properties of chloromethylated cross-linked poly(styrene-co-divinylbenzene) macroporous resins, functionalised with chloromethyl, amino and phenylboronic acid groups, towards rebaudioside A and stevioside were studied. The results revealed that the resins with amino and phenylboronic acid groups preferred to adsorb stevioside rather than rebaudioside A, and their adsorption kinetics fitted a pseudo-second-order model. Isothermal equilibrium curves of rebaudioside A and stevioside showed a good fitness with the Langmuir and Freundlich models. The adsorption of rebaudioside A and stevioside onto resins was a spontaneous and exothermic process as indicated by the negative values in free energy and enthalpy. Results from the resin-packed column demonstrated that an effluent rich in rebaudioside A (purity 98%) was obtained prior to the breakthrough point of stevioside.

Topics: Adsorption; Boronic Acids; Diterpenes, Kaurane; Glucosides; Hydrogen-Ion Concentration; Kinetics; Polystyrenes; Sweetening Agents

The purpose of this research was to apply Aspergillus aculeatus solid fermentation extracts to convert stevioside and rebaudioside C, followed by identifying and purifying the new conversion product.. The product was identified by high performance liquid chromatography, chromatography-mass spectrometry and Infrared spectrum. The new product and rebaudioside A exited in the supernatant were purified by alcohol and macroporous resin.. The Aspergillus aculeatus enzyme extracts could convert the stevioside and rebaudioside C to the new product within 10 hours. The conversion rate was 98.0% in 24 hours. The conversion product existed in deposit was identified as steviol. The purity and recovery percent of steviol were 95.2% and 84.0% respectively. Because stevioside could occur to deposit, the rebaudioside A existed in supernatant was purified easily. We used the resin chromatography to purify RA and the recovery could reach 80.5%.. Aspergillus aculeatus enzyme extracts could convert stevioside efficiently and specifically, and we could obtain rebaudioside A and steviol at the same time.

Topics: Aspergillus; Biotransformation; Diterpenes, Kaurane; Fermentation; Glucosides

The objective of the present study is to develop a method for rapid determination of the content of stevioside (ST) and rebaudioside A (RA) in Stevia Rebaudiana leaves. One hundred and five samples of stevia from different areas containing ST of 0.27%-1.40% and RA of 0.61%-3.98% were used. The 105 groups' NIRS diagram was processed by different methods including subtracting a straight line (SLS), multiplicative scatter correction (MSC), first derivative (FD), second derivative (SD) and so on, and then all data were analyzed by partial least square (PLS). The study showed that SLS can be used to extracted spectra information thoroughly to analyze the contents of ST, the correlation coefficients of calibration (Re), the root-mean-square errors of calibration (RMSEC) and prediction (RMSEP), and the residual predictive deviation (RPD) were 0.986, 0.341, 1.00 and 2.8, respectively. The correlation coefficients of RA was 0.967, RMSEC was 1.50, RMSEP was 1.98 and RPD was 4.17. The results indicated that near infrared spectroscopy (NIRS) technique offers effective quantitative capability for ST and RA in Stevia Rebaudiana leaves. Then the model of stevia dried leaves was used to compare with the stevia powder near infrared model whose correlation coefficients of ST was 0.986, RMSEC was 0.32, RMSEP was 0.601 and RPD was 2.86 and the correlation coefficients of RA was 0.968, RMSEC was 1.50, RMSEP was 1.48 and RPD was 4.2. The result showed that there was no significant difference between the model of dried leaves and that of the powders. However, the dried leaves NIR model reduces the unnecessary the steps of drying and grinding in the actual detection process, saving the time and reducing the workload.

Topics: Calibration; Diterpenes, Kaurane; Glucosides; Least-Squares Analysis; Plant Leaves; Spectroscopy, Near-Infrared; Stevia; Sweetening Agents

With the mainstream emergence of natural sweeteners such as stevia, which is available in different commercial formulations, suitability for yogurt needs to be validated. The present study aimed to determine the appropriate concentration level of 3 processed stevia sweeteners/supplements in commercial plain low-fat yogurt flavored with natural vanilla. Three different levels of sucrose, aspartame, an erythritol and 95% rebaudiana A stevia sweetener, a 95% pure mix of maltodextrin and steviol glycosides, and a cold water stevia extract were used in the study. The just-about-right level for each sweetener and consumer acceptability of each naturally flavored low-fat vanilla yogurt were evaluated. Results from penalty analysis demonstrated that only 0.7% of stevia containing maltodextrin and 95% steviol glycoside was necessary, whereas higher levels (between 4.0 to 5.5%) were more appropriate for stevia containing erythritol and 95% rebaudiana A or cold water extract of stevia, respectively. The concentrations of stevia sweeteners used influenced the perceived sweetness and sourness. In general, consumers disliked the yogurt sweetened with stevia or aspartame, and neither disliked nor liked the yogurt sweetened with sucrose, which was largely driven by perceived sourness of the base yogurt. The findings underline the importance of careful selection of stevia type and concentration as well as optimizing yogurt cultures and fermentation conditions before product launch.

Topics: Aspartame; Diterpenes, Kaurane; Fermentation; Glucosides; Stevia; Sucrose; Sweetening Agents; Taste; Vanilla; Yogurt

Two new diterpene glycosides in addition to five known glycosides have been isolated from a commercial extract of the leaves of Stevia rebaudiana. Compound 1 (rebaudioside KA) was shown to be 13-[(O-β-d-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid 2-O-β-d-glucopyranosyl-β-d-glucopyranosyl ester and compound 2, 12-α-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester. Five additional known compounds were identified, rebaudioside E, rebaudioside M, rebaudioside N, rebaudioside O, and stevioside, respectively. Enzymatic hydrolysis of stevioside afforded the known ent-kaurane aglycone 13-hydroxy-ent-kaur-16-en-19-oic acid (steviol) (3). The isolated metabolite 1 possesses the ent-kaurane aglycone steviol (3), while compound 2 represents the first example of the isomeric diterpene 12-α-hydroxy-ent-kaur-16-en-19-oic acid existing as a glycoside in S. rebaudiana. The structures of the isolated metabolites 1 and 2 were determined based on comprehensive 1D- and 2D-NMR (COSY, HSQC, and HMBC) studies. A high-quality crystal of compound 3 has formed, which allowed the acquisition of X-ray diffraction data that confirmed its structure. The structural similarities between the new metabolites and the commercially available stevioside sweeteners suggest the newly isolated metabolites should be examined for their organoleptic properties. Accordingly rebaudiosides E, M, N, O, and KA have been isolated in greater than gram quantities.

Topics: Diterpenes, Kaurane; Glucosides; Minnesota; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Plant Leaves; Stevia; Sweetening Agents

Stevia (Stevia rebaudiana) is an important medicinal plant that yields diterpenoid steviol glycosides (SGs). SGs are currently used in the preparation of medicines, food products and neutraceuticals because of its sweetening property (zero calories and about 300 times sweeter than sugar). Recently, some progress has been made in understanding the biosynthesis of SGs in Stevia, but little is known about the molecular mechanisms underlying this process. Additionally, the genomics of Stevia, a non-model species, remains uncharacterized. The recent advent of RNA-Seq, a next generation sequencing technology, provides an opportunity to expand the identification of Stevia genes through in-depth transcript profiling.. We present a comprehensive landscape of the transcriptome profiles of three genotypes of Stevia with divergent SG compositions characterized using RNA-seq. 191,590,282 high-quality reads were generated and then assembled into 171,837 transcripts with an average sequence length of 969 base pairs. A total of 80,160 unigenes were annotated, and 14,211 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. Gene sequences of all enzymes known to be involved in SG synthesis were examined. A total of 143 UDP-glucosyltransferase (UGT) unigenes were identified, some of which might be involved in SG biosynthesis. The expression patterns of eight of these genes were further confirmed by RT-QPCR.. RNA-seq analysis identified candidate genes encoding enzymes responsible for the biosynthesis of SGs in Stevia, a non-model plant without a reference genome. The transcriptome data from this study yielded new insights into the process of SG accumulation in Stevia. Our results demonstrate that RNA-Seq can be successfully used for gene identification and transcript profiling in a non-model species.

