Nanocurcumin-Based Sugar-Free Formulation: Development and Impact on Diabetes and Oxidative Stress Reduction.

The objective of this study is the development of innovative nanocurcumin-based formulations designed for the treatment and prevention of oxidative stress and diabetes. Nanocurcumin was obtained through a micronization process and subsequently encapsulated within biopolymers derived from corn starch and fenugreek mucilage, achieving encapsulation rates of 75% and 85%, respectively. Subsequently, the encapsulated nanocurcumin was utilized in the formulation of sugar-free syrups based on Stevia rebaudiana Bertoni. The stability of the resulting formulations was assessed by monitoring particle size distribution and zeta potential over a 25-day period. Dynamic light scattering (DLS) revealed a particle size of 119.9 nm for the fenugreek mucilage-based syrup (CURF) and 117 nm for the corn starch-based syrup (CURA), with polydispersity indices PDIs of 0.509 and 0.495, respectively. The dissolution rates of the encapsulated nanocurcumin were significantly enhanced, showing a 67% improvement in CURA and a 70% enhancement in CURF compared with crude curcumin (12.82%). Both formulations demonstrated excellent antioxidant activity, as evidenced by polyphenol quantification using the 2.2-diphenyl 1-pycrilhydrazyl (DPPH) assay. In the evaluation of antidiabetic activity conducted on Wistar rats, a substantial reduction in fasting blood sugar levels from 392 to 187 mg/mL was observed. The antioxidant properties of CURF in reducing oxidative stress were clearly demonstrated by a macroscopic observation of the rats' livers, including their color and appearance.

Unveiling the potential: Extracellular vesicles from plant cell suspension cultures as a promising source.

Extracellular vesicles are secreted by all eukaryotic cells and they have an important role in intercellular signaling. Plant extracellular vesicles (PEVs) are a novel area of research that has gained attention due to their potential implications in biomolecule transport and therapeutic applications. PEVs are lipid bilayer-enclosed structures that contain a diverse cargo of biomolecules such as proteins and lipids. Moreover, it is known that PEVs have a noticeable therapeutic potential for various conditions such as inflammation and oxidative stress. However, there are critical problems such as removing the endosomes and plant-derived biomolecules that decrease the standardization and therapeutic efficacy of PEVs. In our study, the aim was to characterize plant cell suspension-derived extracellular vesicles (PCSEVs) obtained from two different plant cell suspension cultures: Stevia rebaudiana and Vaccaria hispanica. These vesicles were isolated using ultrafiltration and characterized with nanoparticle tracking analysis (NTA) and atomic force microscopy (AFM). The molecular composition of PCSEVs was profiled and the cellular uptake assay was performed. Our results demonstrated that PCSEVs have a spherical shape, less than 200 nm. In the fatty acid analysis, the primary components in PCSEVs were palmitic acid, linoleic acid, and cis-vaccenic acid. The protein content of Stevia rebaudiana-derived EVs (SDEVs) was largely associated with proteins involved in extracellular structures and functions. Conversely, Vaccaria hispanica-derived EVs (HDEVs) displayed a higher presence of cytosolic proteins. These findings contribute to the understanding of PCSEVs and open up potential avenues in extracellular vesicle research, pointing to promising prospects for future innovations in various fields.

The Origin of The Problem: Characterization of Paraguayan Septoria steviae, Causal Agent of Septoria Leaf Spot of Stevia Based on Multi-Locus Sequence Analysis.

