Exercise and Stevia Rebaudiana (R) Extracts Attenuate Diabetic Cardiomyopathy in Type 2 Diabetic Rats: Possible Underlying Mechanisms.

BACKGROUND AND AIMS: In the current work, we studied the effects of exercise and stevia rebaudiana (R) extracts on diabetic cardiomyopathy (DCM) in type 2 diabetic rats and their possible underlying mechanisms. METHODS: Thirty-two male Sprague Dawley rats were randomly allocated into 4 equal groups; a) normal control group, b) DM group, type 2 diabetic rats received 2 ml oral saline daily for 4 weeks, c) DM+ Exercise, type 2 diabetic rats were treated with exercise for 4 weeks and d) DM+ stevia R extracts: type 2 diabetic rats received methanolic stevia R extracts. By the end of the experiment, serum blood glucose, HOMA-IR, insulin and cardiac enzymes (LDH, CK-MB), cardiac histopathology, oxidative stress markers (MDA, GSH and CAT), myocardial fibrosis by Masson trichrome, the expression of p53, caspase-3, α-SMA and tyrosine hydroxylase (TH) by immunostaining in myocardial tissues were measured. RESULTS: T2DM caused a significant increase in blood glucose, HOMA-IR index, serum CK-MB and LDH, myocardial damage and fibrosis, myocardial MDA, myocardial α-SMA, p53, caspase-3, Nrf2 and TH density with a significant decrease in serum insulin and myocardial GSH and CAT (p< 0.05). On the other hand, treatment with either exercise or stevia R extracts significantly improved all studied parameters (p< 0.05). Moreover, the effects of stevia R was more significant than exercise (p< 0.05). CONCLUSION: Both exercise and methanolic stevia R extracts showed cardioprotective effects against DCM and Stevia R offered more cardioprotective than exercise. This cardioprotective effect of these lines of treatment might be due to attenuation of oxidative stress, apoptosis, sympathetic nerve density and fibrosis and upregulation of the antioxidant transcription factor, Nrf2.

Engineered ZnO and CuO Nanoparticles Ameliorate Morphological and Biochemical Response in Tissue Culture Regenerants of Candyleaf (Stevia rebaudiana).

Sustainable production of secondary metabolites in medicinal plants by artificial culturing on the industrial scale has gained worldwide importance. Engineered nanoparticles (ENPs) play a pivotal role in the elicitation of compounds of medicinal value. This investigation explores the influence of ZnO and CuO ENPs on in vitro roots formation, non-enzymatic antioxidant activities, and production of steviol glycosides (SGs) in regenerants of Candyleaf, Stevia rebaudiana. ENPs were applied in 0, 2, 20, 200, and 2000 mg/L of concentration in the MS medium containing plant shoots. The percentage of rooting induced was 91% and 94% by applying ZnO ENPs (2 mg/L) and CuO ENPs (20 mg/L), respectively. Moreover, at 2 mg/L of ZnO and 20 mg/L of CuO ENPs, the high performance liquid chromatography studies determined the significantly greatest content of SGs; rebaudioside A (4.42 and 4.44) and stevioside (1.28 and 1.96). Phytochemical studies including total flavonoid content, total phenolic content, and 2,2-diphenyl-1-picryl hydrazyl-free radical scavenging activity were calculated highest by the regenerants grown in 2 mg/L of ZnO and 20 mg/L of CuO ENPs dosage. Both ZnO and CuO ENPs at 200 mg/L and 2000 mg/L of concentration induced adverse effects on plant biomass, antioxidant activities, and SGs content up to 1.22 and 1.77 for rebaudioside A and 0.21 and 0.25 for stevioside. Hence, the biochemical and morphophysiological responses of Candyleaf were elicited as a defense against ZnO and CuO ENPs applied under threshold limit. This artificial biotechnological technique holds great promise for continued production of natural antioxidants on commercial scale and our study has further strengthened this impact.

Stevia rebaudiana Bertoni bioactive effects: From in vivo to clinical trials towards future therapeutic approaches.

