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Stevia rebaudiana is zero-calorie bio-sweetener and furnishes the best nutrition out of it. The study was conducted to characterize the extracts from water, ethanol, methanol, and supercritical fluid for Steviosides/SGs. The compounds found in appreciable quantities were Stevioside (665.34 ± 27.27 to 1107.95 ± 50.96 mg/kg), Rebaudioside A (383.38 ± 17.25 to 792.15 ± 38.02 mg/kg) and Steviol (357.26 ± 14.64 to 485.25 ± 22.32 mg/kg). Stevia cookies were prepared by replacing sucrose with Stevia powder (10%), water extract (1%), and supercritical extract (3%). Bio-efficacy rats modeling trials were done to check their impact against hyperglycemia and hypercholesterolemia. Blood glucose level was reduced from 4.54 to 7.00%, insulin level was increased by 4.27 to 6.13% due to diets enriched with stevia powder and extracts as compared to the control. In hypercholesterolemic rats, up surged cholesterol level was reported to reduce as a function of Stevia powder and extracts from 1.28 to 5.47%. Substantial increment in HDL, and reduction in LDL and triglycerides was recorded in Stevia leaves powder and extracts treatments by 2.79–6.66%, 2.68–4.16%, and 3.12–5.36, respectively. Stevia cookies administration in rats showed non-significant impact on the liver, renal, and hepatological parameters, while the safety of cookies was improved. Therefore, it can be concluded from the outcomes that in addition to sweetness, Stevia health health-beneficial effects make it suitable to be included in cookies.

Stevioside is a secondary metabolite of diterpenoid glycoside production in plants. It has been used as a natural sweetener in various foods because of its high sweetness and low-calorie content. In this study, we constructed a Saccharomyces cerevisiae strain for the complete synthesis of stevioside using a metabolic engineering strategy. Firstly, the synthesis pathway of steviol was modularly constructed in S. cerevisiae BY4742, and the precursor pathway was strengthened. The yield of steviol was used as an indicator to investigate the expression effect of different sources of diterpene synthases under different combinations, and the strains with further improved steviol yield were screened. Secondly, glycosyltransferases were heterologously expressed in this strain to produce stevioside, the sequence of glycosyltransferase expression was optimized, and the uridine diphosphate-glucose (UDP-Glc) supply was enhanced. Finally, the results showed that the strain SST-302III-ST2 produced 164.89 mg/L of stevioside in a shake flask experiment, and the yield of stevioside reached 1104.49 mg/L in an experiment employing a 10 L bioreactor with batch feeding, which was the highest yield reported. We constructed strains with a high production of stevioside, thus laying the foundation for the production of other classes of steviol glycosides and holding good prospects for application and promotion. [ABSTRACT FROM AUTHOR]

Steviol and isosteviol were prepared from the commercially available sweetener stevioside and converted into lipophilic F16 hybrids. Their cytotoxicity was determined in SRB assays and showed to depend on both the substitution pattern of the aromatic substituent as well as on the spacer length. Therefore, compound 25 held an IC50 (A2780) of 180 nM, thus surpassing the activity of comparable rhodamine hybrids. Several of the compounds were also able to overcome drug resistance in the A2780/A2780cis model. Extra staining experiments showed a similar subcellular accumulation pattern of the F16 hybrids as a well-established mitocan, hence proving preferential mitochondrial accumulation but also some other accumulation in other cellular areas. [ABSTRACT FROM AUTHOR]

A series of novel diterpene-type 1,3-aminoalcohols and their regioisomers have been synthesised from natural stevioside in a stereoselective manner. The key intermediate β-keto alcohol was prepared using Wagner–Meerwein rearrangement of the epoxide derived from steviol methyl ester. The primary aminoalcohol was formed via Raney-nickel-catalysed hydrogenation of an oxime, and a versatile library of aminoalcohols was synthesised using a Schiff base with the primary amines. The aminoalcohol regioisomers were prepared from the mesylate of the β-keto alcohols. The corresponding primary aminoalcohol was formed via the palladium-catalysed hydrogenation of hydroxyl-azide, and click reactions of the latter were also carried out. The new compounds were characterised using 1D- and 2D-NMR techniques and HRMS measurements. The in vitro investigations showed high inhibition of cell growth in human cancer cell lines (HeLa, SiHa, A2780, MCF-7 and MDA-MB-231) in the case of naphthalic N-substituted derivatives. The antiproliferative effects were assayed using the MTT method. [ABSTRACT FROM AUTHOR]

Worldwide, the demand for natural and synthetic sweeteners in the food industry as an alternative to refined sugar is increasing. This has prompted more research to be conducted to estimate its safety and effects on health. The gut microbiome is critical in metabolizing selected sweeteners which might affect overall health. Recently, more studies have evaluated the relationship between sweeteners and the gut microbiome. This review summarizes the current knowledge regarding the role played by the gut microbiome in metabolizing selected sweeteners. It also addresses the influence of the five selected sweeteners and their metabolites on GI cancer-related pathways. Overall, the observed positive effects of sweetener consumption on GI cancer pathways, such as apoptosis and cell cycle arrest, require further investigation in order to understand the underlying mechanism. [ABSTRACT FROM AUTHOR]