Septoria leaf spot is a significant disease affecting cultivated stevia, potentially reducing yields by > 50%. The disease is caused by Septoria steviae, first identified in 1978 in Japan as a new pathogen of stevia. Understanding the origin of S. steviae could clarify how it spread to new production areas. To investigate this, twelve isolates of Septoria sp. were obtained from stevia's native range in the Amambay forests and field plantings in Paraguay from 2018 to 2020. These isolates underwent colony morphology and molecular characterization of Actin, ß-Tubulin, Calmodulin, ITS, LSU, RPB2, and TEF1α loci. GenBank sequences from S. steviae isolates collected in France, Japan, and the United States (USA) were included. Multi-locus sequence phylogenetic analysis generated a maximum likelihood (ML) tree. The morphological characteristics of Paraguayan isolates were similar to previously reported S. steviae type cultures from Japan. The ML analysis showed that Paraguayan isolates formed a monophyletic group with S. steviae isolates from France, Japan, and the USA. During blotter tests, pycnidia and cirri of S. steviae were observed on multiple stevia seed surfaces from different sources. Further characterization confirmed viable pathogenic conidia of S. steviae. This observation suggests that S. steviae could be associated with stevia seed, possibly spreading from the center of origin to other countries. This research is the first to genetically characterize S. steviae from Paraguay and propose its potential spread mechanism from the center of origin to the rest of the world.

Copper nanoparticles biosynthesis by Stevia rebaudiana extract: biocompatibility and antimicrobial application.

The growth of material science and technology places a high importance on the creation of better processes for the synthesis of copper nanoparticles. So that, an easy, ecological, and benign process for producing copper nanoparticles (CuNPs) has been developed using candy leaf (Stevia rebaudiana) leaves aqueous extract for the first time. UV-visible spectroscopy, dynamic light scattering (DLS), X-ray diffraction (XRD), high-resolution transmission electron microscope (HR-TEM), Fourier transmission infrared (FTIR), and zeta potential were applied to demonstrate strong characterization for the biosynthesized stevia-CuNPs. The UV-visible absorbance at 575 nm of surface plasmon resonance (SPR) was 1.2. The particle size mean diameter was recorded as 362.3 nm with - 10.8 mV zeta potential. The HR-TEM scanning revealed 51.46-53.17 nm and spherical-shaped stevia-CuNPs surrounded by coat-shell proteins. The cytotoxicity and cytocompatibility activity assay revealed that stevia-CuNPs was safe in lower concentrations and had a significant cell viability reduction in higher concentrations. The produced stevia-CuNPs were applied as antimicrobial agents against eight pathogenic bacteria and five fungi strains. The inhibitory action of the stevia-CuNPs was more pronounced in bacteria than in fungi, and they likewise demonstrated further inhibition zones in Staphylococcus aureus (50.0 mm) than in Aspergillus flavus (55.0 mm). With inhibition zone sizes of 50.0 mm and 47.0 mm and 50 µg/ml minimum inhibitory concentration, S. aureus and A. flavus were the most inhibited pathogens. The minimum lethal effect (MLC) estimate for S. aureus was 50 µg/ml, whereas 75 µg/ml for A. flavus. The stevia-CuNPs mode of action was characterized as bactericidal/fungicidal as the ratio of MIC to MLC was estimated to be equal to or less than 2. After all, stevia-CuNPs could be used as an alternative to commercial antibiotics to solve the problem of multidrug-resistant (MDR) microorganisms.

Identifying Potential Sources of Phthalate Contamination in the Leaves of Stevia Rebaudiana (Bertoni) and the Development of Removal Technology.

Steviosides extracted from the leaves of the plant Stevia rebaudiana are increasingly used in the food industry as natural low-calorie sweeteners. Phthalates in food are often assumed to arise from food containers or packaging materials. Here, experiments were carried out to identify the potential sources of DMP, DBP, DIBP, and DEHP in the leaves of stevioside through investigation of their content in native stevioside tissues, soils, and associated agronomic materials. The results show that phthalate contamination was present in all the samples tested, and the influence of regional factors at the provincial level on the content of plasticizers in stevia leaves was not significant. Phthalates in stevia leaves can be absorbed into the plant body through leaves and roots. Using resin removal, the phthalate content in stevioside glycosides was reduced to less than 0.05 ppm, and some indicators were far lower than the limit standard in EU food.

Efficient Conversion of Stevioside to Rebaudioside M in Saccharomyces cerevisiae by a Engineering Hydrolase System and Prolonging the Growth Cycle.