Topics: Diterpenes, Kaurane; Genotype; Glucosides; Glucosyltransferases; Metabolic Networks and Pathways; Microsatellite Repeats; Plant Leaves; Plant Proteins; Polymorphism, Single Nucleotide; RNA, Plant; Sequence Analysis, RNA; Stevia; Transcriptome

A high level of sweetness and health-promoting properties make steviol glycosides an interesting alternative to sugars or artificial sweeteners. The radical oxygen species scavenging activity of these compounds may influence the stability of labile particles present in food. Model buffer solutions containing steviol glycosides, a selected food antioxidant (vitamin C or anthocyanins), and preservative were analyzed during storage. The addition of steviol glycosides at concentrations of 50, 125, and 200 mg/L increased the stability of both ascorbic and dehydroascorbic acid (degradation rates decreased up to 3.4- and 4.5-fold, respectively); the effect was intensified by higher sweetener concentrations and higher acidity of the solutions. Glycosides used alone did not affect the stability of anthocyanins; however, they enhanced the protective effect of sugars; half-life times increased by ca. 33% in the presence of sucrose (100 g/L) and by ca. 52% when both sucrose (100 g/L) and glycosides (total 200 mg/L) were used. Steviol glycosides concentrations remained stable during experiments.

Topics: Anthocyanins; Ascorbic Acid; Diterpenes, Kaurane; Drug Stability; Glucosides; Half-Life; Plant Extracts; Stevia

Stevia rebaudiana Bertoni (Asteraceae) is an important medicinal plant and is much used due to its zero calories sweetening property. Stevia leaves as well as its extracts and pure compounds are currently used in the preparation of several medicines, food products and neutraceuticals.. To study the genetic and metabolic variability in S. rebaudiana among accessions of different geographical regions of India using random amplified polymorphic DNA (RAPD) markers and high-performance thin layer chromatography (HPTLC) analysis.. The RAPD analysis of Stevia rebaudiana (11 accessions) was carried out using 20 random operon primers. Dendrogram was constructed for cluster analysis based on the unweighted pair group method with arithmetic means (UPGMA) using Winboot. The HPTLC analysis of all samples was carried out on silica using acetone:ethyl acetate:water (5:4:1, v/v/v) for fingerprinting and quantification of stevioside and rebaudioside A at 360 nm after spraying with anisaldehyde sulphuric acid.. Ten out of 20 primers screened were found most informative; amplification products of the genotypes yielded a total of 87 scorable bands (67 polymorphic), whereas genetic similarity (GS) coefficient (0.01-0.08) and polymorphism (67.24-92.40%) showed huge variability. Similarly, HPTLC analysis showed large variation among different samples with respect to their presence or absence of metabolite and their concentration.. Out of the 11 Stevia accessions, Delhi and Mohali varieties showed much relatedness with each other and were concluded to be the superior genotype in context to RAPD and HPTLC analysis. The information obtained here could be valuable for devising strategies for cultivating this medicinal plant.

Topics: Chromatography, Thin Layer; Cluster Analysis; Diterpenes, Kaurane; Genetic Variation; Genotype; Glucosides; India; Random Amplified Polymorphic DNA Technique; Stevia

The extract prepared from the leaves of Stevia rebaudiana BERTONI (Asteraceae) contains sweet steviol glycosides, mainly stevioside and rebaudioside A. Highly purified stevia extracts have become popular worldwide as a natural, low-calorie sweetener. They contain various types of steviol glycosides, and their main components are stevioside and rebaudioside A. The content of each steviol glycoside is quantified by comparing the ratios of the molecular weights and the chromatographic peak areas of the samples to those of stevioside or rebaudioside A standards of the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) Joint Expert Committee on Food Additives (JECFA) and other specifications. However, various commercial standard reagents of stevioside and rebaudioside A are available. Their purities are different and their exact purities are not indicated. Therefore, the measured values of stevioside and rebaudioside A contained in a sample vary according to the standard used for the quantification. In this study, we utilized an accurate method, quantitative NMR (qNMR), for determining the contents of stevioside and rebaudioside A in standards, with traceability to the International System of Units (SI units). The purities of several commercial standards were determined to confirm their actual values.

Topics: Diterpenes, Kaurane; Glucosides; Magnetic Resonance Spectroscopy; Reference Standards; Stevia; Sweetening Agents

This study was aimed at identifying the effect of harvest time, experimental site and crop age on the no-calorie sweetener steviol glycosides (SG) and on the antioxidant properties of stevia leaf extracts. The experiment was conducted over two growing seasons at two sites in the northeastern plain of Italy.. The results showed that all analysed factors played an important role in defining the SG profile and the antioxidant properties of stevia extracts. A high level of phenols (78.24 mg GAE g⁻¹ DW by Folin-Ciocalteu method) and high antioxidant activity (812.6 µmol Fe²⁺ g⁻¹ DW by FRAP assay) were observed. The inhibition of DPPH free radicals was evaluated and an IC₅₀ mean value of 250 µg mL⁻¹ was obtained. Significant relationships among the total antioxidant capacity and the analysed compounds were found.. The results showed the possibility of obtaining, in the tested environments, very high SG yields thanks to the long-day conditions during the spring/summer season. The harvest time played a key role in determining the stevia quality, influencing the rebaudioside A/stevioside ratio. The strong antioxidant properties make very interesting the possibility of using stevia extracts to improve functional food properties.

Topics: Antioxidants; Chromatography, High Pressure Liquid; Crops, Agricultural; Diterpenes, Kaurane; Food Additives; Glucosides; Glycosides; Italy; Non-Nutritive Sweeteners; Oligosaccharides; Phenols; Phytochemicals; Plant Extracts; Plant Leaves; Seasons; Soil; Spatio-Temporal Analysis; Stevia

A promising method of micropropagation of Stevia rebaudiana Bertoni has been developed with an aim to increase the biomass, survivability of the plantlets and stevioside production, using chlorocholine chloride (CCC). Microshoots transferred to the MS medium containing different combinations CCC and IBA were found to be most effective in terms of growth pattern, hardening ability of the plantlets and stevioside content, compared to MS medium containing either IBA or CCC. Among other combinations tested, MS medium supplemented with 3 mg/l CCC and 3 mg/l IBA was found most effective in inducing significant changes like reduced shoot length, increased number of roots, higher leaf size, increased biomass and chlorophyll retaining capacity, higher survival percentage and most importantly the elevated stevioside content. Collectively, the major observations of this research indicate that application of CCC in micropropagation of S. rebaudiana Bertoni is a promising approach and has commercial prospects.

Topics: Breeding; Chlormequat; Chromatography, High Pressure Liquid; Culture Media; Diterpenes, Kaurane; Glucosides; Plant Extracts; Plant Growth Regulators; Plant Roots; Plant Shoots; Seeds; Stevia; Tissue Culture Techniques

This study examined the effects of three different NaCl concentrations (60, 90, and 120 mM) on the growth, physiological responses, and steviol glycoside composition of Stevia rebaudiana Bertoni for 4 weeks. The results showed that the total dry weight decreased by 40% at 120 mM NaCl but remained the same at 60 and 90 mM NaCl. As salt concentration increased, chlorophyll contents decreased markedly by 10-70%, whereas the increments of the antioxidant enzyme activities were 1.0-1.6, 1.2-1.3, and 2.0-4.0 times, respectively, for superoxide dismutase, peroxidase, and catalase. The proline contents in salt-treated plants were 17-42 times higher than that in control. Moreover, leaf possessed significantly higher K⁺ content and K⁺/Na⁺ ratio than stem and root for all salt treatments. In addition, 90-120 mM NaCl treatment notably decreased the content of rebaudioside A (RA) and stevioside (ST) by 16.2-38.2%, whereas the increment of the ratio of RA/ST of salt-treated plants was 1.1-1.4 times. These results indicate that S. rebaudiana is moderately tolerant to salt stress. Hypohaline soil can be utilized in the plantation of S. rebaudiana and may be profitable for optimizing the steviol glycoside composition.

