steviol 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

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

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

The diterpenoids present highly varied oxidation patterns subjected to fascinating skeletal rearrangements, and possess antitumor, antibacterial, antihyperglycemic, anti-inflammatory activities and so on. While total synthesis is a possible solution to obtain required diterpenoid derivatives, the need for multisteps to build complex "privileged structures" is always prohibitive by semi-synthesis from naturally abundant related molecules. From this perspective, stevioside and its hydrolysis products steviol and isosteviol are good leads in the field of medicinal chemistry for diterpenoid drug discovery. Stevioside has a complex diterpenoid glycoside molecule comprised of an aglycone, steviol with the ent-kaurane skeleton and three molecules of glucose. Hydrolyzed under alkaline or acidic condition, stevioside generates ent-kaurane diterpenoid steviol or ent-beyerane diterpenoid isosteviol, respectively. Except for direct applications, they are widely used to provide ent-kaurane or ent-beyerane core structures for further medicinal chemistry study. Besides, these molecules also serve as model molecules, starting materials or catalysts in the field of synthetic chemistry. In this review, their biological activities and medicinal chemistry work are comprehensively summarized which are very helpful for diterpenoid drug exploration.

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Chemistry Techniques, Synthetic; Diterpenes, Kaurane; Drug Discovery; Glucosides; Humans; Hydrolysis; Hypoglycemic Agents

The safety of steviol glycoside sweeteners has been extensively reviewed in the literature. National and international food safety agencies and approximately 20 expert panels have concluded that steviol glycosides, including the widely used sweeteners stevioside and rebaudioside A, are not genotoxic. However, concern has been expressed in recent publications that steviol glycosides may be mutagenic based on select studies representing a small fraction of the overall database, and it has been suggested that further in vivo genotoxicity studies are required to complete their safety profiles. To address the utility of conducting additional in vivo genotoxicity studies, this review evaluates the specific genotoxicity studies that are the sources of concern, and evaluates the adequacy of the database including more recent genotoxicity data not mentioned in those publications. The current database of in vitro and in vivo studies for steviol glycosides is robust and does not indicate that either stevioside or rebaudioside A are genotoxic. This, combined with a lack of evidence for neoplasm development in rat bioassays, establish the safety of all steviol glycosides with respect to their genotoxic/carcinogenic potential.

Topics: Animals; Carcinogens; Databases, Factual; Diterpenes, Kaurane; Glucosides; Humans; Mutagenicity Tests; Mutagens; Rats; Sweetening Agents

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

Extracts of the leaves of the stevia plant (Stevia rebaudiana Bertoni) are used to sweeten food and beverages in South America, Japan and China. The components responsible for the sweet properties of the plant are glycosides of steviol, primary stevioside (ent-13-hydroxykaur-16-en-18-oic acid), which is 250-300 times sweeter than sucrose and rebaudiosides A and C. Stevioside and steviol have been subjected to extensive genetic testing. The majority of the findings show no evidence of genotoxic activity. Neither stevioside nor its aglycone steviol have been shown to react directly with DNA or demonstrate genotoxic damage in assays relevant to human risk. The mutagenic activity of steviol and some of its derivatives, exhibited in strain TM677, was not reproduced in the same bacteria having normal DNA repair processes. The single positive in vivo study measuring single-strand DNA breaks in Wistar rat tissues by stevioside, was not confirmed in experiments in mice and appears to be measuring processes other than direct DNA damage. Neither stevioside nor steviol-induced clastogenic effects at extremely high dose levels in vivo. Application of a Weight-of-Evidence approach to assess the genetic toxicology database concludes that these substances do not pose a risk of genetic damage following human consumption.

Topics: Animals; Bacteria; Diterpenes, Kaurane; DNA Damage; Glucosides; Humans; Mutagenicity Tests; Mutagens; Rats; Rats, Wistar; Risk Factors; Sweetening Agents

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

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

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

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

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

Studies on the effects of non-nutritive sweeteners (NNSs) among pregnant women are scarce and have produced mixed results. One of the major challenges is to accurately assess NNS intake, especially in countries that have implemented policies to prevent obesity and where many foods and beverages have been progressively reformulated to partially or totally replace sugar with NNSs. This study aimed to develop and assess the relative validity of a food frequency questionnaire (FFQ) for use in pregnant women. We developed an FFQ to examine the intake of seven NNSs (acesulfame-k, aspartame, cyclamate, saccharin, sucralose, steviol glycosides, and D-tagatose). This questionnaire was piloted in 29 pregnant women (median age = 31.2 y; 25th-75th percentile: 26.9-34.7) to assess NNS intake over the previous month, compared to 3-day dietary records (3-DR). The validity of this dietary method was assessed using Spearman's correlation coefficient, Lin´s concordance correlation coefficient (CCC), and Bland-Altman plots. Spearman's correlations between the FFQ on NNSs and 3-DR ranged from 0.50 for acesulfame K to 0.83 for saccharin. CCC ranged between 0.22 and 0.66. The Bland-Altman plots showed an overestimation of saccharin, sucralose, and steviol glycosides intake by the FFQ on NNSs compared with 3-DR, and an underestimation of acesulfame K and aspartame. Overall, the NNSs most frequently consumed were sucralose, and none of the participants exceeded the acceptable daily intake for any of the NNSs evaluated. The FFQ on NNSs seems to be reasonably valid in the assessment of NNSs among pregnant women.

