Stevioside Research Articles

Modification of glycosyltransferase SrUGT76G1 based on computer-aided screening of mutation sites for efficient synthesis of rebaudioside M

Steviol glycosides (SGs) from Stevia rebaudiana Bertoni is a widely used sweetener with the advantage of high sweet and low in calories. Stevioside (ST) and Rebaudioside A (Reb A) with a bitter aftertaste are the main sweetening components in SGs. Reb M is a highly potency sweetener with almost no bitter aftertaste but is found in low content in S. rebaudiana Bertoni. In this study, the pre- and post-catalytic differences of stevioside glycosyltransferase SrUGT76G1 were investigated through long-time molecular dynamics simulations. SrUGT76G1 was mutated to enhance its efficiency in catalyzing the synthesis of Reb D to Reb M by identifying the enzyme mutation site through an integrated three-in-one approach that combines hinge region analysis, coevolutionary conservation, and energy calculations. The kcat and kcat/Km values of the M88V mutant with Reb D as substrate were 2.18-fold and 4.85-fold higher than those of wild-type SrUGT76G1, respectively. In addition, the M88V mutant coupled with sucrose synthase StSUS1 increased the yield of Reb M synthesis from Reb D to 92.40%. The present study provides an effective method for rapid search for mutation sites in the enzyme and for increasing the yield of Reb M.

Influence of solvent type and leaf moisture on extraction efficiency of major steviol glycosides in stevia (var. Levent 93) plant

AbstractSteviol glycosides (SGs) are natural sweeteners derived from stevia leaves, which have various applications in food and beverage products. The extraction efficiency of SGs depends on several factors, such as solvent type, solid to solvent ratio, centrifugal force, and temperature. This study aimed to compare the effects of different solvents (ethanol and water) and leaf moisture (dry and fresh) on the extraction efficiency of major steviol glycosides (SGs) of rebaudioside A (Reb A), stevioside (ST), and rebaudioside C (Reb C) in stevia (var. Levent 93) plant. A Box–Behnken design was used to optimise the extraction parameters for each extraction type. The results showed that ethanol was a more effective solvent than water, however, aqueous extraction was more environmentally friendly and low-cost, and could also yield high concentrations of SGs when fresh leaves were used. The major SGs had similar concentrations in ethanolic extraction, but Reb A was twice as high as ST in aqueous extraction. Reb C was influenced by the solid to solvent ratio in both extraction types, but more so in water extraction. Temperature had a positive effect on ST and Reb C in water extraction of fresh leaves, but not in water extraction of dry leaves or ethanolic extraction. The results of this study contribute to the optimisation of SGs extraction from stevia leaves and provide insights for future research on the effects of different solvents and extraction parameters on the quality and purity of SGs.

Effect of Stevioside (Stevia rebaudiana) on Entamoeba histolytica Trophozoites.

Human amoebiasis still represents a major health problem worldwide. Metronidazole has been used as the most common drug to treat the disease; however, it is also known that the drug causes undesirable side effects. This has led to the search for new pharmacological alternatives which include phytochemical compounds with antiamoebic effects. We analyzed the amoebicidal activity of stevioside (STV), a diterpene glycoside present in Stevia rebaudiana, on trophozoites of E. histolytica. Different concentrations of STV were tested, and an inhibitory concentration of 50% of cell viability (IC50) was determined with an exposition of 9.53 mM for 24 h. Trophozoites exposed to STV showed morphological changes evidenced by the decrease in the basic structures related to the movement and adherence to the substrate, as well as ultrastructural features characterized by a loss of regularity on the cell membrane, an increase in cytoplasmic granularity, and an increase in apparent autophagic vacuoles. Also, the decrease in cysteine protease expression and the proteolytic activity of trophozoites to degrade the cell monolayer were analyzed. A histological analysis of hamster livers inoculated with trophozoites and treated with STV showed changes related to the granulomatous reaction of the liver parenchymal tissue. Our results constitute the first report related to the possible use of STV as a therapeutic alternative in amoebiasis.