Topics: China; Diterpenes, Kaurane; Glucosides; Plant Leaves; Plant Roots; Plant Stems; Salinity; Soil; Stevia; Stress, Physiological; Sweetening Agents

Stevioside is a natural non-caloric sweetener isolated from Stevia rebaudiana Bertoni leaves. We have proposed its effect on attenuation of tumour necrosis factor α (TNF-α) and interleukin 1β (IL-1β) release in lipopolysaccharide (LPS)-stimulated monocytes. In this study, the anti-inflammatory and immunomodulatory activities of stevioside and its metabolite, steviol, on human colon carcinoma cell line (Caco-2) were evaluated.. Stevioside and steviol, in the doses used in this study, had no cytotoxicity on Caco-2 cells. Anti-inflammatory activities of these two compounds were observed by potentially suppressed LPS-mediated TNF-α, IL-1β and IL-6 release. In addition, stevioside and steviol showed immunomodulatory effects on IκBα activation and nuclear factor kappa B (NF-κB) suppression in western blotting.. Stevioside and steviol attenuate LPS-induced pro-inflammatory cytokine productions by affecting cytokine gene expression via IκBα/NF-κB signalling pathway.

Topics: Anti-Inflammatory Agents; Caco-2 Cells; Colon; Cytokines; Diterpenes, Kaurane; Epithelial Cells; Gene Expression; Glucosides; Humans; I-kappa B Proteins; Immunologic Factors; Inflammation; Inflammation Mediators; Intestinal Mucosa; Lipopolysaccharides; NF-kappa B; NF-KappaB Inhibitor alpha; Phytotherapy; Plant Extracts; Signal Transduction; Stevia

This study was conducted to compare the effects of foliar spray and rhizosphere irrigation with purple phototrophic bacteria (PPB) on growth and stevioside (ST) yield of Stevia. rebaudiana. The S. rebaudiana plants were treated by foliar spray, rhizosphere irrigation, and spray plus irrigation with PPB for 10 days, respectively. All treatments enhanced growth of S. rebaudiana, and the foliar method was more efficient than irrigation. Spraying combined with irrigation increased the ST yield plant (-1) by 69.2% as compared to the control. The soil dehydrogenase activity, S. rebaudiana shoot biomass, chlorophyll content in new leaves, and soluble sugar in old leaves were affected significantly by S+I treatment, too. The PPB probably works in the rhizosphere by activating the metabolic activity of soil bacteria, and on leaves by excreting phytohormones or enhancing the activity of phyllosphere microorganisms.

Topics: Chlorophyll; Chlorophyll A; Diterpenes, Kaurane; Glucosides; Oxidoreductases; Plant Leaves; Rhizosphere; Rhodopseudomonas; Soil; Solubility; Stevia

Meeting the rising consumer demand for natural food ingredients, steviol glycosides, the sweet principle of Stevia rebaudiana Bertoni (Bertoni), have recently been approved as food additives in the European Union. As regulatory constraints require sensitive methods to analyze the sweet-tasting steviol glycosides in foods and beverages, a HILIC-MS/MS method was developed enabling the accurate and reliable quantitation of the major steviol glycosides stevioside, rebaudiosides A-F, steviolbioside, rubusoside, and dulcoside A by using the corresponding deuterated 16,17-dihydrosteviol glycosides as suitable internal standards. This quantitation not only enables the analysis of the individual steviol glycosides in foods and beverages but also can support the optimization of breeding and postharvest downstream processing of Stevia plants to produce preferentially sweet and least bitter tasting Stevia extracts.

Topics: Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Food Analysis; Glucosides; Glycosides; Plant Leaves; Reference Standards; Sensitivity and Specificity; Stevia; Sweetening Agents; Tandem Mass Spectrometry

Catalytic hydrogenation of the three ent-kaurane diterpene glycosides isolated from Stevia rebaudiana, namely rubusoside, stevioside, and rebaudioside-A has been carried out using Pd(OH)₂ and their corresponding dihydro derivatives have been isolated as the products. Synthesis of reduced steviol glycosides was performed using straightforward chemistry and their structures were characterized on the basis of 1D and 2D NMR spectral data and chemical studies. Also, we report herewith the sensory evaluation of all the reduced compounds against their corresponding original steviol glycosides and sucrose for the sweetness property of these molecules.

Topics: Catalysis; Diterpenes, Kaurane; Fungal Proteins; Glucosides; Glycosides; Humans; Hydrogenation; Hydrolysis; Hydroxides; Oxidation-Reduction; Palladium; Polygalacturonase; Sweetening Agents; Taste

A direct, versatile method for the determination of steviol and nine steviol glycosides in food products has been developed by using electrospray ionisation liquid chromatography-mass spectrometry in the negative-ion mode. Ten stevia compounds were readily separated on an amino column by using a gradient separation. Data for analyte quantification were collected in the selected ion monitoring mode, giving the method limit of detection of 0.01-0.34 µg g⁻¹ and repeatability at the limit of quantitation of 2%-15% relative standard deviation. Thirty-four commercially available food products were tested by using the optimised method, and in these products rebaudioside A and stevioside comprised 52%-100% of the total steviol glycosides. Multiple reaction monitoring data were collected to provide analyte confirmation. Stability data for rebaudioside A stored at room temperature, 40°C and 60°C over a period of 1-14 days are shown.

Topics: Beverages; Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Food Analysis; Food Handling; Food Storage; Food, Preserved; Glucosides; Glycosides; Hot Temperature; Limit of Detection; Molecular Structure; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; Sweetening Agents; Tandem Mass Spectrometry; Time Factors

Stevioside, a noncaloric sweetener isolated from Stevia rebaudiana, exhibits anti-inflammatory and immunomodulatory effects through interference of nuclear factor (NF)-kappa B pathway. We investigated whether this anti-inflammatory property of stevioside could improve muscle regeneration following cardiotoxin-induced muscle injury. Adult male Wistar rats received stevioside orally at an accepted daily dosage of 10 mg kg⁻¹ for 7 days before cardiotoxin injection at the tibialis anterior (TA) muscle of the right hindlimb (the left hindlimb served as control), and stevioside administration was continued for 3 and 7 days. TA muscle was examined at days 3 and 7 postinjury. Although stevioside treatment had no significant effect in enhancing muscle regeneration as indicated by the absence of decreased muscle inflammation or improved myofibrillar protein content compared with vehicle treated injured group at day 7 postinjury, the number of MyoD-positive nuclei were increased (P < 0.05), with a corresponding decrease in NF-κB nuclear translocation (P < 0.05). This is the first study to demonstrate that stevioside could enhance satellite cell activation by modulation of the NF-κB signaling pathway in regenerating muscle following injury. Thus, stevioside may be beneficial as a dietary supplementation for promoting muscle recovery from injury. However, its pharmacological effect on muscle function recovery warrants further investigation.