Topics: Adult; Aspartame; Chile; Female; Glucosides; Humans; Non-Nutritive Sweeteners; Pilot Projects; Pregnancy; Pregnant Women; Saccharin; Surveys and Questionnaires

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

Steviol glycosides obtained from

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

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

Steviol glycosides, the active sweet components of stevia plant, have been recently found to possess a number of therapeutic properties, including some recorded anticancer ones against various cancer cell types (breast, ovarian, cervical, pancreatic, and colon cancer). Our aim was to investigate this anticancer potential on the two most commonly used breast cancer cell lines which differ in the phenotype and estrogen receptor (ER) status: the low metastatic, ERα+ MCF-7 and the highly metastatic, ERα-/ERβ+ MDA-MB-231. Specifically, glycosides' effect was studied on cancer cells': (a) viability, (b) functionality (proliferation, migration, and adhesion), and (c) gene expression (mRNA level) of crucial molecules implicated in cancer's pathophysiology. Results showed that steviol glycosides induced cell death in both cell lines, in the first 24 hr, which was in line with the antiapoptotic BCL2 decrease. However, cells that managed to survive showcased diametrically opposite behavior. The low metastatic ERα+ MCF-7 cells acquired an aggressive phenotype, depicted by the upregulation of all receptors and co-receptors (ESR, PGR, AR, GPER1, EGFR, IGF1R, CD44, SDC2, and SDC4), as well as VIM and MMP14. On the contrary, the highly metastatic ERα-/ERβ+ MDA-MB-231 cells became less aggressive as pointed out by the respective downregulation of EGFR, IGF1R, CD44, and SDC2. Changes observed in gene expression were compatible with altered cell functions. Glycosides increased MCF-7 cells migration and adhesion, but reduced MDA-MB-231 cells migratory and metastatic potential. In conclusion, the above data clearly demonstrate that steviol glycosides have different effects on breast cancer cells according to their ER status, suggesting that steviol glycosides might be examined for their potential anticancer activity against breast cancer, especially triple negative breast cancer (TNBC).

Topics: Diterpenes, Kaurane; ErbB Receptors; Estrogen Receptor alpha; Estrogen Receptor beta; Glucosides; Glycosides; Matrix Metalloproteinase 14; Neoplasms; Proto-Oncogene Proteins c-bcl-2; Receptors, Estrogen; RNA, Messenger

Government regulatory actions and public policies to reduce sugar consumption were recently implemented in Brazil. To evaluate their potential impact on the supply of products containing high-intensity sweeteners (HIS) and on dietary exposure to these substances, this study aimed to create a comprehensive database on HIS declared in Brazilian commercial products and estimate their intake through consumption of these products. The occurrence of HIS was evaluated through labeling information of 1869 commercial products available in the Brazilian market, collected between January 2021 and August 2021, and the daily intake was estimated for eight HIS (acesulfame K, advantame, aspartame, cyclamate, steviol glycosides, neotame, saccharin and sucralose) using a deterministic approach by multiplying the maximum permitted levels of HIS in foods and beverages by the consumption data of these products. The consumption data were obtained from the report of Household Budget Survey (POF/IBGE), conducted from 2017 to 2018 through a 24-hour dietary recall applied to 46,164 individuals aged 10 years and over, which included only average data (i.e. average consumption for the general population or subgroups). The most frequent HIS in the investigated products were sucralose (26.8 %; n = 938) and acesulfame K (21.7 %; n = 759), and although the combination of sweeteners is a common practice in the food industry, there was a predominance of only one substance in the investigated products (46.7 %; n = 873). The estimated intake of HIS for average consumers was below the Acceptable Daily Intake (ADI) and does not suggest a toxicological concern. A similar scenario was observed for high consumers, except for cyclamate and steviol glycosides, which corresponded to 144 % and 131 % of their respective ADIs in the general population. To our knowledge, this is the most comprehensive database on HIS in Brazil and the most recent exposure assessment performed nationally.

Topics: Aspartame; Brazil; Cyclamates; Dietary Sugars; Diterpenes, Kaurane; Glucosides; Humans; Non-Nutritive Sweeteners; Saccharin; Sweetening Agents; Thiazines

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

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

Topics: Diterpenes, Kaurane; Glucosides; Glycosides; Microbacterium; Molecular Structure; Phylogeny

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

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

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

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

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

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

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

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

Steviol glycosides, a natural sweetener, may perform bioactivities via steviol, their main metabolite in human digestion. The metabolising kinetics, i.e. glucuronidation kinetics and interaction between steviol glycosides or their metabolites and metabolising enzyme, are important for understanding the bioactivity and cytotoxicity. The present study investigated kinetics of steviol glucuronidation in human liver microsome and a recombinant human UDP-glucuronosyltransferases isomer, UGT2B7, along with molecular docking to analyse interaction between UGT2B7 and steviol or glucose. The active pocket of UGT2B7 is consisted of Arg352, Leu347, Lys343, Phe339, Tyr354, Lys355 and Leu353. The influence of stevioside, rebaudioside A, glucose and some chemotherapy reagents on the glucuronidation was also studied. The predicted hepatic clearence suggested that steviol could be classified as high-clearence drug. The steviol glycosides did not affect the glucuronidation of steviol notably.

Topics: Diterpenes, Kaurane; Glucose; Glucosides; Glucuronosyltransferase; Humans; Kinetics; Microsomes, Liver; Models, Molecular; Protein Binding; Protein Conformation; Recombinant Proteins

To purify and characterize a specific enzyme from a commercial pectinase for the production of steviol from stevioside (Ste) without adding organic solvent and to improve steviol production.. βglyPX contains great potential for industrial steviol production from Ste.

Topics: beta-Glucosidase; Diterpenes, Kaurane; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Glucosides; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; Polygalacturonase; Temperature

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

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

Curcuminoids from rhizomes of Curcuma longa possess various biological activities. However, low aqueous solubility and consequent poor bioavailability of curcuminoids are major limitations to their use. In this study, curcuminoids extracted from turmeric powder using stevioside (Ste), rebaudioside A (RebA), or steviol glucosides (SG) were solubilized in water. The optimum extraction condition by Ste, RebA, or SG resulted in 11.3, 9.7, or 6.7mg/ml water soluble curcuminoids. Curcuminoids solubilized in water showed 80% stability at pH from 6.0 to 10.0 after 1week of storage at 25°C. The particle sizes of curcuminoids prepared with Ste, RebA, and SG were 110.8, 95.7, and 32.7nm, respectively. The water soluble turmeric extracts prepared with Ste, RebA, and SG showed the 2,2-diphenyl-1-picrylhydrazyl radical scavenging (SC50) activities of 127.6, 105.4, and 109.8μg/ml, and the inhibition activities (IC50) against NS2B-NS3(pro) from dengue virus type IV of 14.1, 24.0 and 15.3μg/ml, respectively.