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

Effects of cytokinins, gibberellic acid 3, and gibberellic acid 4/7 on in vitro growth, morphological traits, and content of steviol glycosides in Stevia rebaudiana.

Steviol glycosides (SGs) and gibberellic acids share a part of their biosynthesis pathways. Despite the widespread studies on the effect of gibberellic acid 3 (GA3), the effect of gibberellic acid 4 and 7 (GA4/7) on Stevia rebaudiana has never been studied. This study aimed at a comparative evaluation of different hormone effects, i.e., 1 mg L-1 GA4/7, 1 mg L-1 GA3, or 0.5 mg L-1 kinetin and 0.5 mg L-1 6-benzylaminopurine (BAP) (KB 0.5), on in vitro propagation, growth, morphological properties, and content of SGs in leaf samples of stevia. In comparison with the control group (hormone-free), the treatments of KB 0.5 or GA3 produced the highest biomasses and largest leaf areas. The three hormonal treatments produced a similar number of leaves, the ratio of fresh to dry weight, and leaf length. GA4/7-treated explants produced the highest ratio of leaf area to leaf length. The effect of GA4/7 on shoot elongation was greater than that of the control or even GA3. While the effect of GA3 on rebaudioside-A (Reb-A) production was similar to that of the control (16.2 and 18.04 mg g-1, respectively), GA4/7 resulted in a lower amount of it (13.31 mg g-1). Except for GA4/7, which induced more stevioside accumulation, the treatments' effects were comparable to that of the control. The ratio of stevioside to Reb-A was the highest for GA4/7 (2.62), followed by GA3 (1.93), and then the two others. Sum of Reb-A and stevioside content was not changed by the use of any of the treatments.

Comparative metabolic and ionomic profiling of two cultivars of Stevia rebaudiana Bert. (Bertoni) grown under salinity stress.

This study provides a comprehensive investigation on the impact of increasing NaCl concentrations on hydroponically grown Stevia rebaudiana cultivars (Shoutian-2 and Fengtian). Growth parameters including plant height, biomass and physiological responses including osmotic potential were measured. In addition, the levels of steviol glycosides, elements and primary metabolites were measured and statistically evaluated. The cultivar Fengtian grew faster, accumulated less Na+ and compatible organic solutes, and more K+ in the leaves, as compared to the cv. Shoutian-2. Metabolite analysis identified 81 differentially accumulated metabolites, indicating an alteration in the metabolite phenotype of both cultivars upon exposure to salinity A general increase in many amino acids, amines, sugars and sugar phosphates with a concurrent decrease in most organic acids; including tricarboxylic acid (TCA) cycle intermediates, was observed. In the more salt tolerant cv. Fengtian, the levels of hexose phosphates and metabolites involved in cellular protection increased in response to salinity. These metabolites remained unchanged in the sensitive cv. Shoutian-2. Interestingly, salt treatment notably increased the rebaudioside A concentration by 53% while at the same time stevioside decreased by 38% in Fengtian which has important implications for controlling the relative amounts of reboudioside A and stevioside. The findings of this study leads to the conclusion that mild salinity stress can increase the yield of sweetener compounds, which is dependent on the cultivar and the level of salinity stress.

Effect of decapitation and exogenous application of gibberellic acid (GA3) and cytokinin (CK) on some physiological characteristics of stevia.

Stevia (Stevia rebaudiana B.) has auxiliary buds that often remain dormant for a long time and sometimes remain dormant until the plants change at the reproductive stage. This study was designed out to investigate whether decapitation and exogenous application of plant growth regulators enhance the productivity of stevia through breaking the apical dominance and increasing physiological characteristics. Experiment was carried out as a factorial in randomized complete block design with three replications. Factors were consisted two agricultural practices (Decapitation and No-decapitation) and eight foliar spray including without spray as control, water spray, GA3 (300, 600 and 900 µm) and CK (100, 200 and 400 µm). The results of the present investigation indicated a positive response on number of branches and leaves, leaves and stem fresh weight and total dry weight, in both harvests not only from the decapitation of apical buds but also from foliar application of CK (400 µM). Thus, it can be concluded that the decapitation practices in conjunction with foliar application of CK (400 µM) could be used to increase the dry-leaf yield of stevia. However, further studies are required to standardize the dose of CK (400 µM) to improve the yield and quality of stevia.