Stevioside is a secondary metabolite of diterpenoid glycoside production in plants. It has been used as a natural sweetener in various foods because of its high sweetness and low-calorie content. In this study, we constructed a Saccharomyces cerevisiae strain for the complete synthesis of stevioside using a metabolic engineering strategy. Firstly, the synthesis pathway of steviol was modularly constructed in S. cerevisiae BY4742, and the precursor pathway was strengthened. The yield of steviol was used as an indicator to investigate the expression effect of different sources of diterpene synthases under different combinations, and the strains with further improved steviol yield were screened. Secondly, glycosyltransferases were heterologously expressed in this strain to produce stevioside, the sequence of glycosyltransferase expression was optimized, and the uridine diphosphate-glucose (UDP-Glc) supply was enhanced. Finally, the results showed that the strain SST-302III-ST2 produced 164.89 mg/L of stevioside in a shake flask experiment, and the yield of stevioside reached 1104.49 mg/L in an experiment employing a 10 L bioreactor with batch feeding, which was the highest yield reported. We constructed strains with a high production of stevioside, thus laying the foundation for the production of other classes of steviol glycosides and holding good prospects for application and promotion.

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A new family of diterpene-type aminotriol derivatives has been synthesised from stevioside in a stereoselective manner. The key intermediate spiro-epoxide was prepared through the methyl ester of the allilyc diol derived from steviol. The oxirane ring was opened with primary and secondary amines, providing a versatile library of aminotriols. The corresponding primary aminotriol was formed by palladium-catalysed hydrogenation, and an N,O-heterocyclic compound was synthesised in a regioselective reaction. All new compounds were characterised by 1D- and 2D-NMR techniques and HRMS measurements. In our in vitro investigations, we found that the aromatic N-substituted derivatives exhibited high inhibition of cell growth on human cancer cell lines (HeLa, SiHa, A2780, MCF-7 and MDA-MB-231). The antiproliferative activities were assayed by the MTT method. Furthermore, the introduction of an additional hydroxy group slightly increased the biological activity. The drug-likeness of the compounds was assessed by in silico and experimental physicochemical characterisations, completed by kinetic aqueous solubility and in vitro intestinal-specific parallel artificial membrane permeability assay (PAMPA-GI) measurements. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The present research was undertaken in order to investigate if some compounds used as natural sweeteners (steviol and sucrose) can be used as EPR dosimeters. The samples were exposed to low doses of gamma ionizing radiation used in the practices of radiodiagnostic medicine and interventional radiology. The EPR spectra of the gamma irradiated compounds reveal large signals, with multiple lines, which are characteristic for the free radicals in the solid state. From the analysis of the dependence of EPR signal of the absorbed dose, it was observed that by γ-irradiation the amount of generated radicals shows a linear dependence, which mean that there exist a possibility to use these two compounds as EPR dosimeters. [ABSTRACT FROM AUTHOR]

Background: Steviol glycosides, constituents of Stevia rebaudiana Bertoni, are high-intensity sweeteners that enhance glucose-induced insulin secretion and decrease hyperglycemia in mice and rats. Given the extreme sweetness and bitter aftertaste of these compounds, the non-tasting aglycon steviol might be a promising alternative food additive that also harbors anti-hyperglycemic effects. Furthermore, it has been suggested that steviol glucuronide is the main bioactive metabolite of steviol and steviol glycosides that appears in blood. Aim: To investigate whether steviol and/or steviol glucuronide reduce hyperglycemia in a high-fat diet (HFD)-induced diabetic mouse model. Methods: Steviol was administered acute per os (p.o.), chronic via drinking water and intravenous (i.v.) to HFD-induced diabetic mice. The effect on glucose homeostasis was assessed using glucose tolerance tests (GTTs). Following chronic and i.v. steviol administration, steviol and steviol glucuronide plasma levels were determined. Steviol glucuronide was administered i.v. and the effect on glycemia was assessed with GTTs. Results: Acute and chronic oral steviol administration did not lower glycemia. This is due to the very limited and short-lived bioavailability of steviol in plasma. Only after high dosage i.v. steviol, glycemia was transiently reduced shortly after administration (Δ 15 min: 76.4 ± 30.9 mg dL−1, p = 0.035). In contrast, i.v. steviol glucuronide resulted in a 16.4 ± 5.9% lower AUCGlucose(0–120 min) compared to mice that received vehicle (p = 0.040). Moreover, steviol glucuronide enhanced the glucose-induced calcium oscillation frequency in insulin-secreting pancreatic islets. Conclusion: Our findings demonstrate that steviol is not a useful derivative of steviol glycosides to improve hyperglycemia in type 2 diabetes. On the other hand, steviol glucuronide has significant anti-hyperglycemic effects in living mice with type 2 diabetes.