Topics: Animals; Cardiotoxins; Diterpenes, Kaurane; Glucosides; Humans; Male; Muscle, Skeletal; NF-kappa B; Plant Extracts; Rats; Regeneration; Satellite Cells, Skeletal Muscle; Signal Transduction; Stevia; Sweetening Agents

Topics: Diterpenes, Kaurane; Food Safety; Glucosides; Nutritive Value; Plant Extracts; Stevia; Sweetening Agents

Stevia rebaudiana extracts and plant materials are increasingly used as natural sweeteners. Polyphenolic and stevioside compounds contained in S. rebaudiana extracts were separated by comprehensive LC. A polyamine column operated in normal phase mode was used for the first dimension separation (D1), and a UHPLC C18 column operated in reversed phase mode was used for the second dimension separation (D2). The sub-2 μm column (2.1 mm × 30 mm, maintained at 70°C) and the UHPLC pump employed for D2 elution allowed a separation/cycle time of 20 s, with a backpressure oscillating between 805 and 922 bar at 3.4 mL/min. The reduced D2 cycle time allowed 3-12 D2 samplings for each peak eluted by D1. Polyphenolic and stevioside compounds were identified by combining the information coming from the position of the compounds in the 2D plot and UV spectra with that of reference materials.

Topics: Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Flavonoids; Glucosides; Glycosides; Phenols; Plant Extracts; Stevia

Prebiotics such as inulin (Inu)-type fructans and alternative natural sweeteners such as stevioside (Ste) become more popular as food ingredients. Evidence is accumulating that carbohydrates and carbohydrate-containing biomolecules can be considered true antioxidants, capable of scavenging reactive oxygen species (ROS). Here, we report on the ROS scavenging abilities of Inu and Ste in comparison with other sugars, sugar derivatives and arbutin. It is found that Inu and Ste are superior scavengers of both hydroxyl and superoxide radicals, more effective than mannitol and sucrose. Other compounds, such as 1-kestotriose, trehalose, raffinose and L-malic acid, also showed good reactivity to at least one of the two oxygen free radicals. The strong antioxidant properties of Inu and Ste are discussed. Within the plant vacuole, these compounds could play a crucial role in antioxidant defense mechanisms to help survive stresses. Addition to food assists in natural sweetening, food stabilization and maximizes health impact.

Topics: Antioxidants; Arbutin; Diterpenes, Kaurane; Food Additives; Free Radical Scavengers; Glucosides; Inulin; Malates; Mannitol; Oligosaccharides; Oxidative Stress; Plant Extracts; Prebiotics; Reactive Oxygen Species; Stevia; Sweetening Agents

Three novel diterpene glycosides were isolated for the first time from the commercial extract of the leaves of Stevia rebaudiana, along with several known steviol glycosides, namely stevioside, rebaudiosides A-F, rubusoside and dulcoside A. The new compounds were identified as 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-15-en-19-oic acid, 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-16β-hydroxy-ent-kauran-19-oic acid and 13-methyl-16-oxo-17-nor-ent-kauran-19-oic acid-β-D-glucopyranosyl ester on the basis of extensive 2D NMR and MS spectroscopic data as well as chemical studies.

Topics: Diterpenes; Diterpenes, Kaurane; Glucosides; Glycosides; Magnetic Resonance Spectroscopy; Molecular Structure; Plant Extracts; Plant Leaves; Stevia

From the commercial extract of the leaves of Stevia rebaudiana, two new minor diterpene glycosides having α-glucosyl linkage were isolated besides the known steviol glycosides including stevioside, steviolbioside, rebaudiosides A-F, rubusoside and dulcoside A. The structures of the two compounds were identified as 13-[(2-O-(3-α-O-d-glucopyranosyl)-β-d-glucopyranosyl-3-O-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester (1), and 13-[(2-O-β-d-glucopyranosyl-3-O-(4-O-α-d-glucopyranosyl)-β-d-glucopyranosyl-β-d-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-d-glucopyranosyl ester (2), on the basis of extensive NMR and MS spectral data as well as chemical studies.

Topics: Diterpenes; Diterpenes, Kaurane; Glucosides; Glycosides; Magnetic Resonance Spectroscopy; Molecular Structure; Stevia

The stability of the two steviol glycosides stevioside and rebaudioside A and the possible formation of the aglycon steviol in different soft drinks were analyzed in samples spiked with stevioside or rebaudioside A after 24, 48, and 72 h storage times at 80 °C. Degradation of up to 70% was observed, and stevioside was less stable than rebaudioside A. Stevioside and rebaudioside A and their degradation products were analyzed by high-performance liquid chromatography with ultraviolet detection (UV-HPLC) on a HILIC analytical column, and the identity of the degradation products was confirmed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS(n)) in negative mode. A UV-HPLC method was developed using a C18 analytical column to exclude the presence of the aglycon steviol, which gave a positive response in the forward mutation assay using the sensitive Salmonella typhimurium TM677 strain. The recoveries of steviol with this method ranged from 95.9 to 109.2%, and the calibration curves were linear from 1 to 100 μg/mL with R(2) = 0.9999. The limit of detection was 1 μg/mL. Confirmation by LC-ESI-MS(n) resulted in a LOD of 6 ng/mL. The absence of steviol in the degraded samples could be unambiguously confirmed by UV-HPLC and by LC-ESI-MS(n).

Topics: Carbonated Beverages; Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Food Additives; Glucosides; Spectrometry, Mass, Electrospray Ionization; Sweetening Agents

Stevia rebaudiana leaves contain non-cariogenic and non-caloric sweeteners (steviol-glycosides) whose consumption could exert beneficial effects on human health. Steviol-glycosides are considered safe; nonetheless, studies on animals highlighted adverse effects attributed to the aglycone steviol. The aim of the present study was to develop and validate two different ultra-high-performance liquid chromatography methods with electrospray ionization mass spectrometry (UHPLC-MS) to evaluate steviol-glycosides or steviol in Stevia leaves and commercial sweetener (Truvia). Steviol-glycosides identity was preliminarily established by UV spectra comparison, molecular ion and product ions evaluation, while routine analyses were carried out in single ion reaction (SIR) monitoring their negative chloride adducts. Samples were sequentially extracted by methanol, cleaned-up by SPE cartridge and the analytes separated by UHPLC HSS C18 column (150 mm x 2.1 mm I.D., 1.8 microm). The use of CH2Cl2 added to the mobile phase as source of Cl- enhance sensitivity. The LLOD for stevioside, rebaudioside A, steviolbioside and steviol was 15, 50, 10 and 1 ng ml(-1), respectively. Assay validation demonstrated good performances in terms of accuracy (89-103%), precision (<4.3%), repeatability (<5.7%) and linearity (40-180 mg/g). Stevioside (5.8+/-1.3%), rebaudioside A (1.8+/-1.2%) and rebaudioside C (1.3+/-1.4%) were the most abundant steviol-glycosides found in samples of Stevia (n=10) from southern Italy. Rebaudioside A was the main steviol-glycosides found in Truvia (0.84+/-0.03%). The amounts of steviol-glycosides obtained by the UHPLC-MS method matched those given by the traditional LC-NH2-UV method. Steviol was found in all the leaves extract (2.7-13.2 mg kg(-1)) but was not detected in Truvia (<1 microg kg(-1)). The proposed UHPLC-MS methods can be applied for the routine quality control of Stevia leaves and their commercial preparations.

Topics: Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Glucosides; Mass Spectrometry; Plant Leaves; Solid Phase Extraction; Stevia; Sweetening Agents

Use of natural noncaloric sweeteners in commercial foods and beverages has expanded recently to include compounds from the plant Stevia rebaudiana. Little is known about the responses of rodents, the animal models for many studies of taste systems and food intake, to stevia sweeteners. In the present experiments, preferences of female Sprague-Dawley rats and C57BL/6J mice for different stevia products were compared with those for the artificial sweetener saccharin. The stevia component rebaudioside A has the most sweetness and least off-tastes to human raters. In ascending concentration tests (48-h sweetener vs. water), rats and mice preferred a high-rebaudioside, low-stevioside extract as strongly as saccharin, but the extract stimulated less overdrinking and was much less preferred to saccharin in direct choice tests. Relative to the extract, mice drank more pure rebaudioside A and showed stronger preferences but still less than those for saccharin. Mice also preferred a commercial mixture of rebaudioside A and erythritol (Truvia). Similar tests of sweet receptor T1R3 knockout mice and brief-access licking tests with normal mice suggested that the preferences were based on sweet taste rather than post-oral effects. The preference response of rodents to stevia sweeteners is notable in view of their minimal response to some other noncaloric sweeteners (aspartame and cyclamate).