Rebaudioside (Reb) M is an important sweetener with high sweetness, but its low content in Stevia rebaudiana and low catalytic capacity of the glycosyltransferases in heterologous microorganisms limit its production. In order to improve the catalytic efficiency of the conversion of stevioside to Reb M by Saccharomyces cerevisiae, several key issues must be resolved including knocking out endogenous hydrolases, enhancing glycosylation, and extending the enzyme catalytic process. Herein, endogenous glycosyl hydrolase SCW2 was knocked out in S. cerevisiae. The glycosylation process was enhanced by screening glycosyltransferases, and UGT91D2 from S. rebaudiana was identified as the optimum glycosyltransferase. The UDP-glucose supply was enhanced by overexpressing UGP1, and co-expressing UGT91D2 and UGT76G1 achieved efficient conversion of stevioside to Reb M. In order to extend the catalytic process, the silencing information regulator 2 (SIR2) which can prolong the growth cycle of S. cerevisiae was introduced. Finally, combining these modifications produced 12.5 g/L Reb M and the yield reached 77.9% in a 5 L bioreactor with 10.0 g/L stevioside, the highest titer from steviol glycosides to Reb M reported to date. The engineered strain could facilitate the industrial production of Reb M, and the strategies provide references for the production of steviol glycosides.

Transcriptomic Analyses Reveal Insights into the Shared Regulatory Network of Phenolic Compounds and Steviol Glycosides in Stevia rebaudiana.

Stevia rebaudiana (Bertoni) is a highly valuable crop for the steviol glycoside content in its leaves, which are no-calorie sweeteners hundreds of times more potent than sucrose. The presence of health-promoting phenolic compounds, particularly flavonoids, in the leaf of S. rebaudiana adds further nutritional value to this crop. Although all these secondary metabolites are highly desirable in S. rebaudiana leaves, the genes regulating the biosynthesis of phenolic compounds and the shared gene network between the regulation of biosynthesis of steviol glycosides and phenolic compounds still need to be investigated in this species. To identify putative candidate genes involved in the synergistic regulation of steviol glycosides and phenolic compounds, four genotypes with different contents of these compounds were selected for a pairwise comparison RNA-seq analysis, yielding 1136 differentially expressed genes. Genes that highly correlate with both steviol glycosides and phenolic compound accumulation in the four genotypes of S. rebaudiana were identified using the weighted gene co-expression network analysis. The presence of UDP-glycosyltransferases 76G1, 76H1, 85C1, and 91A1, and several genes associated with the phenylpropanoid pathway, including peroxidase, caffeoyl-CoA O-methyltransferase, and malonyl-coenzyme A:anthocyanin 3-O-glucoside-6″-O-malonyltransferase, along with 21 transcription factors like SCL3, WRK11, and MYB111, implied an extensive and synergistic regulatory network involved in enhancing the production of such compounds in S. rebaudiana leaves. In conclusion, this work identified a variety of putative candidate genes involved in the biosynthesis and regulation of particular steviol glycosides and phenolic compounds that will be useful in gene editing strategies for increasing and steering the production of such compounds in S. rebaudiana as well as in other species.

Nitrogen starvation modulates the sensitivity of rhizobacterial community to drought stress in Stevia rebaudiana.

Alterations in water regimes or nitrogen (N) availability lead to shifts in the assemblage of rhizosphere microbial community; however, how the rhizosphere microbiome response to concurrent changes in water and N availability remains largely unclear. Herein, we investigated the taxonomic and functional characteristics of rhizobacteria associated with stevia (Stevia rebaudiana Bertoni) under varying combinations of water and N levels. Community diversity and predicted functions of rhizobacteria were predominantly altered by drought stress, with N-starvation modulating these effects. Moreover, N fertilization simplified the ecological interactions within rhizobacterial communities and heightened the relative role of stochastic processes on community assembly. In terms of rhizobacterial composition, we observed both common and distinctive changes in drought-responsive bacterial taxa under different N conditions. Generally, the relative abundance of Proteobacteria and Bacteroidetes phyla were depleted by drought stress but the Actinobacteria phylum showed increases. The rhizobacterial responses to drought stress were influenced by N availability, where the positive response of δ-proteobacteria and the negative response of α- and γ-proteobacteria, along with Bacteroidetes, were further heightened under N starvation. By contrast, under N fertilization conditions, an amplified negative or positive response to drought were demonstrated in Firmicutes and Actinobacteria phyla, respectively. Further, the drought-responsive rhizobacteria were mostly phylogenetically similar, but this pattern was modulated under N-rich conditions. Overall, our findings indicate an N-dependent specific restructuring of rhizosphere bacteria under drought stress. These changes in the rhizosphere microbiome could contribute to enhancing plant stress tolerance.