Stevia rebaudiana Bertoni is famous for sweetness due to the content of steviol glycosides (SG). This study aimed to determine the effect of SG (stevioside, rebaudioside A) and steviol on adipogenesis and lipogenesis in the 3T3-L1 model. Test compounds were also analysed for their ability to affect glucose uptake in hypertrophied insulin-resistant 3T3-L1 adipocytes. The most active compound in modulating adipogenesis, lipogenesis and insulin resistance was steviol, SG metabolism product. Steviol (10 μM, 100 μM) significantly down-regulated the expression of adipogenic transcription factors (PPARγ, C/EBPα, SREBP1) and lipogenic genes (FAS, aP2, LPL), which caused decreased lipid accumulation and triglyceride content in adipocytes. Treatment of insulin-resistant adipocytes with steviol (1 μM) and stevioside (1 μM, 10 μM) increased GLUT-4 transcript level and improved glucose uptake. Steviol also lowered resistin gene expression, which may mitigate insulin resistance in hypertrophied adipocytes. This study promotes SG as potential health-promoting ingredients in low-calorie functional foods.

Some sweet tasting plant secondary metabolites are non-caloric or low nutritive compounds that have traditional use in food formulations. This mini-review focuses on conventional and advanced cultivation regimes of plants that accumulate sweet tasting or sweet taste modulating secondary metabolites of potential economic importance, in particular mogrosides (Siraitia grosvenorii), phyllodulcin (Hydrangea macrophylla), glycyrrhizin (Glycyrrhiza glabra), steviol glycosides (Stevia grosvenorii), and rubusoside (Rubus suavissimus). Consequential obstacles during the cultivation of Hydrangea macrophylla cultivars outside their natural habitat in a protected cultivation environment are addressed. Culturing at non-habitat locations facilitates short transportation routes of plant material for processing, which can be a key to an economically and environmentally compatible usage. The biosynthetic pathways, as far as known, are shortly mentioned. The proved or hypothetical degradation pathways of the compounds to minimalize environmental contamination are another focal point. [ABSTRACT FROM AUTHOR]

Sweeteners are widespread primarily in the food industry. An attractive alternative to sugar. Their prevalence was driven primarily by food shortage during the war. The intense sweetener is permitted for consumption by specifying the ratio of ADI. Due to the chemical structure stands out semi-synthetic and synthetic substances. The most common sweeteners are saccharin, acesulfame-K, steviol glycosides, aspartame, xylitol. Sweet taste is guaranteed've been using a minimum amount of their low calorie. The aim of this review was to decribe the most common sweeteners The article emphasizes both advantages and diadvantages of sucrose's substitutes intake.

The strain which degraded steviol glycosides to steviol (STE) was screened and isolated from soil samples. A phylogenetic tree was constructed and used to determine the taxonomic status of the strain. 16S rDNA sequence was ultimately used to identify the strain as Microbacterium barkeri XJ. The transformation product was detected and identified as STE by HPLC/LC-MS/IR analysis. The bioconversion rate of 1% (v/v) steviol glycosides (stevioside, rebaudioside A, rebaudioside C) into STE in basic medium were 100% within 24 h, 84 h and 144 h, respectively. The results indicated XJ was more effective than mixed flora in the bioconversion of steviol glycosides to STE. [ABSTRACT FROM AUTHOR]

Abstract Background Steviol glycosides such as stevioside have attracted the attention of the food and beverage industry. Recently, efforts were made to produce these natural sweeteners in microorganisms using metabolic engineering. Nonetheless, the steviol titer is relatively low in metabolically engineered microorganisms, and therefore a steviol-biosynthetic pathway in heterologous microorganisms needs to be metabolically optimized. The purpose of this study was to redesign and reconstruct a steviol-biosynthetic pathway via synthetic-biology approaches in order to overproduce steviol in Escherichia coli. Results A genome-engineered E. coli strain, which coexpressed 5′ untranslated region (UTR)-engineered geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, and kaurene synthase, produced 623.6 ± 3.0 mg/L ent-kaurene in batch fermentation. Overexpression of 5′-UTR–engineered, N-terminally modified kaurene oxidase of Arabidopsis thaliana yielded 41.4 ± 5 mg/L ent-kaurenoic acid. Enhanced ent-kaurenoic acid production (50.7 ± 9.8 mg/L) was achieved by increasing the cellular NADPH/NADP+ ratio. The expression of a fusion protein, UtrCYP714A2-AtCPR2 derived from A. thaliana, where trCYP714A2 was 5′-UTR–engineered and N-terminally modified, gave 38.4 ± 1.7 mg/L steviol in batch fermentation. Conclusions 5′-UTR engineering, the fusion protein approach, and redox balancing improved the steviol titer in flask fermentation and bioreactor fermentation. The expression engineering of steviol-biosynthetic enzymes and the genome engineering described here can serve as the basis for producing terpenoids—including steviol glycosides and carotenoids—in microorganisms.