Topics: Animals; Aspartame; Cyclamates; Diterpenes, Kaurane; Drinking; Feeding Behavior; Female; Food Preferences; Glucosides; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Rats; Rats, Sprague-Dawley; Saccharin; Species Specificity; Stevia; Sweetening Agents; Taste; Taste Threshold

Topics: Animals; Diterpenes, Kaurane; Glucosides; Humans; Stevia; Sweetening Agents

The toxicokinetics and metabolism of rebaudioside A, stevioside, and steviol were examined in rats for comparative purposes to determine whether toxicological studies conducted previously with stevioside would be applicable to the structurally-related glycoside, rebaudioside A. Single, oral doses of the radiolabelled compounds were extensively and rapidly absorbed with plasma concentration-time profiles following similar patterns for stevioside and rebaudioside A. Elimination of radioactivity from plasma was essentially complete within 72h. All plasma samples had similar metabolite profiles; the predominant radioactive component in all samples was steviol, with lower amounts of steviol glucuronide(s) and low levels of one or two other metabolites. Rebaudioside A, stevioside, and steviol were metabolized and excreted rapidly, with the majority of the radioactivity eliminated in the feces within 48h. Urinary excretion accounted for less than 2% of the administered dose for all compounds in both intact and bile duct-cannulated rats, and the majority of the absorbed dose was excreted via the bile. After administration of the compounds to intact and bile duct-cannulated rats, radioactivity in the feces was present primarily as steviol. The predominant radioactive compound detected in the bile of all cannulated rats was steviol glucuronide(s), indicating de-conjugation in the lower intestine. Overall, the data on toxicokinetics and metabolism indicate that rebaudioside A and stevioside are handled in an almost identical manner. These studies support the use of toxicological safety studies conducted with stevioside for the safety assessment of rebaudioside A.

Topics: Animals; Bile; Carbon Radioisotopes; Diterpenes, Kaurane; Feces; Female; Gastrointestinal Tract; Glucosides; Male; Rats; Rats, Sprague-Dawley; Sex Characteristics

The Coca-Cola Company and Cargill, Inc. have initiated the development and commercialization of the Stevia rebaudiana (Bertoni) derived sweetener rebaudioside A. Efforts were focused on high purity rebaudioside A (>97% by HPLC), commonly known as rebiana. In the course of the development program, extensive stability studies were carried out on rebiana, all supporting good stability for use in all food and beverage applications, including conditions where rebiana-sweetened beverages were exposed to light. Our findings on rebiana light stability refute those of an earlier study that suggested rebaudioside A to be unstable to sunlight exposure, while the structurally homologous stevioside is stable. We replicated the earlier study and found no significant photodegradation for either rebaudioside A or stevioside.

Topics: Beverages; Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Drug Stability; Glucosides; Light

Recently, we showed that rebaudioside A potently stimulates the insulin secretion from isolated mouse islets in a dose-, glucose- and Ca(2+)-dependent manner. Little is known about the mechanisms underlying the insulinotropic action of rebaudioside A. The aim of this study was to define the signalling system by which, rebaudioside A acts. Isolated mouse islets were used in the cAMP[(125)I] scintillation proximity assay to measure total cAMP level, and in a luminometric method to measure intracellular ATP and ADP concentrations. Conventional and permeabilized whole-cell configuration of the patch-clamp technique was used to verify the effect of rebaudioside A on ATP-sensitive K(+)-channels from dispersed single beta cells from isolated mouse islets. Insulin was measured by radioimmunoassay from insulinoma MIN6 cells. In the presence of 16.7 mM glucose, the addition of the maximally effective concentration of rebaudioside A (10(-9) M) increased the ATP/ADP ratio significantly, while it did not change the intracellular cAMP level. Rebaudioside A (10(-9) M) and stevioside (10(-6) M) reduced the ATP-sensitive potassium channel (K(ATP)) conductance in a glucose-dependent manner. Moreover, rebaudioside A stimulated the insulin secretion from MIN6 cells in a dose- and glucose-dependent manner. In conclusion, the insulinotropic effect of rebaudioside A is mediated via inhibition of ATP-sensitive K(+)-channels and requires the presence of high glucose. The inhibition of ATP-sensitive K(+)-channels is probably induced by changes in the ATP/ADP ratio. The results indicate that rebaudioside A may offer a distinct therapeutic advantage over sulphonylureas because of less risk of causing hypoglycaemia.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Analysis of Variance; Animals; Cell Line; Cyclic AMP; Diterpenes, Kaurane; Female; Glucose; Glucosides; Glyburide; Insulin; Insulin Secretion; Insulin-Secreting Cells; KATP Channels; Mice; Patch-Clamp Techniques; Potassium Channel Blockers; Stimulation, Chemical

A perennial schrub, stevia, and its extracts are used as a natural sweetener and have been shown to possess antimicrobial properties. Stevia contains high levels of sweetening glycosides including stevioside which is thought to possess antimicrobial and antifungal properties. Little is known about the nutritional value of the schrub in livestock. This study determined the potential use of the shrub as a prebiotic animal feed supplement in light of the recent ban on the use of antibiotics in animal feed and the role of its constituent stevioside in the effects of the shrub. Male Cobb broiler chicks were fed a basal broiler diet without antibiotic but with performance enhancing enzyme mix (positive control), a basal diet without antibiotic and enzymes (negative control), or diets in which 2% of the negative control diet was replaced with either dried ground stevia leaves or 130 ppm pure stevioside during 2 week starter and 2 week grower periods. Body weight gains, feed conversion, abdominal fat deposition, plasma hormone and metabolites and caecal short chain fatty acids (SCFA) were measured in the broilers at 2 and 4 weeks of age. There was no significant effect of the treatments on feed intake during the starter period but birds fed diet supplemented with stevia leaves and stevioside consumed more feed (p < 0.05) than those fed the positive control diet during the grower period. Weight gain by birds fed the positive control and stevioside diets was higher (p < 0.05) than those fed other diets only during the starter period. Feed/gain ratio of birds fed the positive control and stevioside diets was superior (p < 0.05) to others. There was no effect of the treatments on nutrient retention and water content of the excreta. Dietary stevia leave and stevioside decreased total concentration of SCFA and changed their profile in the ceca. There was no effect of the treatments on pancreas weight. Dietary stevia reduced blood levels of glucose, triglycerides and triiodothyronine (T(3)) but had no effect on non-esterified fatty acids. In contrast, stevioside only decreased T(3). Both the stevia leaves and stevioside diets significantly increased abdominal fat content. It is concluded that dietary enzyme growth promoters are beneficial to the broilers only during the starter stage and that inclusion of stevia leaves or stevioside has no beneficial effect on the performance of broilers.

Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Anti-Bacterial Agents; Body Composition; Cecum; Chickens; Diterpenes, Kaurane; Eating; Fatty Acids; Fatty Acids, Volatile; Glucosides; Male; Nutritive Value; Plant Leaves; Probiotics; Random Allocation; Stevia; Weight Gain

A high-performance thin-layer chromatographic (HPTLC) method was developed and validated as per ICH (International Conferences on Harmonization) guidelines for simultaneous quantification of three steviol glycosides, i.e. steviolbioside, stevioside and rebaudioside-A in Stevia rebaudiana leaves. For achieving good separation, mobile phase of ethyl acetate-ethanol-water (80:20:12, v/v/v) on pre-coated silica gel 60 F254 HPTLC plates were used. The densitometric quantification of steviol glycosides was carried out at lambda=510 nm in reflection-absorption mode after spraying with acetic anhydride:sulphuric acid:ethanol reagent. The calibration curves were linear in the range of 160-960 ng/spot for steviolbioside, 1-6 microg/spot for stevioside and 0.5-3 microg/spot for rebaudioside-A with good correlation coefficients (0.998-0.999). The method was found to be reproducible for quantitative analysis of steviol glycosides in S. rebaudiana leaves collected from ten different locations and will serve as a quality control indicator to monitor the commercial production of stevioside and its allied molecules during different stages of its processing.