Topics: Curcuma; Curcumin; Diterpenes, Kaurane; Glucosides; Plant Extracts

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

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

Topics: Archaeal Proteins; Diterpenes, Kaurane; Enzyme Stability; Glucosidases; Glucosides; Hot Temperature; Hypoglycemic Agents; Kinetics; Recombinant Proteins; Sulfolobus solfataricus; 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

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

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

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

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

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

Enzymatic hydrolysis and transgalactosylation of stevioside (St) were investigated using a β-galactosidase from Sulfolobus sp. The hydrolysis yielded steviol as the main final product. Under the optimal transgalactosylation conditions, the highest conversion of stevioside was 87.3% with lactose as a donor, several galactosylated products (St-Gals) were obtained. Metal ions such as Na(+), K(2+), Ca(2+), Ba(2+), Mn(2+) and Mg(2+) (2 mM) did not affect the transgalactosylation activity, while Fe(2+), Fe(3+) and Cu(2+) reduced the transgalactosylation activity of β-galactosidase to 64%, 33% and 18%, respectively.

Topics: beta-Galactosidase; Diterpenes, Kaurane; Glucosides; Hydrolysis; Lactose; Sulfolobus

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

Cyst enlargement in autosomal dominant polycystic kidney disease (ADPKD) is associated with cAMP-activated proliferation of cyst-lining epithelial cells and transepithelial fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel leading to renal failure for which no effective treatment is currently available. We previously reported that steviol retards Madin-Darby canine kidney (MDCK) cyst enlargement by inhibiting CFTR channel activity and promoting proteasomal-mediated CFTR degradation. It is imperative to examine the effect of steviol in animal models of ADPKD. Therefore, we examined the effect of steviol on renal cyst growth in an orthologous mouse model of human ADPKD (Pkd1(flox/flox):Pkhd1-Cre). The results showed that daily treatment with both 200mg/kg BW of steviol and 1000mg/kg BW of stevioside for 14 days markedly decreased kidney weight and cystic index in these mice. However, only steviol markedly reduced blood urea nitrogen and creatinine values. Steviol also reduced cell proliferation but had no effect on cell apoptosis. In addition, steviol suppressed CFTR and mTOR/S6K expression in renal cyst-lining epithelial cells. Interestingly, steviol was found to stimulate AMP-activated protein kinase (AMPK). Our findings indicate that steviol slows cyst progression in ADPKD mouse model, in part, through the activation of AMPK which subsequently inhibits CFTR chloride channel expression and inhibits renal epithelial cell proliferation via mTOR/S6K pathway. Most importantly, steviol could markedly improve kidney function in a mouse model of ADPKD. Steviol thus has potential application for further development as a therapeutic compound for the treatment of polycystic kidney disease.

Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Cell Proliferation; Cystic Fibrosis Transmembrane Conductance Regulator; Diterpenes, Kaurane; Enzyme Activation; Epithelial Cells; Glucosides; Humans; Mice; Mice, Mutant Strains; Polycystic Kidney Diseases

The aim of this work is to propose an HPLC method for analysing major steviol glycosides as well as to optimise the extraction and clarification conditions for obtaining these compounds. Toward this aim, standards of stevioside and rebaudioside A with purities ⩾99.0%, commercial samples from different companies and Stevia rebaudiana Bertoni leaves from Paraguay supplied by Insobol, S.L., were used. The analytical method proposed is adequate in terms of selectivity, sensitivity and accuracy. Optimum extraction conditions and adequate clarification conditions have been set. Moreover, this methodology is safe and eco-friendly, as we use only water for extraction and do not use solid-phase extraction, which requires solvents that are banned in the food industry to condition the cartridge and elute the steviol glycosides. In addition, this methodology consumes little time as leaves are not ground and the filtration is faster, and the peak resolution is better as we used an HPLC method with gradient elution.

Topics: Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Glucosides; Glycosides; Plant Leaves; Solid Phase Extraction

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

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

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

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

We have applied a diversity-oriented approach for the synthesis of skeletally diverse and stereochemically complex templates for small-molecule library production by performing Beckmann rearrangement and Beckmann fragmentation reactions on the bicyclo[3.2.1]octane rings of steviol and isosteviol, aglycones derived from the diterpene natural product stevioside. The optimization of these two reaction pathways is presented along with the successful application of a photo-Beckmann rearrangement. This work also led to the discovery of cyano-Prins-type and Thorpe-Ziegler-type cyclization reactions.

Topics: Biological Products; Combinatorial Chemistry Techniques; Cyclization; Diterpenes, Kaurane; Glucosides; Nuclear Magnetic Resonance, Biomolecular; Octanes

This study aimed to develop an economically viable enzyme for the optimal production of steviol (S) from stevioside (ST). Of 9 commercially available glycosidases tested, S-producing β-glucosidase (SPGase) was selected and purified 74-fold from Penicillium decumbens naringinase by a three-step column chromatography procedure. The 121-kDa protein was stable at pH 2.3-6.0 and at 40-60 °C. Hydrolysis of ST by SPGase produced rubusoside (R), steviolbioside (SteB), steviol mono-glucoside (SMG), and S, as determined by HPLC, HPLC-MS, and (1)H- and (13)C-nuclear magnetic resonance. SPGase showed higher activity toward steviol mono-glucosyl ester, ST, R, and SMG than other β-linked glucobioses. The optimal conditions for S production (30 mM, 64 % yield) were 47 mM ST and 43 μl of SPGase at pH 4.0 and 55 °C. This is the first report detailing the production of S from ST hydrolysis by a novel β-glucosidase, which may be useful for the pharmaceutical and agricultural areas.

Topics: beta-Glucosidase; Diterpenes, Kaurane; Enzyme Stability; Fungal Proteins; Glucosides; Kinetics; Penicillium; Substrate Specificity

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

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

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

The structure activity relationship between the C₁₆-C₁₇ methylene double bond on the aglycone of steviol glycosides and the corresponding impact on their sweet taste has been reported here for the first time. It has been observed that converting stevioside and rebaudioside A to their corresponding ketones by switching the doubly bonded methylene on C-17 for a ketone group actually removes the sweet taste properties of these molecules completely. Regenerating the original molecules tends to restore the sweet taste of both the steviol glycosides. Thus this C₁₆-C₁₇ methylene double bond in rebaudioside A and stevioside can be regarded as a pharmacophore essential for the sweetness property of these molecules.