In vitro propagation of Indonesian stevia (Stevia rebaudiana) genotype using axenic nodal segments.

OBJECTIVE: The high industrial demand for Stevia cultivation (Stevia rebaudiana) has increased due to its high stevioside content derived from the leaves. However, the low germination rate makes the cultivation of the plant become the main obstacle. Therefore, an efficient cultivation technique is required. This present work aims to analyze the effect of five combinations of Kinetin (Kin) and benzyladenine (BA) on stevia micropropagation using nodal segment explants. RESULTS: The micropropagation of stevia was performed using Murashige and Skoog (MS) medium supplemented with BA and Kin. We analyzed different organogenesis and callogenesis responses. In addition, the number of shoots and root formed during in vitro culture were also observed. Our results demonstrated that all treatments with Kin, both alone and in combination with BA, resulted in the development of callus on all nodal segment explants. Explants treated in MS with 1 mg L-1 BA exhibited the best average of shoot number (36.27). In contrast, the treatment without PGR resulted in the best root formation (2.6). The overall results suggested that different combination of BA and Kin resulted in distinct organogenesis responses, where 1 mg L-1 of BA was potentially used for boosting the number of shoots in micropropagation of stevia accession Mini.

The use of plant extracts and bacteriophages as an alternative therapy approach in combatting bacterial infections: the study of lytic phages and Stevia rebaudiana.

Introduction: In the light of the problem of antibiotic resistance, the use of combined alternative therapies in combatting bacteria-related disorders has gained popularity. Bacteriophages are one element implemented in new combination therapy. Stevia rebaudiana is known to have antimicrobial activity and regarded as potentially having a synergistic effect with bacteriophages. Therefore, possible interactions of lytic bacteriophages (MS2, T4 and Phi6) with acetone and methanol S. rebaudiana extracts (SRa and SRm) in the bacterial environment were examined. Material and Methods: The interactions were tested using a microdilution method, phage-extract co-incubation assay, static interaction (synography) and dynamic growth profile experiments in a bioreactor. Results: The interactions of the tested factors in a static environment differed from those in a dynamic environment. Dynamic conditions altered the effect of the extracts in a concentration-dependent manner. How different the effect of the SRa extract was to that of the SRm extract on bacterial growth in a dynamic environment depended on the species of the phage and bacterial host. The greatest differences were observed for E. coli strains and their phages, whereas Pseudomonas syringae and the Phi6 phage reacted very similarly to both extracts. Differences also emerged for the same extract in different E. coli strains and their phages. Conclusion: Every extract type should be tested on a case-by-case basis and experiment outcomes should not be generalised before gathering data. Moreover, many varied experiments should be performed, especially when examining such multifactorial mixtures. The tested mixtures could potentially be used in multidrug-resistant bacterial infection treatments.

Optimal extraction of polysaccharides from Stevia rebaudiana roots for protection against hydrogen peroxide-induced oxidative damage in RAW264.7 cells.

Stevia rebaudiana boasts a wide range of medical and food applications and contains polysaccharides that exert beneficial effects against oxidative stress. In this study, we optimised the extraction of a polysaccharide (SRRP) from S. rebaudiana roots by employing a Box-Behnken design and response surface methodology. The optimal extraction conditions were as follows: 93.57 min, 71.67 °C, and a water-to-raw material ratio of 21.40 mL/g. Under these conditions, 14.00 ± 0.35% of crude polysaccharide was obtained. Treatment of RAW264.7 cells with SRRP prior to the addition of H2O2, a major contributor to oxidative damage, significantly increased cell viability. In addition, SRRP increased the levels of superoxide dismutase, catalase, and glutathione and reduced the levels of malondialdehyde in RAW264.7 cells. Therefore, SRRP can provide effective protection against H2O2-induced oxidative damage. These findings indicate the potential of SRRP as a natural antioxidant in the food and pharmaceutical industries.