Enhanced Overwintering Survival of Stevia by QoI Fungicides Used for Management of Sclerotium rolfsii.

Stevia (Stevia rebaudiana) is a herbaceous perennial under evaluation as a new crop in the southeastern United States. Stem rot caused by Sclerotium rolfsii is common in stevia plantings in North Carolina, with symptoms including wilting, root and stem necrosis, and plant death. Fungicide efficacy trials for management of S. rolfsii were conducted over 2 years. Fungicides evaluated included azoxystrobin, flutolanil, and tebuconazole applied at three timings. Azoxystrobin applied to transplants 1 week prior to planting had the lowest area under the disease progress curve values across all trials. Fungicide plots were also used to evaluate overwintering of stevia. End-of-season stand counts were compared with spring emergence counts to quantify overwintering survival. In spring 2015, plots treated with azoxystrobin in 2014 had greater overwintering survival (78%) than other fungicide treatments (38%) and the control (38%). Similar results were obtained at two locations in spring 2016 in plots treated with azoxystrobin or pyraclostrobin in 2015. Successful overwintering of stevia directly impacts the profitability of second- and third-year harvests and enhances the likelihood of long-term establishment of stevia as a viable crop. Future studies will be directed at elucidating the mechanism of the enhanced overwintering survival of plants treated with quinone outside inhibitor fungicides.

Effect of zinc oxide (ZnO) nanoparticles on physiology and steviol glycosides production in micropropagated shoots of Stevia rebaudiana Bertoni.

This study aims to address the effects of different concentrations (0, 0.1, 1.0, 10, 100 or 1000 mg L-1) of engineered zinc oxide (ZnO) nanoparticles (34 nm in size) on growth parameters, steviol glycosides (rebaudioside A and stevioside) production and antioxidant activities in the tissue culture grown shoots of Stevia rebaudiana Bertoni. The highest percentage of shoot formation (89.6%) at 1 mg L-1 of ZnO nanoparticles concentration suggests a positive influence of ZnO nanoparticles on S. rebaudiana growth as compared to other treatments with or without ZnO nanoparticles. Additionally, HPLC results illustrate a significant enhancement of steviol glycosides (almost doubled as compared to the control) in micropropagated shoots grown under an oxidative stress of 1 mg L-1 of ZnO nanoparticles. This finding is further affirmed by an increased 2,2-diphenyl-1-picryl hydrazyl (DPPH) scavenging activity, total anti-oxidant capacity, total reducing power, total flavonoid content and total phenolic content, with an ascending oxidative pressure and generation of reactive oxygen species (ROS). However, the antioxidant activities, formation of secondary metabolites and the physiological parameters showed a sudden decline after crossing a threshold of 1 mg L-1 concentration of ZnO nanoparticles and falls to a minimum at 1000 mg L-1, elucidating maximum phytotoxic effect of ZnO nanoparticles at this concentration. This is the first study evaluating both the favorable and adverse effects of ZnO nanoparticles employed to a highly valuable medicinal plant, S. rebaudiana.

Stevia preferences in Wistar rats / Preferencia gustativa de las ratas Wistar por la estevia