Topics: Diterpenes, Kaurane; Glucosides; Sweetening Agents

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

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

In a search for potential cancer chemopreventive agents from natural resources, stevioside (1), a sweetener, and six related compounds, including two aglycones steviol (6) and isosteviol (7), were screened in an in vitro assay for inhibitory effects on Epstein-Barr virus early antigen activation. Compounds 1, 6 and 7 showed significant activity in this assay and also exhibited strong inhibitory effects in a two-stage carcinogenesis test using mouse skin induced by 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The inhibitory effects of these three compounds were greater than that of glycyrrhizin. Furthermore, these three compounds significantly inhibited mouse skin carcinogenesis initiated by peroxynitrite and promoted by TPA. Their activities were comparable to that of curcumin. These results suggested that 1, as well as 6 and 7, could be valuable as chemopreventive agents for chemical carcinogenesis.

Topics: Animals; Antineoplastic Agents; Carcinogenicity Tests; Chemoprevention; Curcumin; Diterpenes, Kaurane; Glucosides; Glycyrrhizic Acid; Herpesvirus 4, Human; Mice; Skin Neoplasms

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

Stevioside and its major metabolite, steviol, have been reported to affect ion transport in many types of tissues, such as the kidney, pancreas, and intestine. The effect of stevioside, steviol, and its analogs on intestinal Cl(-) secretion was investigated in a human T84 epithelial cell line. Short-circuit current measurements showed that steviol and analogs isosteviol, dihydroisosteviol, and isosteviol 16-oxime inhibited in a dose-dependent manner forskolin-induced Cl(-) secretion with IC(50) values of 101, 100, 9.6, and 50 microM, respectively, whereas the parent compound stevioside had no effect. Apical Cl(-) current measurement indicated that dihydroisosteviol targeted the cystic fibrosis transmembrane regulator (CFTR). The inhibitory action of dihydroisosteviol was reversible and was not associated with changes in the intracellular cAMP level. In addition, dihydroisosteviol did not affect calcium-activated chloride secretion and T84 cell viability. In vivo studies using a mouse closed-loop model of cholera toxin-induced intestinal fluid secretion showed that intraluminal injection of 50 microM dihydroisosteviol reduced intestinal fluid secretion by 88.2% without altering fluid absorption. These results indicate that dihydroisosteviol and similar compounds could be a new class of CFTR inhibitors that may be useful for further development as antidiarrheal agents.

Topics: Animals; Cell Line, Tumor; Cell Survival; Cholera Toxin; Diterpenes, Kaurane; Dose-Response Relationship, Drug; Glucosides; Humans; Intestinal Secretions; Jejunum; Mice; Mice, Inbred ICR; Plant Extracts

This work describes the preparation of tetracyclic diterpenoids and determination of their plant growth regulator properties. Stevioside (2) was used as starting material and the derivatives 13-hydroxy-ent-kaur-16-en-19-oic acid (steviol, 3), ent-7alpha,13-dihydroxy-kaur-16-en-19-oic acid (4), 13-hydroxy, ent-kaur-16,17-epoxi-19-oic acid (steviol epoxide, 5), 17-hydroxy-16-ketobayeran-19-oic acid (17-hydroxyisosteviol, 6), 17-hydroxy-16-hydroxyiminobayeran-19-oic acid (7), 16-ketobayeran-19-oic acid (isosteviol, 9), 16,17-dihydroxybeyeran-19-oic acid (8), and 16-hydroxyiminobayeran-19-oic acid (isosteviol oxime, 10) were obtained by simple chemical procedures. Another derivative, ent-7alpha,13-dihydroxycaur-15-en-19-oic acid (4), was obtained by biotransformation of steviol (3) by Penicillium citrinum. In order to determine the plant growth regulator activity the compounds were submitted to the lettuce hypocotyl and barley aleurone bioassays. All compounds showed significant activities in both bioassays. Steviol (3) and isosteviol (9) were also tested in field-grown grapes resulting in an increase in berry weight and size.

Topics: Diterpenes, Kaurane; Glucosides; Hordeum; Lactuca; Molecular Structure; Plant Growth Regulators; Structure-Activity Relationship

Stevioside, isolated from Stevia rebaudiana, is a commercial sweetener. It was previously demonstrated that stevioside attenuates NF-kappaB-dependent TNF-alpha and IL-1beta synthesis in LPS-stimulated monocytes. The present study examined the effects of stevioside and its metabolite, steviol, on human colon carcinoma cell lines. High concentrations of stevioside (2-5 mM) and steviol (0.2-0.8 mM) decreased cell viability in T84, Caco-2, and HT29 cells. Stevioside (2 mM) potentiated TNF-alpha-mediated IL-8 release in T84 cells. However, steviol (0.01-0.2 mM) significantly suppressed TNF-alpha-induced IL-8 release in all three cell lines. In T84 cells, steviol attenuated TNF-alpha-stimulated IkappaB --> NF-kappaB signaling. Chloride transport was stimulated by steviol (0.1 mM) > stevioside (1 mM) at 30 min. Two biological effects of steviol in the colon are demonstrated for the first time: stimulation of Cl(-) secretion and attenuation of TNF-alpha-stimulated IL-8 production. The immunomodulatory effects of steviol appear to involve NF-kappaB signaling. In contrast, at nontoxic concentrations stevioside affects only Cl(-) secretion.

Topics: Caco-2 Cells; Diterpenes, Kaurane; Glucosides; HT29 Cells; Humans; I-kappa B Proteins; Interleukin-8; Intestinal Mucosa; Intestines; NF-kappa B; NF-KappaB Inhibitor alpha; Signal Transduction; Sweetening Agents; Tumor Necrosis Factor-alpha

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

Stevioside (250-mg capsules) was given thrice daily for 3 days to 10 healthy subjects. Blood samples were collected and blood pressure measured after nocturnal fasting, before and at different time points during the third day of the administration of stevioside. No significant differences were found between the control and the stevioside condition for blood pressure and blood biochemical parameters. The 24-hr urinary volume and urinary excretion of electrolytes were not significantly different. Likewise, no significant difference was found for mean blood glucose and insulin between control and stevioside conditions. Thus, oral stevioside is not directly effective as a hypotensive or hypoglycemic agent in healthy subjects at the dose administered in this study. Stevioside, free steviol, and steviol metabolites were analyzed in blood, feces, and urine after 3 days of stevioside administration. No uptake was found of stevioside by the gastrointestinal tract or the amounts taken up were very low and below the detection limit of the UV detector. Stomach juice did not degrade stevioside. All the stevioside reaching the colon was degraded by micro-organisms into steviol, the only metabolite found in feces. In blood plasma, no stevioside, no free steviol or other free steviol metabolites were found. However, steviol glucuronide (SV glu) was found in maximum concentrations of 33 micro g/ml (21.3 micro g steviol equivalents/ml). In urine, no stevioside or free steviol were present, but SV glu was found in amounts of up to 318 mg/24-hr urine (205 mg steviol equivalents/24 hrs). No other steviol derivatives were detected. In feces, besides free steviol, no other steviol metabolites or conjugates were detected. Steviol was excreted as SV glu in urine.