Unlocking the Therapeutic Potential of Stevia rebaudiana Bertoni: A Natural Antiglycating Agent and Non-Toxic Support for HDF Cell Health.

Sugar carbonyl groups interact with protein amino groups, forming toxic components referred to as advanced glycation end products (AGEs). The glycation system (BSA, a model protein, and fructose) was incubated for five weeks at 37 °C in the presence and absence of Stevia leaf extract. The results indicated that the leaf extract (0.5 mg/mL) decreased the incidence of browning (70.84 ± 0.08%), fructosamine (67.27 ± 0.08%), and carbonyl content (64.04 ± 0.09%). Moreover, we observed an 81 ± 8.49% reduction in total AGEs. The inhibition of individual AGE (argpyrimidine, vesper lysine, and pentosidine) was ~80%. The decrease in the protein aggregation was observed with Congo red (46.88 ± 0.078%) and the Thioflavin T (31.25 ± 1.18%) methods in the presence of Stevia leaf extract. The repercussion of Stevia leaf extract on DNA glycation was examined using agarose gel electrophoresis, wherein the DNA damage was reversed in the presence of 1 mg/mL of leaf extract. When the HDF cell line was treated with 0.5 mg/mL of extract, the viability of cells decreased by only ~20% along with the same cytokine IL-10 production, and glucose uptake decreased by 28 ± 1.90% compared to the control. In conclusion, Stevia extract emerges as a promising natural agent for mitigating glycation-associated challenges, holding potential for novel therapeutic interventions and enhanced management of its related conditions.

Influence of the Abiotic Elicitors Ag Salts of Aspartic Acid Derivatives, Self-Organized in Nanofibers with Monomeric and Dimeric Molecular Structures, on the Antioxidant Activity and Stevioside Content in Micropropagated Stevia rebaudiana Bert.

The use of nanomaterials in biotechnology for the in vitro propagation of medical plants and the accumulation of certain biologically active metabolites is becoming an efficient strategy. This study aimed to evaluate the influence of the concentration (0, 1, 10, 50, and 100 mg L-1) of two types of nanofibers on the growth characteristics, the antioxidant status, and the production of steviol glycosides in micropropagated Stevia rebaudiana Bert. plantlets. The nanofibers were synthesized by aspartic acid derivatives (L-Asp) Ag salts self-organized into nanofibers with two different molecular structures: monomeric, containing one residue of L-Asp with one hydrophilic head which bonds one Ag ion (NF1-Ag salt); and dimeric, containing two residues of L-Asp with two hydrophilic heads which bond two Ag ions (NF2-Ag salt). An increase in the shoots from the explants' number and length, biomass accumulation, and micropropagation rate was achieved in the plants treated with the NF1-Ag salt in concentrations from 1 to 50 mg L-1 after 30 days of in vitro proliferation compared to the NF2-Ag salt. In contrast, the plants grown on MS media supplemented with NF2-Ag salt exhibited an increase in the level of stevioside, rebaudioside A, and mono- (CQA) and dicaffeoylquinic (DCQA) acids as compared to the NF1-Ag salt.

Integration of biochar with nitrogen in acidic soil: A strategy to sequester carbon and improve the yield of stevia via altering soil properties and nutrient recycling.