Topics: Calibration; Chromatography, Thin Layer; Diterpenes, Kaurane; Glucosides; Glycosides; Molecular Structure; Reproducibility of Results; Sensitivity and Specificity; Stevia

Pressurised fluid extraction using water or methanol was employed for the extraction of stevioside from Stevia rebaudiana Bertoni. The extraction method was optimised in terms of temperature and duration of the static or the dynamic step. Extracts were analysed by liquid chromatography followed by UV and mass-spectrometric (MS) detections. Thermal degradation of stevioside was the same in both solvents within the range 70-160 degrees C. Methanol showed better extraction ability for isolation of stevioside from Stevia rebaudiana leaves than water within the range 110-160 degrees C. However, water represents the green alternative to methanol. The limit of detection of stevioside in the extract analysed was 30 ng for UV detection and 2 ng for MS detection.

Topics: Diterpenes, Kaurane; Glucosides; Methanol; Plant Extracts; Solvents; Stereoisomerism; Stevia; Water

The effects of a hot water extract of the stem of Stevia rebaudiana on the smooth muscle of isolated guinea pig ileum were investigated. The butyl alcohol layer of the extract antagonized the contractions of the isolated guinea pig ileum induced by histamine (1 x 10(-5) M) and acetylcholine (1 x 10(-5) M) in a concentration-dependent manner. The butyl alcohol layer of the extract also showed inhibition of CaCl(2) (1 x 10(-3)-3.8 x 10(-1) M)-induced contractions. The antagonism of the extract was considered to be non-specific, but this action might be related to an influx of extracellular Ca(2+). With column chromatography preparation, the active component was assumed to be as stevioside. The antagonistic effects exerted by the stem extract of Stevia rebaudiana contributed to the gastroprotective activity of the extract in animals fed dietary histamine.

Topics: Acetylcholine; Animals; Butanols; Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Glucosides; Guinea Pigs; Hot Temperature; Ileum; Male; Mass Spectrometry; Molecular Weight; Muscle Contraction; Muscle, Smooth; Plant Extracts; Plant Shoots; Stevia; Water

The purpose of the present study was to compare the effect of the oral treatment (gavage) with Stevia rebaudiana (Bert.) Bertoni (SRB) and stevioside (STV) on glycaemia and gluconeogenesis of 15-h fasted rats. For this purpose, the rats received SRB (20 mg/kg x day), STV (5.5 mg/kg x day) or an equal volume of water (controls) during 15 days. To measure hepatic gluconeogenesis, liver perfusion and isolated hepatocytes were used. Glycaemia and gluconeogenesis from L-alanine (5 mM), L-glutamine (5 mM) and L-lactate (2 mM) were decreased (P < 0.05) after pre-treatment with SRB. However, the treatment with STV did not influence glycaemia and gluconeogenesis. Moreover, to get further information about the mechanism by which SRB leaves inhibit gluconeogenesis their potential role as a PPARgamma agonist was investigated. The data showed absence of activation of PPARgamma receptors. In summary, our results showed that the reduction of glycaemia promoted by the treatment with SRB leaves was mediated, at least in part, by an inhibition of hepatic gluconeogenesis. However, this effect did not involve stevioside and the activation of PPARgamma receptors.

Topics: Administration, Oral; Animals; Diabetes Mellitus, Type 1; Diterpenes, Kaurane; Gluconeogenesis; Glucose; Glucosides; Hypoglycemic Agents; Liver; Male; Phytotherapy; Plant Extracts; Plant Leaves; Rats; Rats, Wistar; Stevia

A simple and rapid method for the simultaneous determination of nine kinds of sweeteners (acesulfame potassium, AK; sucralose, SUC; saccharin, SA; cyclamate, CYC; aspartame, APM; dulcin, DU; glycyrrhizic acid, GA; stevioside, STV; rebaudioside A, REB) in various foods by high-performance liquid chromatography-electrospray mass spectrometry (LC/ESI-MS) was developed. The LC separation was performed on a ZORBAX Eclipse XDB-C18 (2.1 mm x 150 mm) with a mobile phase of 5 mmol/L dibutylammonium acetate (DBAA) and acetonitrile-water (8: 2). Mass spectral acquisition was done in the negative ion mode by applying selected ion monitoring (SIM). The sweeteners were extracted from foods with 0.08 mol/L phosphate buffer (pH 7.0)- ethanol (1:1), and the extract was cleaned up on a Sep-pak Vac C18 cartridge after the addition of tetrabutylammonium bromide and phosphate buffer (pH 3.0). The recovery of the nine kinds of sweeteners from five kinds of foods fortified at the level 0.01 g/kg, 0.05 g/kg and 0.20 g/kg was 75.7-109.2%, and the between-day SD values were 0.5-10.9%. The quantification limits of AK, SA, CYC, APM and STV were 0.001 g/kg, and those of SUC, DU, GA and REB were 0.005 g/kg. A recovery test from each cleaned-up sample solution was necessary to detect ionization suppression.

Topics: Aspartame; Chromatography, High Pressure Liquid; Cyclamates; Diterpenes, Kaurane; Food Analysis; Glucosides; Glycyrrhizic Acid; Phenylurea Compounds; Saccharin; Spectrometry, Mass, Electrospray Ionization; Sucrose; Sweetening Agents; Thiazines

The natural sweetening agent stevioside and its aglycone metabolite, steviol, have been shown to inhibit transepithelial transport of para-aminohippurate (PAH) in isolated rabbit renal proximal tubules by interfering with basolateral entry. The aim of the present study was to determine which of the cloned basolateral organic anion transporters were involved in the renal transport of stevioside and steviol. This question was addressed in Xenopus laevis oocytes expressing human organic anion transporter 1 (hOAT1), 3 (hOAT3), and winter flounder OAT (fOat1). The parent compound, stevioside, had no inhibitory effect on either PAH (hOAT1) or ES (estrone sulfate; hOAT3) uptake. In contrast, steviol showed significant, dose-dependent inhibition of PAH and ES uptake in hOAT1- or hOAT3-expressing oocytes, respectively. The IC(50) of steviol for hOAT1-mediated PAH transport was 11.1 microM compared with 62.6 microM for hOAT3-mediated ES uptake. The Michaelis-Menten inhibition constants (K(i)) for steviol transport mediated by hOAT1 and hOAT3 were 2.0 +/- 0.3 and 5.4 +/- 2.0 microM, respectively. Trans-stimulation of PAH efflux by steviol was assessed to determine whether steviol itself was transported by hOAT1 or hOAT3. A low concentration of 1 microM steviol increased the efflux of [(3)H]PAH (trans-stimulated) via both hOAT1 and hOAT3. In addition, it was shown by electrophysiology that steviol entry induced inward current in fOat1-expressing oocytes. In conclusion, stevioside had no interaction with either hOAT1 or hOAT3, whereas hOAT1, hOAT3, and fOat1 were all shown to be capable of steviol transport and thus, can play a role in its renal transport and excretion.