Topics: Adult; Biotransformation; Blood Glucose; Blood Pressure; Diterpenes, Kaurane; Electrolytes; Feces; Female; Gastric Juice; Glucosides; Humans; Insulin; Male; Urination; Urine

Stevioside is a natural sweetener obtained from the leaves of Stevia rebaudiana. It is a glycoside of steviol and other glycosides of the same aglycone are also found in the plant. Stevioside is usually the major component in commercial products, but it is not the one with the best organoleptic properties. It has a bitter aftertaste and, for this reason, attempts have been made in order to modify its molecule. In this work, the commercial and purified stevioside were modified by hydrolytic enzymes from Gibberella fujikuroi. A screening was carried out on six strains of the fungus in order to select the most active. The production of the enzymes by the fungi was induced by its culture in a medium containing stevioside as the sole carbon source and the enzymatic extract was then used in the experiments. The products obtained were analyzed by HPLC-UV and HPLC-MS/MS. The results showed a significant increase in the concentration of rebaudioside A in the final product, which has better organoleptic properties than stevioside.

Topics: Catalysis; Cell-Free System; Chromatography, High Pressure Liquid; Diterpenes, Kaurane; Gibberella; Glucosides; Hydrolysis; Kinetics; Mass Spectrometry

Stevioside, a natural noncaloric sweetener isolated from Stevia rebaudiana Bertoni, possesses anti-inflammatory and antitumor promoting properties; however, no information is available to explain its activity. The aim of this study was to elucidate the anti-inflammatory and immunomodulatory activities of stevioside and its metabolite, steviol. Stevioside at 1 mM significantly suppressed lipopolysaccharide (LPS)-induced release of TNF-alpha and IL-1beta and slightly suppressed nitric oxide release in THP-1 cells without exerting any direct toxic effect, whereas steviol at 100 microM did not. Activation of IKKbeta and transcription factor NF-kappaB were suppressed by stevioside, as demonstrated by Western blotting. Furthermore, only stevioside induced TNF-alpha, IL-1beta, and nitric oxide release in unstimulated THP-1 cells. Release of TNF-alpha could be partially neutralized by anti-TLR4 antibody. This study suggested that stevioside attenuates synthesis of inflammatory mediators in LPS-stimulated THP-1 cells by interfering with the IKKbeta and NF-kappaB signaling pathway, and stevioside-induced TNF-alpha secretion is partially mediated through TLR4.

Topics: Anti-Inflammatory Agents; Cell Line; Diterpenes, Kaurane; Glucosides; Humans; I-kappa B Kinase; Immunity; Interleukin-1; Monocytes; NF-kappa B; Nitric Oxide; Signal Transduction; Sweetening Agents; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Stevioside (250 mg capsules) was given three times daily to 10 healthy subjects. Steviol glucuronide (steviol 19-O-beta-D-glucopyranosiduronic acid; MM, 494.58; melting point, 198-199 degrees C) was characterized in the 24 h urine as the only excretion product of oral stevioside by MS, NMR, IR, and UV spectroscopy. This is the first report on the unambiguous identification of steviol glucuronide in human urine.

Topics: Diterpenes, Kaurane; Glucosides; Glucuronides; Humans; Magnetic Resonance Spectroscopy; Mass Spectrometry; Spectrophotometry, Infrared; Spectrophotometry, Ultraviolet

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

Our previous studies have shown that both stevioside and steviol inhibited transepithelial transport of para-aminohippurate (PAH) in isolated rabbit renal proximal tubules by interfering with organic anion transport system. The current study examined the direct interactions of stevioside and steviol with specific organic anion transporters.. S2 cells expressing human organic anion transporters (hOAT1, hOAT2, hOAT3, and hOAT4) and an intact renal epithelium were used to determine the inhibitory effect of stevioside and steviol on organic anion transport.. Stevioside at 0.5-1 mM showed no interaction with any OAT. In contrast, steviol markedly inhibited substrate uptake in all S2hOAT cells. Steviol had low IC50 for hOAT1 (11.4 microM) and hOAT3 (36.5 microM) similar to that of probenecid, whereas IC50 for hOAT2 (1000 microM) and hOAT4 (285 microM) was much higher. Results obtained in mouse renal cortical slices were very similar; that is, stevioside was without inhibitory effect and steviol was a potent inhibitor of PAH and estrone sulfate (ES) transport.. Stevioside has no interaction with human or mouse OATs. In contrast, steviol interacts directly with human OATs, in particular, hOAT1 and hOAT3, with a potency approximating probenecid, suggesting that the inhibition of OAT-mediated transport by steviol could alter renal drug clearance.

Topics: Animals; Cell Line; Cell Survival; Diterpenes, Kaurane; Estrone; Glucosides; Glutarates; In Vitro Techniques; Kidney; Kidney Cortex; Kidney Tubules, Proximal; Kinetics; Mice; Mice, Inbred C57BL; Organic Anion Transporters; p-Aminohippuric Acid

The main toxicological concern of stevioside, a highly potent sweetener from S. rebaudiana, is its main metabolite, steviol. To determine very low levels of steviol in in vivo experiments, a sensitive liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) method was developed for quantifying steviol in rat plasma after oral administration of a single dose of stevioside (0.5 g/kg). The sample preparation uses liquid-liquid extraction with tert-butyl methyl ether in an acidic environment. The retention time of steviol was 10.5 min. The assay was linear over the range 2-1000 ng/mL with a lower limit of detection of 1 ng/mL. The intra- and inter-day precision were <5 and <7%, respectively, and the accuracy was in the range 95-108%. The steviol concentration profile in rat plasma was determined.