In the present, study we analyzed the electronic properties of Steviol, the Stevia rebaudiana metabolite, and its interaction with antiapoptotic protein BCL-2. The ionization potential and electrophilicity index values were evaluated in the framework of the DFT, and these values suggest that Steviol may form ligand-receptor interactions. Also, the bond dissociation energy and the electrostatic potential distribution of Steviol reveal its antioxidant behavior. Docking studies were performed to evaluate the feasibility of this molecule to interact with antiapoptotic protein BCL-2. However, no hydrogen bonds were found in the pocket site, instead six interactions, including alkyl and π-alkyl type were formed, suggesting that the possible most feasible mechanism for anticancer activity would be through free radicals scavenging. [ABSTRACT FROM AUTHOR]

Plants under different environmental regimes exhibit phenotypic plasticity, sometimes producing more secondary metabolites when microenvironmental conditions are manipulated but these responses may be species, cultivar and/or genotype dependent. To test the hypothesis of whether in vitro plants of S. rebaudiana Bertoni would accumulate higher amounts of steviol glycosides when plants were growing under salt and drought stress, cultivar ST2100 plants were used. We thus applied 25 to 100 mM NaCl and polyethylene glycol 6000 (PEG) at 2.5% to 10.0% (w/v) to generate different Murashige and Skoog (Physiol Plant 15:473–497, 1962) media. Microplant cultures were also profiled for stevioside, rebaudioside A and steviol via LC–MS. Essential oil chemicals and fatty acids were assessed using GC–MS. Finally, a chemometric analysis of ethanolic extracts produced from treated and control plants is presented from MSE fragmentation data and various phenolic acids were tentatively identified using ion fragmentation patterns. Increasing amounts of both NaCl and PEG led to poor growth and development in cultures of S. rebaudiana. For example, the 25 and 50 mM NaCl-treated plants had fewer roots in comparison to controls and at even higher concentrations (75 and 100 mM NaCl), plants did not to root. Poor in vitro organogenesis was more pronounced with PEG. For instance, when plants were placed on a 10% PEG-medium, the ability for shoot regeneration was lost and callus became more apparent. Increasing levels of NaCl and PEG were also correlated to lowered levels of rebaudioside A and stevioside. In relation to the control plants that had 0.054 mg g−1 FW of steviol, the 25 mM NaCl treatment group had highest levels of this compound, recorded at 0.156 mg g−1 FW. All other salt treatments led to trace amounts of this chemical (0.005–0.009 mg g−1 FW) and it was not detected in any of the PEG-treated plants, except for the controls. The PCA loadings plots exposed stevioside, rebaudioside E and a steviol glycoside derivative as the MS signals that contributed to discriminant clusters segregating controls from the NaCl-treated groups. For PEG, segregation in the PCA is mostly influenced by dicaffeoylquinic acid as a marker ion, separating the controls from the treatment groups. PEG-treatments caused more prominent changes to the essential oil chemistry of Stevia plants. This was evident when 7.5 or 10% PEG was applied as sabinene, α-terpinolene, n-amyl isovalerate, 7-octen-4-ol, α-bergamotene, junipene, (+)-calarene, α-cadinol, β-pinene, α-bergamotene, (+)-calarene and junipene became undetectable. Changes of this nature may be undesirable when aromatic oils of S. rebaudiana are targetted for commercial markets as our data suggest adjustment to stresses may negatively impact volatile compounds leading to a loss of bioactive aromatic compounds. This study reports, for the first time, the effects of salinity and drought conditions in vitro on changed essential oil profiles of S. rebaudiana, providing new insights into the effects of stress on the essential oil chemistry of S. rebaudiana. Key message: Stevia responses to salt and drought stresses in vitro lead to lowered measured steviol glycosides (i.e stevioside, rebaudioside A and steviol) and significantly changes the essential oil terpenoid profiles [ABSTRACT FROM AUTHOR]

Introduction: Steviol stevioside, which has been used in the production of food products as a low-calorie sweeten, is one of the main glycoside groups in the leaves of the Stevia plants. It is useful for human health. Objective: The main objection of the present study was to find out some major chemical compositions of the dried Stevia rebaudiana Bertoni leaves and an effective, affordable, and environmentally friendly method to reach the high extraction yield of total steviosides from them. Therefore, a novel extraction, ultrasound-assisted extraction (UAE), was carried out to extract total steviosides from dried leaves of S. rebaudiana plant with ethanol of 70% (v/v) as a solvent. Methods: Some major chemical compositions of the dried S. rebaudiana leaves were analyzed according to the AOAC (Association of Official Analytical Chemists) and total steviosides content (TSC) was measured by anthrone-sulphuric acid colorimetric assay with main influencing factors including material/solvent ratio, extraction temperature, and extraction time. Results: The results referred that some chemical compounds such as protein, lipid, fibre, sugar, etc. existed in this material. The best extraction conditions were the sample/solvent ratio of 1:100 (g/ml), extraction temperature of 75oC, and extraction time of 30 min. Conclusion: The highest amount of total steviosides content of 8.894 % was obtained at the optimal extraction condition. Consequently, these results demonstrated that the parameters of UAE were applied successfully for producing total glycosides. [ABSTRACT FROM AUTHOR]