Topics: Animals; Biological Transport; Diterpenes, Kaurane; Female; Glucosides; Humans; Organic Anion Transport Protein 1; Organic Anion Transporters, Sodium-Independent; Stevia; Sweetening Agents; Xenopus laevis

The intake of dietary fructose has undergone a marked increase around the world, especially the developed countries, in recent times. Stevioside, a glycoside contained in the leaves of Stevia rebaudiana Bertoni (Compositae), was used to screen the effect induced by a diet containing 60% fructose on insulin resistance in rats. Single oral administration of stevioside for 90 min decreased plasma glucose concentrations in a dose-dependent manner in rats receiving fructose-rich chow for four weeks. In addition, insulin action on glucose disposal rate was measured using the glucose-insulin index, the product of the areas under the curve of glucose, and insulin during the intraperitoneal glucose tolerance test. Oral administration of stevioside (5.0 mg/kg) in rats given four weeks of fructose-rich chow for 90 min reversed the value of glucose-insulin index, indicating that stevioside has the ability to improve insulin sensitivity in this insulin-resistant animal model. Time for the loss of plasma glucose lowering response to tolbutamide (10.0 mg/kg, i. p.) in fructose-rich chow fed rats was also markedly delayed by repeated stevioside treatment three times daily compared to the vehicle-treated group. The plasma glucose-lowering activity of tolbutamide was introduced to account for varying levels of endogenous insulin secretion, and is widely used as the indicator of insulin resistance development. Thus, it provided the supportive data that repeated oral administration of stevioside delayed the development of insulin resistance in rats on a high-fructose diet. Increased insulin sensitivity by stevioside administration was further identified using the plasma glucose-lowering action of exogenous insulin in streptozotocin-induced diabetic rats (STZ-diabetic rats). Oral administration of stevioside at 0.2 mg/kg three times daily into STZ-diabetic rats for ten days increased the response to exogenous insulin. Taken together, this demonstrated that oral administration of stevioside improves insulin sensitivity, and seems suitable as an adjuvant for diabetic patients and/or those that consume large amounts of fructose.

Topics: Animals; Blood Glucose; Diabetes Mellitus, Experimental; Dietary Carbohydrates; Diterpenes, Kaurane; Fructose; Glucose Tolerance Test; Glucosides; Insulin Resistance; Male; Phytotherapy; Plant Extracts; Rats; Rats, Wistar; Stevia; Tolbutamide

A method for the simultaneous determination of stevioside (Stev), rebaudioside A (RebA) and glycyrrhizic acid (GA) in foods was developed. These sweeteners were extracted from foods, except for dried fishes and shellfishes, by dialysis against Tris-HCl buffer (pH 9.0). Dried fishes and shellfishes were extracted with Tris-HCl buffer--methanol (2:8). The extracts were cleaned up with an Oasis MAX cartridge. The cartridge was washed with 0.05 mol/L sodium acetate (pH 4.0)--methanol (19:1), and the three sweeteners were eluted with 0.1 mol/L phosphoric acid--acetonitrile (1:1). Stev, RebA and GA in the eluate were chromatographed on a Develosil RPAQUEOUS-AR-5 (4.6 mm i.d. x 250 mm) column with 0.02 mol/L phosphoric acid-acetonitrile--methanol (90:55:5) as a mobile phase and monitored at 210 nm for Stev and RebA, and at 254 nm for GA. The recoveries of Stev, RebA and GA from 8 kinds of foods spiked at the level of 0.1 g/kg were 81.7-101%, 81.5-100% and 78.6-95.0%, respectively. The determination limits were 0.01 g/kg in samples.

Topics: Chromatography, High Pressure Liquid; Diterpenes; Diterpenes, Kaurane; Food Analysis; Glucosides; Glycyrrhizic Acid; Sweetening Agents; Terpenes

A simple and highly sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) has been developed for the determination of steviol (SV) using dihydroisosteviol (DHISV) as an internal standard (IS). SV and DHISV were derivatized by reaction of the acids with 4-(bromomethyl)-7-methoxycoumarin in an aprotic solvent (DMF or acetone). The resulting ester derivatives were separated on an ODS column (250 x 4.6 mm i.d., 5 microm particle size) using fluorescence detection with excitation at 321 nm and emission at 391 nm. The mobile phase consisted of acetonitrile/water (80:20 v/v) with a flow rate of 1 mL min(-)(1). A linear relationship was observed for concentrations between 0.5 and 50 microg/mL of SV, and the detection limit was 100 pg. For application of this method to samples of beer fortified with stevioside, a simple procedure for extraction of the beer with diethyl ether and derivatization in DMF was applied. Whereas beer samples spiked with SV gave a linear response over the range 0.1-15 microg/mL beer, no SV could be detected in beer samples enriched in stevioside that had been stored for over 3 years. The application of the method to plant samples involved preparation of an acid fraction containing the SV analyte, derivatization, and sample cleanup using small silica columns and thin-layer chromatography. A sensitive determination of 594 ng of steviol present in 100 mg of dry plant material was performed with high precision and accuracy.

Topics: Beer; Chromatography, High Pressure Liquid; Diterpenes; Diterpenes, Kaurane; Glucosides; Microchemistry; Plant Leaves; Pyrones; Sensitivity and Specificity; Spectrometry, Fluorescence; Stevia; Time Factors

Stevia mixture, sweeteners extracted from the leaves of Stevia rebaudiana Bertoni, consists mainly of stevioside and rebaudioside A (glycosides of the diterpene derivative steviol). The aim of this study was to investigate human intestinal metabolism of stevia mixture and its alpha-glucose derivative (known in Japan as enzymatically modified stevia) by LC/MS/ESI analysis. Degradation was examined by incubating stevia mixture, enzymatically modified stevia, stevioside, rebaudioside A, alpha-monoglucosylstevioside, alpha-monoglucosylrebaudioside A and the aglycone, steviol with pooled human faecal homogenates (obtained from five healthy volunteers) for 0, 8 and 24 h under anaerobic conditions. Stevia mixture, enzymatically modified stevia, stevioside and rebaudioside A (0.2 mg/ml) were completely eliminated within 24 h, whereas no degradation of steviol (0.08 and 0.2 mg/ml) appeared to be found during the incubation period. Stevia mixture, stevioside and rebaudioside A appeared to be hydrolyzed to steviol by human intestinal microflora: this observation is consistent with previous rat metabolism studies. Similarly, enzymatically modified stevia appeared to be metabolized via stevia components and, finally, to steviol. This study suggests that there are apparently no species differences in intestinal metabolism of stevia mixture between rats and humans.

Topics: Adult; Anaerobiosis; Animals; Bacteria, Anaerobic; Chromatography, Ion Exchange; Chromatography, Liquid; Digestive System; Diterpenes; Diterpenes, Kaurane; Feces; Gas Chromatography-Mass Spectrometry; Glucosides; Humans; Hydrolysis; Kinetics; Male; Molecular Weight; Plant Extracts; Rats; Species Specificity; Stevia; Structure-Activity Relationship; Sweetening Agents; Terpenes

Stevioside orally administered to pigs was completely converted into steviol by the bacteria of the colon. However, no stevioside or steviol could be detected in the blood of the animals, even not after converting steviol into the (7-methoxycoumarin-4-yl)methyl ester of steviol, a very sensitive fluorescent derivative with a detection limit of about 50 pg. The intestinal transport characteristics of stevioside, rebaudioside A and steviol were also studied in the Caco-2 system. Only a minor fraction of stevioside and rebaudioside A was transported through the Caco-2 cell layer giving a Papp value of 0.16x10(-6) and 0.11x10(-6) cm/s, respectively. The Papp value for the absorptive transport of steviol was about 38.6x10(-6) cm/s while the Papp value for the secretory transport of steviol was only about 5.32x10(-6) cm/s suggesting carrier-mediated transport. The discrepancy between the relatively high absorptive transport of steviol and the lack of steviol in the blood may be explained by the fact that in the Caco-2 study, steviol is applied as a solution facilitating the uptake, whereas in the colon steviol probably is adsorbed to the compounds present in the colon of which the contents is being concentrated by withdrawal of water.