Topics: Administration, Oral; Animals; Blood Chemical Analysis; Chromatography, High Pressure Liquid; Diterpenes; Diterpenes, Kaurane; Glucosides; Male; Metabolic Clearance Rate; Microchemistry; Plasma; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization

Microbial transformation of isosteviol (2), a beyerane-type diterpenoid obtained from stevioside (1) by acid hydrolysis, yielded 7beta-hydroxyisosteviol (3), 11beta-hydroxyisosteviol (5), and 12beta-hydroxyisosteviol (6) by the fungus Aspergillus niger, 17-hydroxyisosteviol (7) by the fungus Glomerella cingulata, and 3 and 7-oxoisosteviol (4) by the fungus Mortierella elongate. The five metabolites, 3-7, along with 1 and 2 were evaluated for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells as a primary screening test for inhibitors of tumor promoters. All the diterpenes tested showed potent inhibitory effects, with the five metabolites 3-7 exhibiting more potent effects.

Topics: Antigens, Viral; Antineoplastic Agents, Phytogenic; Biotransformation; Diterpenes; Diterpenes, Kaurane; Dose-Response Relationship, Drug; Glucosides; Herpesvirus 4, Human; Humans; Mortierella; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Virus Activation

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

A new group of steviolbioside amide dimers 2a-g, derivatives 2h-i and their related steviol and isosteviol amide dimers 3a and 4a were prepared by reacting aliphatic alkylamine and alkyldiamines with PyBOP and DIEA. The synthesized compounds had cytotoxic effects on cancer and human embryonic lung cells. Compounds 3a, 4a, 2b and 2h were cytotoxic to cancer cells and to a lesser extent to human embryo lung cells. Compounds 2f, 2g and 4 of this series had favorable antibacterial effects, and were superior to penicillin G at inhibiting growth of Bacillus subtilis (BCRC 10029). The cytotoxicity and antibacterial effects may depend on the dimerization and derivative moieties in relation to the respective aglycons.

Topics: Amides; Amines; Anti-Bacterial Agents; Antineoplastic Agents; Bacillus subtilis; Cell Line, Tumor; Diterpenes, Kaurane; Escherichia coli; Glucosides; Humans; Microbial Sensitivity Tests; Organophosphorus Compounds; Pseudomonas aeruginosa; Structure-Activity Relationship; Triazoles

In intubation experiments (643-1168 mg per animal), most of the stevioside administered to chickens was recovered unchanged in the excreta, and only about 2% was converted into steviol. Neither stevioside nor steviol could be found in the blood. In chronic studies (667 mg of stevioside/kg of feed) with laying hens and meat-type chickens, no significant differences were found in feed uptake, weight gain, and feed conversion as the result of stevioside administration. The egg production and egg composition of laying hens were not influenced. Most of the stevioside taken up was found untransformed in the excreta, and about 21.5% or 7.3% was converted to steviol by meat-type chickens or laying hens, respectively. No stevioside or steviol could be detected in the blood or in the eggs of the different groups of animals. In anaerobic incubation experiments with chicken excreta, only a 20% conversion of stevioside into steviol was found. No harmful effects were observed in the chronic stevioside supplementation experiments nor in the intubation experiments in which very high stevioside doses were given.

Topics: Animal Nutritional Physiological Phenomena; Animals; Chickens; Diet; Diterpenes; Diterpenes, Kaurane; Eating; Eggs; Feces; Female; Glucosides; Oviposition; Sweetening Agents; Weight Gain

At day 7 of incubation, fertile broiler eggs were injected with different amounts of stevioside and steviol of 0.08, 0.8, or 4 mg stevioside/egg and 0.025, 0.25, or 1.25 mg steviol/egg. At hatch (day 21) and 1 week later, not any influence of the different treatments could be found on embryonic mortality, body weight of the hatchlings, deformations (e.g., bone, beak, and head malformations, abnormal feathering, open vent), or abnormal development of the gonads. No stevioside or steviol could be detected in the blood of the hatchlings. The hatchlings developed normally. It is concluded that prenatal exposure to stevioside and steviol is not toxic for the chicken embryo.

Topics: Animals; Chick Embryo; Chickens; Cricetinae; Diterpenes; Diterpenes, Kaurane; Female; Glucosides; Male; Pregnancy; Reproduction; Sweetening Agents

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

The natural sweetener stevioside, which is found in the plant Stevia rebaudiana Bertoni, has been used for many years in the treatment of diabetes among Indians in Paraguay and Brazil. However, the mechanism for the blood glucose-lowering effect remains unknown. To elucidate the impact of stevioside and its aglucon steviol on insulin release from normal mouse islets and the beta-cell line INS-1 were used. Both stevioside and steviol (1 nmol/L to 1 mmol/L) dose-dependently enhanced insulin secretion from incubated mouse islets in the presence of 16.7 mmol/L glucose (P < .05). The insulinotropic effects of stevioside and steviol were critically dependent on the prevailing glucose concentration, ie, stevioside (1 mmol/L) and steviol (1 micromol/L) only potentiated insulin secretion at or above 8.3 mmol/L glucose (P < .05). Interestingly, the insulinotropic effects of both stevioside and steviol were preserved in the absence of extracellular Ca2+. During perifusion of islets, stevioside (1 mmol/L) and steviol (1 micromol/L) had a long-lasting and apparently reversible insulinotropic effect in the presence of 16.7 mmol/L glucose (P < .05). To determine if stevioside and steviol act directly on beta cells, the effects on INS-1 cells were also investigated. Stevioside and steviol both potentiated insulin secretion from INS-1 cells (P < .05). Neither stevioside (1 to 100 micromol/L) nor steviol (10 nmol/L to 10 micromol/L) influenced the plasma membrane K+ adenosine triphosphate ((K+)ATP)-sensitive channel activity, nor did they alter cyclic adenosine monophosphate (cAMP) levels in islets. In conclusion, stevioside and steviol stimulate insulin secretion via a direct action on beta cells. The results indicate that the compounds may have a potential role as antihyperglycemic agents in the treatment of type 2 diabetes mellitus.