Steviol is a natural diterpenoid glycoside isolated from Stevia rebaudiana Bertoni leaves and widely used as a non-caloric sweetener. In addition to their sweet taste, Steviol glycosides may also have some therapeutic benefits. There are few reports on the cytotoxicity of Steviol in human cells. Our objective was to test this sweetener under and at average concentrations of consumption, evaluating parameters of cytotoxicity, genotoxicity, and immunotoxicity. For this purpose, we made use of lymphocyte cultures and the analysis of their CD3+, CD4+, and CD8+ subpopulations. In a complementary way, the mechanism of action is proposed here by computational methods. Our results showed that Steviol reduces the number of lymphocytes due to falls of CD4+, CD8+, and CD4+CD8+ subpopulations. Besides, we observed an increase in the level of DNA damage and a gradual incidence of structural changes in the lymphocyte chromosomal sets. It was possible to propose that Steviol modulates gene expression, mainly interfering with the SESN1, NAP1L1, SOX4, and TREX1 genes. Although Steviol is used globally as a sweetener, its use should be cautious, as our study points out that Steviol has cytotoxic, genotoxic and mutagenic effects in the concentrations and conditions tested in the culture of human lymphocyte cells. [ABSTRACT FROM AUTHOR]

BACKGROUND 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. RESULTS: 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 18F‐fluorodeoxyglucose traced micro‐PET experiment revealed that stevioside and steviol could increase the uptake of glucose in the myocardium and brain of the diabetic mice within 60 min, and decrease the accumulation of glucose in the liver and kidney. CONCLUSIONS: 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. [ABSTRACT FROM AUTHOR]

The concentrations of steviol and its derivatives stimulating the growth of wheat plants were measured: 10–8 for stevioside and 10–9 М for steviol and isosteviol. It was found that stevioside increased the activity of amylolytic enzymes and protein content, as well as frost tolerance of the roots of wheat seedlings. Thus, stevioside can be recommended for the development of complex phytopreparations for stimulating the growth processes and increasing the resistance of wheat plants to low temperatures. [ABSTRACT FROM AUTHOR]

Pancreatic cancer has the worst prognosis and lowest survival rate among all cancers. Pancreatic cancer cells are highly metabolically active and typically reprogrammed for aberrant glucose metabolism; thus they respond poorly to therapeutic modalities. It is highly imperative to understand mechanisms that are responsible for high glucose metabolism and identify natural/synthetic agents that can repress glucose metabolic machinery in pancreatic cancer cells, to improve the therapeutic outcomes/management of pancreatic cancer patients. We have identified a glycoside, steviol that effectively represses glucose consumption in pancreatic cancer cells via the inhibition of the translation initiation machinery of the molecular components. Herein, we report that steviol effectively inhibits the glucose uptake and lactate production in pancreatic cancer cells (AsPC1 and HPAF-II). The growth, colonization, and invasion characteristics of pancreatic cancer cells were also determined by in vitro functional assay. Steviol treatment also inhibited the tumorigenic and metastatic potential of human pancreatic cancer cells by inducing apoptosis and cell cycle arrest in the G1/M phase. The metabolic shift by steviol was mediated through the repression of the phosphorylation of mTOR and translation initiation proteins (4E-BP1, eIF4e, eIF4B, and eIF4G). Overall, the results of this study suggest that steviol can effectively suppress the glucose metabolism and translation initiation in pancreatic cancer cells to mitigate their aggressiveness. This study might help in the design of newer combination therapeutic strategies for pancreatic cancer treatment.

Background: Steviol glycosides such as stevioside have attracted the attention of the food and beverage industry. Recently, efforts were made to produce these natural sweeteners in microorganisms using metabolic engineering. Nonetheless, the steviol titer is relatively low in metabolically engineered microorganisms, and therefore a steviol-biosynthetic pathway in heterologous microorganisms needs to be metabolically optimized. The purpose of this study was to redesign and reconstruct a steviol-biosynthetic pathway via synthetic-biology approaches in order to overproduce steviol in Escherichia coli. Results: A genome-engineered E. coli strain, which coexpressed 5′ untranslated region (UTR)-engineered geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, and kaurene synthase, produced 623.6 ± 3.0 mg/L ent-kaurene in batch fermentation. Overexpression of 5′-UTR–engineered, N-terminally modified kaurene oxidase of Arabidopsis thaliana yielded 41.4 ± 5 mg/L ent-kaurenoic acid. Enhanced ent-kaurenoic acid production (50.7 ± 9.8 mg/L) was achieved by increasing the cellular NADPH/NADP+ ratio. The expression of a fusion protein, UtrCYP714A2-AtCPR2 derived from A. thaliana, where trCYP714A2 was 5′-UTR–engineered and N-terminally modified, gave 38.4 ± 1.7 mg/L steviol in batch fermentation. Conclusions: 5′-UTR engineering, the fusion protein approach, and redox balancing improved the steviol titer in flask fermentation and bioreactor fermentation. The expression engineering of steviol-biosynthetic enzymes and the genome engineering described here can serve as the basis for producing terpenoids—including steviol glycosides and carotenoids—in microorganisms. [ABSTRACT FROM AUTHOR]