Topics: Algorithms; Animals; Biological Transport; Caco-2 Cells; Cell Division; Cell Membrane Permeability; Diterpenes; Diterpenes, Kaurane; Epithelial Cells; Feces; Female; Glucosides; Humans; Intestinal Absorption; Kinetics; Swine; Terpenes

Stevia rebaudiana standardized extracts (SSEs) are used as natural sweeteners or dietary supplements in different countries for their content of stevioside or rebaudioside A. These compounds possess up to 250 times the sweetness intensity of sucrose, and they are noncaloric and noncariogenic sweeteners. The aim of this study was to investigate the in vitro transformation of stevioside and rebaudioside A after incubation with human microflora, the influence of these sweeteners on human microbial fecal community and which specific groups metabolize preferentially stevioside and rebaudioside A. The experiments were carried out under strict anaerobic conditions in batch cultures inoculated with mixed fecal bacteria from volunteers. The hydrolysis was monitored by HPLC coupled to photodiode array and mass spectrometric detectors. Isolated bacterial strains from fecal materials incubated in selective broths were added to stevioside and rebaudioside A. These sweeteners were completely hydrolyzed to their aglycon steviol in 10 and 24 h, respectively. Interestingly, the human intestinal microflora was not able to degrade steviol. Furthermore, stevioside and rebaudioside A did not significantly influence the composition of fecal cultures; among the selected intestinal groups, bacteroides were the most efficient in hydrolyzing Stevia sweeteners to steviol.

Topics: Adult; Bacteria; Chromatography, High Pressure Liquid; Diterpenes; Diterpenes, Kaurane; Feces; Female; Glucosides; Humans; Male; Mass Spectrometry; Middle Aged; Plant Extracts; Stevia; Sweetening Agents; Terpenes

Four steviol (ent-kaurene-type diterpenoid) glycosides, stevioside, rebaudiosides A and C, and dulcoside A, have been isolated from Stevia rebaudiana BERTONI. These compounds showed strong inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice. The 50% inhibitory dose of these compounds for TPA-induced inflammation was 54.1-291.6 micro g/ear. Furthermore, at 1.0 and 0.1 mg/mouse of stevioside mixture, the mixture of these compounds markedly inhibited the promoting effect of TPA (1 micro g/mouse) on skin tumor formation initiated with 7,12-dimethylbenz[a]anthracene (50 micro g/mouse).

Topics: Animals; Antineoplastic Agents, Phytogenic; Diterpenes; Diterpenes, Kaurane; Drug Screening Assays, Antitumor; Female; Glucosides; Mice; Mice, Inbred ICR; Skin Neoplasms; Stevia; Tetradecanoylphorbol Acetate

An improved analytical method was developed which may be applied to quality control of stevioside and rebaudioside A contents in dried leaves of Stevia rebaudiana before processing; in a selective sampling program searching for plants of higher yield in diterpene glycosides content; or when a large number of samples are sent to the laboratory for analysis. The procedure developed involves two steps: solvent extraction followed by an isocratic HPLC analysis. The sample, 1 g of dried leaves of S. rebaudiana, is ground and solvent-extracted with EtOH 70% (w/w) in Erlenmeyer flasks by shaking for 30 min in a 70 degrees C water bath. After the extract was cooled, it was filtered and analyzed by HPLC using an NH(2) column (250 x 4.6 mm) and a mixture of acetonitrile/water (80:20, v/v) as mobile phase, pH 5 adjusted with acetic acid. The detection was in the UV range at 210 nm (0.04 AUFS). Quantitation was performed by means of an external standard calibration curve for each analyte which had been obtained from standard solutions of pure stevioside and rebaudioside A. Working under these conditions there were no observed interference effects. The method saves time in sample preparation, and reduces sample handling and chromatographic analysis time, while having little loss of precision [coefficient of variation (CV%) between 1.8% and 3.0%] and recovery [between 98.5% and 100.5%]. The method was applied to 30 samples of S. rebaudiana from Misiones (Northeastern Argentina), and the stevioside content found ranged between 3.78 and 9.75% (weight) whereas Rebaudioside A content ranged between 1.62 and 7.27% (weight).

Topics: Chromatography, High Pressure Liquid; Diterpenes; Diterpenes, Kaurane; Glucosides; Glycosides; Lamiaceae; Reproducibility of Results; Sensitivity and Specificity; Sweetening Agents; Terpenes

The determination of diterpene glycosides from Stevia rebaudiana leaves using capillary electrophoresis is described. Analyses were performed on fused silica capillaries with 20 mM sodium tetraborate buffer, pH 8.3, and 30 mM sodium dodecyl sulfate. The effect of the organic solvent injected with the sample solution on the electrophoretic solution has been confirmed, and an absolute amount of 1.6 nL per injected sample was optimal. Rebaudioside A and steviolbioside were isolated by semipreparative high performance liquid chromatography (HPLC), and their structure was assessed by mass spectrometry.

Topics: Diterpenes; Diterpenes, Kaurane; Electrophoresis, Capillary; Glucosides; Magnoliopsida; Molecular Structure; Plant Extracts; Terpenes

Stevioside and rebaudioside A, two intense natural sweeteners, that are constituents of the South American plant Stevia rebaudiana, were tested for cariogenicity in albino Sprague-Dawley rats. Sixty rat pups colonized with Streptococcus sobrinus were divided into four groups and fed stevioside, rebaudioside A or sucrose added to basal diet 2000 as follows: group 1, 30% sucrose; group 2, 0.5% stevioside; group 3, 0.5% rebaudioside A, and group 4, no addition. All four groups were sacrificed after 5 weeks. S. sobrinus counts were made and caries was evaluated according to Keyes' technique. There were no differences in food and water intake and weight gains between the four groups. There were significant differences in sulcal caries scores (p < 0.02) and S. sobrinus counts (p < 0.05) between group 1 and the other three groups. There were no significant differences between the stevioside, rebaudioside A and no-addition groups. It was concluded that neither stevioside nor rebaudioside A is cariogenic under the conditions of this study.

Topics: Animals; Cariostatic Agents; Colony Count, Microbial; Dental Caries; Dental Plaque; Diet, Cariogenic; Diterpenes; Diterpenes, Kaurane; Evaluation Studies as Topic; Glucosides; Rats; Rats, Sprague-Dawley; Root Caries; Streptococcus sobrinus; Sucrose; Sweetening Agents; Terpenes

Steviol (ent-13-hydroxykaur-16-en-19-oic acid), the aglycone of various plant-derived glycoside sweeteners consumed by human populations, is known to be mutagenic toward Salmonella tymphimurium strain TM677 when metabolically activated using a 9000 x g supernatant fraction derived from the liver of Aroclor 1254-pretreated rats. Mass spectral analysis of this diterpenoid and some analogs revealed characteristic patterns reflecting differential stereochemistry at the C/D rings and variations in the nature of the substituents present. Such information has been used to help identify several in vitro metabolites of steviol in conditions known to produce a mutagenic response, when analyzed by gas chromatography/mass spectrometry. The major pathways of such steviol mammalian metabolism proved to be allylic oxidation and epoxidation. 15-Oxosteviol, a product of oxidation of the major steviol metabolite, 15alpha-hydroxysteviol, was found to be a direct-acting mutagen [corrected].

Topics: Biotransformation; Diterpenes; Diterpenes, Kaurane; Glucosides; Glycosides; Mass Spectrometry; Mutagens; Sweetening Agents; Terpenes

Topics: Chromatography, Gel; Diterpenes; Diterpenes, Kaurane; Glucosides; Glycosides; Magnetic Resonance Spectroscopy; Plants; Terpenes

Contrary to prior indications, the glycosidic sweeteners stevioside and rebaudioside A are degraded to the diterpenoid aglycone steviol by rat intestinal microflora in vitro. Additional studies with steviol-17-[14C] show almost total absorption from the rat lower bowel following intracecal administration.

Topics: Anaerobiosis; Animals; Bile Ducts; Cecum; Diterpenes; Diterpenes, Kaurane; Feces; Glucosides; Glycosides; Intestinal Absorption; Plants; Rats; Terpenes