Topics: Animals; ATP-Binding Cassette Transporters; Calcium; Cyclic AMP; Diterpenes; Diterpenes, Kaurane; Electrophysiology; Glucose; Glucosides; In Vitro Techniques; Insulin; Insulin Secretion; Islets of Langerhans; KATP Channels; Male; Mice; Patch-Clamp Techniques; Potassium Channels; Potassium Channels, Inwardly Rectifying; Terpenes

The developmental toxicity of steviol, a metabolite of stevioside, was studied in hamsters. Pregnant hamsters were intubated with steviol at dose levels of 0, 0.25, 0.5, 0.75 and 1.0 g/kg BW/day on days 6-10 of gestation. Steviol at doses of 0.75 and 1.0 g/kg BW/day were highly toxic to both dams and fetuses. Significant decrease of maternal body-weight gain during the experimental period (days 6-14) and high percentage of maternal mortality indicated the general toxicity of these two high doses. The number of live fetuses per litter and mean fetal weight also significantly decreased in the steviol-treated animals at doses of 0.75 and 1.0 g/kg BW day. The animals treated with an intermediate dose (0.50 g/kg BW/day) exhibited less signs of maternal and developmental toxicity than the two high doses (0.75 and 1.0 g/kg BW/day). One craniomeningocele was found in a fetus under the maternal toxic condition in steviol-treated at a dose of 0.75 g/kg BW/day. Neither the skeleton nor visceral development of the offspring was affected by steviol treatment except delayed ossification of the xiphoid (bifid) and long bones of the limbs and supernumerary thoracic ribs (14th ribs) tended to be increased at doses of 0.5 to 1.0 g/kg BW/day steviol. No dose-related teratogenesis was detected. From the result of the present study concerning maternal toxic condition and embryotoxicity, an oral dose of 0.25 g steviol/kg BW/day is regarded as having no observable effect. This steviol-treated dose is derived from stevioside 625 mg/kg BW/day which is approximately 80 times higher than the suggested acceptable daily intake of stevioside for humans (7.938 mg/kg BW/day).

Topics: Abnormalities, Drug-Induced; Administration, Oral; Animals; Cricetinae; Diterpenes; Diterpenes, Kaurane; Dose-Response Relationship, Drug; Embryonic and Fetal Development; Female; Fetal Viability; Gestational Age; Glucosides; Pregnancy; Sweetening Agents; Terpenes; Weight Gain

Stevioside, a constituent of Stevia rebaudiana, is commonly used as a non-caloric sugar substitute in Japan. The genetic toxicities of stevioside and its aglycone, steviol, were examined with seven mutagenicity tests using bacteria (reverse mutation assay, forward mutation assay, umu test and rec assay), cultured mammalian cells (chromosomal aberration test and gene mutation assay) and mice (micronucleus test). Stevioside was not mutagenic in any of the assays examined. The aglycone, steviol, however, produced dose-related positive responses in some mutagenicity tests, i.e. the forward mutation assay using Salmonella typhimurium TM677, the chromosomal aberration test using Chinese hamster lung fibroblast cell line (CHL) and the gene mutation assay using CHL. Metabolic activation systems containing 9000 g supernatant fraction (S9) of liver homogenates prepared from polychlorinated biphenyl or phenobarbital plus 5,6-benzoflavone-pretreated rats were required for mutagenesis and clastogenesis. Steviol was weakly positive in the umu test using S.typhimurium TA1535/pSK1002 either with or without the metabolic activation system. Steviol, even in the presence of the S9 activation system, was negative in other assays, i.e. the reverse mutation assays using S.typhimurium TA97, TA98, TA100, TA102, TA104, TA1535, TA1537 and Escherichia coli WP2 uvrA/pKM101 and the rec-assay using Bacillus subtilis. Steviol was negative in the mouse micronucleus test. The genotoxic risk of steviol to humans is discussed.

Topics: Animals; Bacillus subtilis; Biotransformation; CHO Cells; Cricetinae; Diterpenes; Diterpenes, Kaurane; Escherichia coli; Glucosides; Male; Mice; Micronucleus Tests; Mutagenicity Tests; Salmonella typhimurium; Sweetening Agents; Terpenes

131I-STEVIOSIDE (1.10 MBq) was injected i.v in Wistar male rats, in distribution in the body and metabolism were studied. The highest concentration of radioactivity was observed in the liver and in the small intestine after 10 and 120 minutes, respectively. At 2 h after injection, the radioactivity eliminated in the bile was 52.0% of the original dose. The results of RP-HPLC analysis of the bile showed that stevioside was degraded in vivo and that steviol appeared as a major metabolite. However, in the urine; RP-HPLC analysis did not show the presence of steviol.

Topics: Animals; Biotransformation; Chromatography, High Pressure Liquid; Diterpenes; Diterpenes, Kaurane; Feces; Glucosides; Injections, Intravenous; Iodine Radioisotopes; Male; Rats; Rats, Wistar; Terpenes; Tissue Distribution

The effects of stevioside and steviol (a product of enzymatic hydrolysis of stevioside) on intestinal glucose absorption were examined in hamster jejunum. By using the everted sac technique, we found that stevioside (1 and 5 mM) had no inhibitory effect on glucose absorption. In contrast, glucose absorption was inhibited 29% by 1 mM steviol. The inhibition of glucose absorption by steviol was related to steviol concentration and incubation time. The possible mechanism of steviol inhibitory action of glucose absorption was also investigated. Reductions in the intestinal mucosal ATP content and absorptive surface area were responsible for the inhibition of glucose absorption by steviol. The decrease in the intestinal mucosal ATP content was accompanied by a decrease in the activities of mitochondrial NADH cytochrome c reductase and cytochrome oxidase. Moreover, no inhibitory effects of steviol on the activity of intestinal Na+,K(+)-ATPase and glucose uptake in the intestinal brush-border membrane vesicles were seen. These results suggest that inhibition of intestinal glucose absorption by steviol in hamsters is due to the reduction in mucosal ATP content and an alteration of the morphology of the intestinal absorptive cells.