The 'sweet herb' (El Caa‐ehe), used by the Guaraní tribes living in the forests of today's eastern Paraguay and southern Brazil, is rapidly emerging as a natural sweetener that is an alternative both to sugar and synthetic sweeteners, well beyond Japan where it has been widely used since the mid‐1970s. Stevia rebaudiana (Bertoni) Bertoni contains very sweet steviol glycosides in its leaves, which do not add calories and do not cause an increase in blood sugar levels. The glycoside that is most abundant in the leaves, stevioside, has high reactive oxygen species quenching activity and several properties that are beneficial to health. Rapid advances in green chemistry technology allow the production of stevia extracts that are devoid of any liquorice‐like after taste. Their high chemical and physical stability enables them to be used in baked and beverage food products and this supports the large‐scale use of stevia as a natural sweetener. Addressing bioeconomic aspects ranging from production to product formulation, this study identifies the last obstacles prior to general adoption of S. rebadudiana as a sweetener that is beneficial for health. © 2018 Society of Chemical Industry and John Wiley & Sons, Ltd [ABSTRACT FROM AUTHOR]

Diterpenoid steviol and its derivatives have gained a growing interest for their broad therapeutic benefits in recent years. In nature, steviol exists primarily in the form of glycosides which are subject to hydrolysis after oral injestion. The present study evaluated the production of steviol using a new commercially available β-glucosidase and its inclusion complex, namely γ-cyclodextrin/steviol (γ-CD/steviol). Firstly, steviol was prepared by sequential hydrolysis from stevioside via steviolbioside, with the highest conversion of stevioside at 100% and a total yield of 64.9% based on the results of single factor experiments. To enhance steviol's aqueous solubility, γ-CD/steviol was prepared by a co-evaporation method. Results showed that the solubility of steviol increased by a factor of 102 times using an 1:1 γ-CD/steviol inclusion complex. Physiochemical properties of the γ-CD/steviol complex were evaluated by HPLC, FT-IR, TGA, XRD, SEM and NMR. At ambient temperature, the γ-CD/steviol complex aqueous solution was extremely stable when assayed in the pH range from 4.01 to 9.18 up to 30 days. [ABSTRACT FROM AUTHOR]

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.

Gibberellins (GAs) are plant hormones widely used in agriculture. At present, GAs are produced by fermentation of the fungus Fusarium fujikuroi . However, fungal growth is too slow, resulting in slow fungal fermentation and a low yield. Here, to develop an alternative production source of GAs, an artificial pathway was engineered in Escherichia coli . By selecting and combining enzymes derived from plants and bacteria, a novel 4-enzyme pathway was successfully constructed to produce GAs using steviol, a readily available and less valuable byproduct during enzymatic refining of rebaudioside A, as a feedstock. Whole-cell biotransformation with E. coli strain expressing the novel pathway produced 71.17 ± 2.00 mg/L GA 1 and a trace amount of GA 3 from steviol in 48 h. This report presents a significant advancement in the fast production of GAs and establishes a method for the metabolism of terpenoids to produce target products in microbial hosts.

Graphical abstract Abstract We describe the in vitro activity of two natural isomeric ent -beyerene diterpenes, several derivatives and synthetic intermediates. Beyerenols 1 and 2 showed EC 50 of 4.6 ± 9.4 and 5.3 ± 9.4 μg/mL against amastigotes of L. (V) brazilensis , with SI of 5.1 and 7.7, respectively. Beyerenol 1 was synthesized from stevioside. In vivo experiments with bereyenols showed cure in 50% of hamsters infected with L. (V) brazilensis topically applied as Cream I (beyerenol 1 , 0.81%, w/w) and Cream III (beyerenol 2 , 1.96%, w/w). These results suggest that beyerenols are potential candidates for cutaneous leishmaniasis chemotherapy by topical application. In vitro assays of amastigotes of L. (V) brazilensis showed EC 50 of 1.1 ± 0.1 and 1.3 ± 0.04 μg/mL, with SI of 3.1 and 3.5 for hydrazone intermediates 10 and 11 , respectively. [ABSTRACT FROM AUTHOR]