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

ANTECEDENTES: la planta Stevia rebaudiana se convertirá en una de las principales fuentes de edulcorantes debido al crecimiento del consumo de productos naturales en el mercado. S. rebaudiana contiene distintos glucósidos diterpenoides, pero los que proporcionan dulzor son el rebaudiosido A y el esteviosido. Estos dos compuestos tienen perfiles farmacocinéticos y metabólicos similares en ratas y humanos. Por otro lado, hay estudios que muestran la existencia de distintos patrones de ingesta voluntaria de edulcorantes en los mamíferos. MÉTODO: se realizaron series de la prueba de libre elección entre dos botellas. Comparamos la ingesta de un rango de concentraciones de edulcorantes frente al agua y frente a sacarina. RESULTADOS: las ratas Wistar prefieren el extracto de estevia y el rebaudiosido A (concentraciones desde 0,001% hasta 0,3%) frente al agua, la ingesta máxima fue a la concentración de 0,1%. También prefieren las soluciones preparadas con el producto comercial Truvia (rebaudiósido A y eritritol) frente al agua, la ingesta máxima fue a la concentración de 2%. CONCLUSIONES: nuestro trabajo proporciona nueva información sobre la preferencia gustativa de las ratas Wistar por distintos compuestos de estevia. Estos resultados ayudarán al diseño de estudios centrados en los efectos comportamentales y fisiológicos del consumo de estevia

Chloroplast ultra structure, photosynthesis and enzyme activities in regenerated plants of Stevia rebaudiana (Bert.) Bertoni as influenced by copper sulphate in the medium.

Stevia rebaudiana (Bert.) Bertoni is an important medicinal plant used as noncaloric commercial sweetener. Plants regenerated with higher levels of copper sulphate in the medium exhibited enhanced activity of peroxidase and polyphenoloxidase (PPO) enzymes. Transmission electron microscopy (TEM) revealed increase in size and number of electron dense inclusions in the chloroplasts of plants regenerated at optimised level of copper sulphate (0.5 microM) in the medium. There was decrease in chlorogenic acid (CGA) content. Chl-a-fluorescence transient pattern (OJIP) showed that the photosynthesis process was more efficient at 0.5 microM CuSO4 in the medium.

Agrobacterium tumefaciens-mediated transgenic plant and somaclone production through direct and indirect regeneration from leaves in Stevia rebaudiana with their glycoside profile.

Agrobacterium tumefaciens (EHA-105 harboring pCAMBIA 1304)-mediated transgenic plant production via direct regeneration from leaf and elite somaclones generation through indirect regeneration in Stevia rebaudiana is reported. Optimum direct regeneration frequency along with highest transformation frequency was found on MS + 1 mg/l BAP + 1 mg/l NAA, while indirect regeneration from callus was obtained on MS + 1 mg/l BAP + 2 mg/l NAA. Successful transfer of GUS-positive (GUS assay and PCR-based confirmation) transgenic as well as four somaclones up to glasshouse acclimatization has been achieved. Inter-simple sequence repeat (ISSR) profiling of transgenic and somaclonal plants showed a total of 113 bands, out of which 49 were monomorphic (43.36 %) and 64 were polymorphic (56.64 %). Transgenic plant was found to be closer to mother plant, while on the basis of steviol, stevioside, and rebaudioside A profile, somaclone S2 was found to be the best and showed maximum variability in ISSR profiling.

Spatio-temporal variation of the diterpene steviol in Stevia rebaudiana grown under different photoperiods.

As part of an ongoing study on the effects of photoperiodism on the metabolism of steviol glycosides (SVglys) in Stevia rebaudiana, the spatio-temporal variations of free steviol (SV) have now been evaluated. For its quantitation, an internal standard method was used, based upon a specific fluorometric detection of SV as its methoxycoumarinyl derivative. The level of free SV in leaves did not exceed 30 µg/g dry wt and was at least 1000-fold smaller than that of its glycosidic conjugates. In other organs, free SV was mainly measured in stem tissue and apices, with relatively large amounts measured in the latter. Similarly to SVglys, the content of free SV was influenced by photoperiod and genotype. In plants grown under long-days (LD) of 16 h, more spatial variations were seen compared to those under short-days (SD) of 8h. In the former, upper leaves contained almost four times more free SV compared to lower ones near the end of vegetative growth. In addition, the correlation between SV and its glycosidic conjugates was more linear under SD. Despite the variability of SV levels, a decrease was noted in all conditions after flower opening, which can be related a decreased transcription of the biosynthetic genes involved.