Topics: Adenosine Triphosphate; Animals; Biological Transport, Active; Cricetinae; Diterpenes; Diterpenes, Kaurane; Electron Transport Complex IV; Glucose; Glucosides; In Vitro Techniques; Intestinal Absorption; Jejunum; Male; Mesocricetus; Mitochondria; NADH Dehydrogenase; Sodium-Potassium-Exchanging ATPase; Sweetening Agents; Terpenes

The effects of stevioside and steviol (a product of enzymatic hydrolysis of stevioside) on intestinal glucose absorption were examined in the hamster jejunum in vitro. By using the jejunal rings technique, we found that stevioside at a high dose of 5 mM had no inhibitory effect on glucose absorption. In contrast, glucose absorption was inhibited 43% by 1 mM steviol. The inhibition of glucose absorption by steviol was related to steviol concentration and incubation time. The inhibitory effect of steviol compared to phlorizin and ouabain was also investigated. Steviol, which caused a decrease in glucose accumulation in the intestinal ring tissues, possibly acts on the brush border membrane as does phlorizin. Furthermore, it was also found that steviol altered the morphology of the intestinal absorptive cells. These results suggest that the possible site of inhibitory action of steviol might be on the mucosal side and/or at the intracellular organelles of intestinal absorptive cells.

Topics: Animals; Cricetinae; Diterpenes; Diterpenes, Kaurane; Dose-Response Relationship, Drug; Glucose; Glucosides; Intestinal Absorption; Jejunum; Male; Mesocricetus; Organelles; Ouabain; Phlorhizin; Terpenes; Time Factors

The influence of stevioside, the sweet glycoside of Stevia rebaudiana leaves, on the glycogen levels of fasted rats was investigated. In one set of experiments, single doses of stevioside (200 mumol) or steviol (200 mumol) were given orally to 24-hours fasted rats, either alone or simultaneously with fructose. Under these conditions both stevioside and steviol increased the initial glycogen deposition in the liver. In another set of experiments, stevioside was given to the rats in the drinking water at the beginning of the fasting periods (5:00 p.m.) of 24 and 48 hours. Two different concentrations were given, 1.0 and 2.0 mM. Increased hepatic glycogen levels were found at 48 hours with stevioside (1.0 mM) and at 24 hours with stevioside (2.0 mM). Steviol had no effect on hepatic glycogen levels when given in the drinking water. It can be concluded that stevioside exerts a stimulatory action on hepatic glycogen synthesis under gluconeogenic conditions.

Topics: Administration, Oral; Animals; Diterpenes; Diterpenes, Kaurane; Fasting; Gluconeogenesis; Glucosides; Glycogen; Liver; Male; Rats; Rats, Wistar; Sweetening Agents; Terpenes

Leaves of Stevia rebaudiana Bertoni have been popularly used as a sweetener in foods and beverages for diabetics and obese people due to their potent sweetener stevioside. In this report, stevioside and steviol were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 and for chromosomal effects on cultured human lymphocytes. Stevioside was not mutagenic at concentrations up to 25 mg/plate, but showed direct mutagenicity to only TA98 at 50 mg/plate. However, steviol did not exhibit mutagenicity in either TA98 or TA100, with or without metabolic activation. No significant chromosomal effect of stevioside and steviol was observed in cultured blood lymphocytes from healthy donors (n = 5). This study indicates that stevioside and steviol are neither mutagenic nor clastogenic in vitro at the limited doses; however, in vivo genotoxic tests and long-term effects of stevioside and steviol are yet to be investigated.

Topics: Animals; Chromosome Aberrations; Diterpenes; Diterpenes, Kaurane; Female; Glucosides; Humans; Molecular Structure; Mutagenicity Tests; Plant Extracts; Rats; Salmonella typhimurium; 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

The effect of several natural products of Stevia rebaudiana on glucose production and oxygen uptake in rat renal cortical tubules was investigated. Steviol, isosteviol and glucosilsteviol decreased glucose production and inhibited oxygen uptake. The sweet principle stevioside, and steviolbioside, however, were without effect on gluconeogenesis and oxygen uptake.

Topics: Animals; Chemical Phenomena; Chemistry; Diterpenes; Diterpenes, Kaurane; Gluconeogenesis; Glucosides; Kidney Tubules; Male; Oxygen Consumption; Rats; Rats, Inbred Strains; Structure-Activity Relationship; Terpenes

Stevioside, a constituent of Stevia rebaudiana, is commonly used as a noncaloric sugar substitute in Japan. Consistent with reports in the literature, we have found that stevioside is not mutagenic as judged by utilization of Salmonella typhimurium strain TM677, either in the presence or in the absence of a metabolic activating system. Similar negative results were obtained with several structurally related sweet-tasting glycosides. However, steviol, the aglycone of stevioside, was found to be highly mutagenic when evaluated in the presence of a 9000 X g supernatant fraction derived from the livers of Aroclor 1254-pretreated rats. Expression of mutagenic activity was dependent on both pretreatment of the rats with Aroclor 1254 and addition of NADPH; unmetabolized steviol was not active. The structurally related species, isosteviol, was not active regardless of metabolic activation. Similarly, chemical reduction of the unsaturated bond linking the carbon-16 and -17 positions of steviol resulted in the generation of two isomeric products, dihydrosteviol A and B, that were not mutagenic. In addition, ent-kaurenoic acid was found to be inactive. It is therefore clear that a metabolite of an integral component of stevioside is mutagenic; structural features of requisite importance for the expression of mutagenic activity include a hydroxy group at position 13 and an unsaturated bond joining the carbon atoms at positions 16 and 17. A potential metabolite of steviol, steviol-16 alpha,17-epoxide, was synthesized chemically and found to be ineffective as a direct-acting mutagen. Thus, although stevioside itself appears innocuous, it would seem prudent to expeditiously and unequivocally establish the human metabolic disposition of this substance.

Topics: Animals; Aroclors; Biotransformation; Chlorodiphenyl (54% Chlorine); Diterpenes; Diterpenes, Kaurane; Glucosides; In Vitro Techniques; Liver; Male; Mutagens; Rats; Salmonella typhimurium; Sweetening Agents; Terpenes

Topics: Diterpenes; Diterpenes, Kaurane; Glucosides; Glycosides; Humans; Sweetening Agents; Terpenes