Stevia rebaudiana Bertoni produces glycosides known as steviosides and they have increased their demand. Biotechnological techniques are used as an alternative to increase the production of these compounds. The aim of this study was to determine the effect of elicitors on the synthesis of steviosides in the calli of S. rebaudiana . To meet this goal, a completely randomized experimental design with four replicates was used for each elicitor. Calli obtained from leaf segments were cultured on medium containing salicylic acid (10 and 100 mM), methyl jasmonate (10 and 100 mM), citric acid (200 mg L −1 ), ascorbic acid (100 mg L −1 ), 2,4-dichlorophenoxyacetic acid (1.5 mg L −1 ), and benzylaminopurine (1.5 mg L −1 ), under red, blue or white light for 24-h, at 25 or 28 °C. The calli cultivated at 28 °C or with 100 mM of salicylic acid increased their stevioside production by 9.8 times compared to that of the leaves of plants grown under greenhouse conditions. The concentration of rebaudioside A exhibited the highest values (34.6 times higher than in leaves) by applying 10 mM of salicylic acid. Steviol accumulated in the presence of antioxidants and growth regulators and by combining 100 mM of salicylic acid and 28 °C. Salicylic acid, certain types of exogenous antioxidants, growth regulators, and temperature may promote the accumulation of steviosides. [ABSTRACT FROM AUTHOR]

Abstract Steviol is a main core-structure of the glycosides in Stevia Rebaudiana. The geometry optimization is carried out for steviol using the B3LYP with 6–311++G (d,p) basis set. The complete structural analysis such as electronic characterization, HOMO and LUMO, DOSs, Molecular Electronic Potential (MEP), contour map, Natural Bond Orbital (NBO) as well as NMR, UV–Vis and IR assignments are performed and discussed with details for mentioned molecule. Highlights • Steviol is a main core-structure of the glycosides in Stevia Rebaudiana. • The geometry optimization is carried out for steviol using the B3LYP with 6–311++G (d,p) basis set. • Full computational structural analysis of steviol molecule such as electronic characterization, HOMO and LUMO, DOSs, Molecular Electronic Potential (MEP), contour map, Natural Bond Orbital (NBO), S1 state excitation structure properties, difference density map, as well as NMR, UV–Vis and IR assignments are calculated and investigated with details. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Chemoselective and affinity-based probes of steviol were applied to the discovery of glycosyltransferase activity and corresponding UDP-glycosyltransferase in Arabidopsis thaliana, which illustrates a simple strategy for rapidly harnessing minable novel biological parts from plants for synthetic biology. [ABSTRACT FROM AUTHOR]

Diabetes-induced injury of myocardium, defined as diabetic cardiomyopathy (DCM), accounts for significant mortality and morbidity in diabetic population. Alleviation of DCM by a potent drug remains considerable interests in experimental and clinical researches because hypoglycemic drugs cannot effectively control this condition. Here, we explored the beneficial effects of isosteviol sodium (STVNa) on type 1 diabetesinduced DCM and the potential mechanisms involved. Male Wistar rats were induced to diabetes by injection of streptozotocin (STZ). One week later, diabetic rats were randomly grouped to receive STVNa (STZ/STVNa) or its vehicle (STZ). After 11 weeks of treatment or 11 weeks treatment following 4 weeks of removal of the treatment, the cardiac function and structure were evaluated and related mechanisms were investigated. In diabetic rats, oxidative stress, inflammation, blood glucose and plasma advanced glycation end products (AGEs) were significantly increased, whereas superoxide dismutase 2 (SOD-2) expression and activity were decreased. STVNa treatment inhibited cardiac hypertrophy, fibrosis and inflammation, showed similar ratio of heart to body weight and antioxidant capacities almost similar to the normal controls, which can be sustained at least 4 weeks. Moreover, STVNa inhibited diabetesinducted stimulation of both extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) signal pathways. However, blood glucose, plasma AGE and insulin levels were not altered by STVNa treatment. These results indicate that STVNa may be developed into a potent therapy for DCM. The mechanism underlying this therapeutic effect involves the suppression of oxidative stress and inflammation by inhibiting ERK and NF-κB without changing blood glucose or AGEs. [ABSTRACT FROM AUTHOR]