The health of agroecosystems is subsiding unremittingly, and the over-use of chemical fertilizers is one of the key reasons. It is hypothesized that integrating biochar, a carbon (C)-rich product, would be an effective approach to reducing the uses of synthetic fertilizers and securing crop productivity through improving soil properties and nutrient cycling. The bamboo biochar at different quantities (4-12 Mg ha-1) and combinations with chemical fertilizers were tested in stevia (Stevia rebaudiana) farming in silty clay acidic soil. The integration of biochar at 8 Mg ha-1 with 100% nitrogen (N), phosphorus (P), and potassium (K) produced statistically (p ≤ 0.05) higher leaf area index, dry leaf yield, and steviol glycosides yield by about 18.0-33.0, 25.8-44.9, and 20.5-59.4%, respectively, compared with the 100% NPK via improving soil physicochemical properties. Soil bulk density was reduced by 5-8% with biochar at ≥ 8 Mg ha-1, indicating the soil porosity was increased by altering the soil macrostructure. The soil pH was significantly (p ≤ 0.05) augmented with the addition of biochar alone or in the combination of N because of the alkaline nature of the used biochar (pH = 9.65). Furthermore, integrating biochar at 8 Mg ha-1 with 100% NPK increased 22.7% soil organic C compared with the sole 100% NPK. The priming effect of applied N activates soil microorganisms to mineralize the stable C. Our results satisfy the hypothesis that adding bamboo biochar would be a novel strategy for sustaining productivity by altering soil physicochemical properties.

Oligosaccharides increased both leaf biomass and steviol glycosides content of Stevia rebaudiana.

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

Antimicrobial and Cytotoxic Activity of Ocimum tenuiflorum and Stevia rebaudiana-Mediated Silver Nanoparticles - An In vitro Study.

Background: Silver nanoparticles (AgNPs) are the nanoparticles of silver between 1 nm and 100 nm in size. In this study, AgNPs were extracted from Ocimum tenuiflorum and Stevia rebaudiana which is a medicinal plant of Indian origin, worshipped by the Hindus and used in Ayurvedic medicine since ancient times. Aim: The aim of the study was to assess the antimicrobial and cytotoxic effect of AgNPs reinforced with the herb O. tenuiflorum and S. rebaudiana against oral pathogens. Materials and Methods: In this in vitro study, the organisms used were Streptococcus mutans, Staphylococcus aureus, Lactobacillus sp., and Candida albicans. Agar well-diffusion method was used to assess the antimicrobial efficacy of the nanoparticles at 25 mL, 50 mL, and 100 mL. To assess the cytotoxic effect, brine shrimp lethality assay was used. Results: Zone of inhibition was found to be highest at 100 mL against S. mutans, S. aureus, Lactobacillus sp., and C. albicans. The cytotoxic activity at 5 mL and 10 mL was 0%. The maximum cytotoxicity was seen at 80 mL where 30% of the Nauplii's died. Conclusion: The findings from this study suggest that AgNPs reinforced with O. tenuiflorum and S. rebaudiana extracts has the potential as an antimicrobial agent and has less cytotoxic effect on brime shrimp and can be used as an alternative to commercially available antimicrobial agents.

Identification, Chemical Synthesis, and Sweetness Evaluation of Rhamnose or Xylose Containing Steviol Glycosides of Stevia (Stevia rebaudiana) Leaves.

Steviol glycosides obtained from Stevia rebaudiana leaves are increasingly used in the food industry as natural low-calorie sweeteners. Among them, the sweetness of major glycosides composed of glucose residues (e.g., stevioside and rebaudioside A) has been widely studied. However, the properties of minor natural products containing rhamnose or xylose residues are poorly investigated. In this study, five unreported steviol glycosides containing rhamnose or xylose were extracted from our developing stevia leaves, and their sweetness was evaluated. The highly glycosylated steviol glycosides were identified, and their structures were examined by fragmentation analysis using mass spectrometry. Chemical synthesis of these glycosides confirmed their structures and allowed sensory evaluation of minor steviol glycosides. Our study revealed that a xylose-containing glycoside, rebaudioside FX1, exhibits a well-balanced sweetness, and thus, it is a promising candidate for natural sweeteners used in the food industry.

Stevia (Stevia rebaudiana) extract ameliorates insulin resistance by regulating mitochondrial function and oxidative stress in the skeletal muscle of db/db mice.