The commercialization of Stevia rebaudiana Bertoni (Asteraceae) extracts as a natural sweetener is driving interest in the use of in vitro propagation systems as an alternative source of steviol glycosides. Out of this suite of chemicals, stevioside is the most abundant but rebaudioside A is the sweetest. We established an in vitro propagation method from germinated seedlings on a Murashige and Skoog (MS) (Physiol Plant 15:473-497, 1962) medium with aims to study the effects of nitrogen and phosphate on the growth and metabolite profiles of S. rebaudiana plants. Generally, NH4NO3 is supplied at a concentration of 20.61 mM in MS medium and together with 18.79 mM KNO3, provide nitrogen to in vitro growing plants. In this study, we used a range of 0.3-72.1 mM NH4NO3 and 9.4-65.8 mM KNO3 and generated six different media with altered nitrogen. Similarly, six different concentrations of KH2PO4, ranging from 0.6 to 4.4 mM were tested for the phosphate treatments and the control medium had 1.25 mM KH2PO4. By reducing the nitrogen and phosphate levels to half, respectively, this led to the tallest plants. Increasing concentrations of nitrogen in the medium significantly lowered the amount of rebaudioside A as plants on the control medium accumulated 270 mg g−1 rebaudioside A compared to those that were on a medium with 3.5 times the nitrogen supply (30 mg g−1 rebaudiose A). Steviol increased with increasing nitrogen available to the microplants. The highest levels of stevioside (740 mg g−1) quantified was linked to microplants on a medium with half the phosphate concentration. To further assess changes to the metabolomic profiles of treated microplants, LC-MS/MS was used in combination with multivariate statistical analyses. Two distinct clusters were revealed after principal component analysis. Steviol hydrate, stevioside hydrate and rebaudioside A contributed significantly to the separation of phosphate-treated plants from those with variable nitrogen concentrations. Chlorogenic acid and its derivatives were linked to changing phosphate concentrations. The clustering suggests different molecular mechanisms at play that are affected by nitrogen and phosphate supply which serve to alter secondary metabolic flux, resulting in different chemical profiles. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Overexpression of aquaporin 2 (AQP2) was observed and suggested to be involved in fluid secretion leading to cyst enlargement in polycystic kidney disease (PKD). The cyst expansion deteriorates the renal function and, therefore, therapies targeting cyst enlargement are of clinical interest. Of note, inhibition of vasopressin function using vasopressin 2 receptor (V2R) antagonist which decreased cAMP production along with AQP2 production and function can slow cyst growth in ADPKD. This finding supports the role of AQP2 in cyst enlargement. Steviol, a major metabolite of the sweetening compound stevioside, was reported to retard MDCK cyst growth and enlargement by inhibiting CFTR activity. Interestingly, its efficacy was found to be higher than that of CFTR inh -172. Since steviol was also found to produce diuresis in rodent, it is likely that steviol might have an additional effect in retarding cyst progression, such as inhibition of AQP2 expression and function. Here, we investigated the effect of steviol on AQP2 function and on cyst growth using an in vitro cyst model (MDCK and Pkd1 −/− cells). We found that steviol could markedly inhibit cyst growth by reducing AQP2 expression in both Pkd1 −/− and MDCK cells. Real-time PCR also revealed that steviol decreased AQP2 mRNA expression level as well. Moreover, a proteasome inhibitor, MG-132, and the lysosomotropic agent, hydroxychloroquine (HCQ) were found to abolish the inhibitory effect of steviol in Pkd1 −/− cells. Increased lysosomal enzyme marker (LAMP2) expression following steviol treatment clearly confirmed the involvement of lysosomes in steviol action. In conclusion, our finding showed for the first time that steviol slowed cyst growth, in part, by reducing AQP2 transcription, promoted proteasome, and lysosome-mediated AQP2 degradation. Due to its multiple actions, steviol is a promising compound for further development in the treatment of PKD. [ABSTRACT FROM AUTHOR]

A proof of principle method using ion mobilitymass spectrometry (IM-MS) and collision induced dissociation (CID) coupled with micro flow ultra high-performance chromatography (UHPLC-IM-MS) has been developed to screen for steviol glycosides. Traveling wave ion mobility was used to determine rotationally averaged collision cross sections in nitrogen buffer gas (TWCCSN2). To explore the evolving applicability of ion mobility screening, the analytical approach was initially developed and applied to the analysis of a steviol/ steviol glycoside spiked chocolate spread extract. Subsequently 55 food commodities were screened using a steviol glycoside TWCCSN2 library. IM analyses produced TWCCSN2 values, enabling the unequivocal identification of the steviol glycosides and isomeric pairs (negating the reliance on product ions). In addition, coeluting isomeric species, comprising (labile fragment ions, doubly charged dimers, and multiply charged species) have been identified and resolved. Isomeric false detections were avoided, with the coeluting isomeric species quantified. A quantitative assessment of TWCCSN2 in the analysis of steviol glycosides was performed. [ABSTRACT FROM AUTHOR]

Steviol is the colonic metabolite of the natural sweetener steviol glycosides. It does not diffuse to the blood and the half maximal inhibitory concentration of steviol is longer compared with that of current chemotherapy agents, including 5-fluorouracil and doxorubicin. The present study demonstrated that steviol inhibits the proliferation of the human osteosarcoma U2OS cell line in a dose- and time-dependent manner, and that the inhibition rate is comparative with that of doxorubicin and 5-fluorouracil. The mechanism of this anticancer activity is also investigated. The results indicated that steviol inhibits U2OS cells through inducing G1 phase cell cycle arrest, downregulating the ability of colony formation via a mitochondrial apoptotic pathway, which was indicated by an increase of the Bax/Bcl-2 ratio and activation of cyclin-dependent kinase inhibitor 1, tumor protein 53 and cyclin-dependent kinase; whereas a Survivin and Caspase 3-independent mechanism was involved. Considering that steviol appears minimally in the plasma during metabolism, and possesses a median lethal dose of 100-fold greater compared with that of 5-fluorouracil, it may become a potential chemotherapy agent. [ABSTRACT FROM AUTHOR]