BACKGROUND: Type 2 diabetes mellitus (T2DM), a growing health problem worldwide, is a metabolic disorder characterized by hyperglycemia due to insulin resistance and defective insulin secretion by pancreatic ß-cells. The skeletal muscle is a central organ that consumes most of the insulin-stimulated glucose in the body, and insulin resistance can damage muscles in T2DM. Based on a strong correlation between diabetes and muscles, we investigated the effects of stevia extract (SE) and stevioside (SV) on the skeletal muscle of diabetic db/db mice. METHODS: The mice were administered saline, metformin  (200 mg/kg/day), SE (200 and 500 mg/kg/day), and SV (40 mg/kg/day) for 35 days. During administration, we checked the levels of fasting blood glucose twice a week and conducted the oral glucose tolerance test (OGTT) and insulin tolerance test (ITT). After administration, we analyzed serum biochemical parameters, triglyceride (TG), total cholesterol (TC), insulin and antioxidant enzymes, and the cross-sectional area of skeletal muscle fibers of db/db mice. Western blots were conducted using the skeletal muscle of mice to examine the effect of SE and SV on protein expression of insulin signaling, mitochondrial function, and oxidative stress. RESULTS: SE and SV administration lowered the levels of fasting blood glucose, OGTT, and ITT in db/db mice. The administration also decreased serum levels of TG, TC, and insulin while increasing those of superoxide dismutase (SOD) and glutathione peroxidase (GPx). Interestingly, muscle fiber size was significantly increased in db/db mice treated with SE500 and SV. In the skeletal muscle of db/db mice, SE and SV administration activated insulin signaling by increasing the protein expression of insulin receptor substrate, Akt, and glucose transporter type 4. Furthermore, SE500 administration markedly increased the protein expression of AMP-activated protein kinase-α, sirtuin-1, and peroxisome proliferator-activated receptor-γ coactivator-1α. SV administration significantly reduced oxidative stress by down-regulating the protein expression of 4-hydroxynonenal, heme oxygenase-1, SOD, and GPx. In addition, SE500 and SV administration suppressed the expression of apoptosis-related proteins in the skeletal muscle of db/db mice. CONCLUSION: SE and SV administration attenuated hyperglycemia in diabetic mice. Moreover, the administration ameliorated insulin resistance by regulating mitochondrial function and oxidative stress, increasing muscle fiber size. Overall, this study suggests that SE and SV administration may serve as a potential strategy for the treatment of diabetic muscles.

Genome-wide identification of SrbHLH transcription factors highlights its potential role in rebaudioside A (RA) biosynthesis in Stevia rebaudiana.

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

Ameliorative effect of Stevia rebaudiana Bertoni on sperm parameters, in vitro fertilization, and early embryo development in a streptozotocin-induced mouse model of diabetes.

Diabetes mellitus (DM) is a common metabolic disease characterized by high blood sugar levels. It is well known that men with diabetes frequently experience reproductive disorders and sexual dysfunction. In fact, sperm quality has a significant effect on fertilization success and embryo development. The current study aimed to investigate the effect of Stevia rebaudiana hydroalcoholic extract on serum testosterone levels, sperm parameters, in vitro fertilization (IVF) success, and in vitro embryonic developmental potential to reach the blastocyst stage in a streptozotocin (STZ)-induced mouse model of diabetes. In this research, 30 male mice were distributed randomly into control, diabetic (streptozotocin 150 mg/kg) and diabetic + Stevia (400 mg/kg) groups. The results revealed a decrease in body and testis weight and elevated blood fasting blood sugar (FBS) levels in the diabetic group, compared with the control. However, Stevia treatment significantly increased body and testis weight, while serum FBS levels were decreased compared with the diabetic group. In addition, Stevia significantly increased blood testosterone levels compared with the diabetic group. Moreover, sperm parameters were improved considerably by Stevia treatment compared with the diabetic group. Furthermore, Stevia administration significantly promoted IVF success rate and in vitro development of fertilized oocytes compared with the diabetic group. In summary, our data indicated that Stevia enhanced sperm parameters, IVF success, and in vitro embryonic developmental competency in diabetic mice, probably because of its antioxidant effects. Therefore, Stevia could ameliorate sperm parameters that, in turn, increase fertilization outcomes in experimental-induced diabetes.