Covalent organic framework adsorption separation of the mother liquor sugars from industrial steviol: A combination of experimental and theoretical analysis

Mother liquor sugar (MLS) is considered a by-product of the industrial production of stevia glycosides. In this work, an efficient purification strategy for separating Steviol glycosides (SvGls) from MLS was developed. A β-ketenamine covalent organic framework (TPAQ-COFs) was prepared by a surfactant (sodium dodecyl sulfate (SDS)) assisted Schiff base reaction. The adsorption capacity for Rebaudioside A (RA), Rebaudioside D (RD), and Stevioside (ST) was studied. The research indicated that after adding SDS, the aggregation phenomenon of TPAQ-COFs was reduced, and the total adsorption of RA, RD, and ST increased by 338.94 mg/g. According to Langmuir model, the adsorption of RA, RD, and ST by TPAQ-COFs were 423.73, 253.16, and 381.68 mg/g, respectively. In addition, density functional theory and electrostatic potential elucidate the adsorption mechanism of SvGls on TPAQ-COFs. In TPAQ-COFs molecules β-ketenamine forms hydrogen bonds with SvGls, as well as abundant Π-Π interaction site is the main reason for its efficient adsorption of SvGls. After 9 cycles, the reuse rates of ST, RA, and RD in TPAQ-COFs were 96.26 %, 96.90 %, and 94.77 %, respectively, demonstrating high reusability and stability. This study lays the foundation for the practical application of a covalent organic framework in the separation and purification of MLS.

Stevioside Ameliorates Palmitic Acid-Induced Abnormal Glucose Uptake via the PDK4/AMPK/TBC1D1 Pathway in C2C12 Myotubes.

Stevioside (SV) with minimal calories is widely used as a natural sweetener in beverages due to its high sweetness and safety. However, the effects of SV on glucose uptake and the pyruvate dehydrogenase kinase isoenzyme (PDK4) as an important protein in the regulation of glucose metabolism, remain largely unexplored. In this study, we used C2C12 skeletal muscle cells that was induced by palmitic acid (PA) to assess the effects and mechanisms of SV on glucose uptake and PDK4. The glucose uptake of C2C12 cells was determined by 2-NBDG; expression of the Pdk4 gene was measured by quantitative real-time PCR; and expression of the proteins PDK4, p-AMPK, TBC1D1 and GLUT4 was assessed by Western blotting. In PA-induced C2C12 myotubes, SV could significantly promote cellular glucose uptake by decreasing PDK4 levels and increasing p-AMPK and TBC1D1 levels. SV could promote the translocation of GLUT4 from the cytoplasm to the cell membrane in cells. Moreover, in Pdk4-overexpressing C2C12 myotubes, SV decreased the level of PDK4 and increased the levels of p-AMPK and TBC1D1. SV was found to ameliorate PA-induced abnormal glucose uptake via the PDK4/AMPK/TBC1D1 pathway in C2C12 myotubes. Although these results warranted further investigation for validation, they may provide some evidence of SV as a safe natural sweetener for its use in sugar-free beverages to prevent and control T2DM.

Stevioside attenuates bleomycin-induced pulmonary fibrosis by activating the Nrf2 pathway and inhibiting the NF-κB and TGF-β1/Smad2/3 pathways

Objective: This study aimed to investigate the effects of stevioside (STE) on pulmonary fibrosis (PF) and the potential mechanisms. Methods: In this study, a mouse model of PF was established by a single intratracheal injection of bleomycin (BLM, 3 mg/kg). The experiment consisted of four groups: control group, BLM group, and STE treatment groups (STE 50 and 100 mg/kg). ELISA and biochemical tests were conducted to determine the levels of TNF-α, IL-1β, IL-6, NO, hydroxyproline (HYP), SOD, GSH, and MDA. Histopathological changes and collagen deposition in lung tissues were observed by HE and Masson staining. Immunohistochemistry was performed to determine the levels of collagen I-, collagen III-, TGF-β1- and p-Smad2/3-positive cells. Western blot analysis was used to measure the expression of epithelial-mesenchymal transition (EMT) markers, including α-SMA, vimentin, E-cadherin, and ZO-1, as well as proteins related to the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, nuclear transcription factor-κB (NF-κB) pathway, and TGF-β1/Smad2/3 pathway in lung tissues. Results: STE significantly alleviated BLM-induced body weight loss and lung injury in mice, decreased HYP levels, and reduced the levels of collagen I- and collagen III-positive cells, thereby decreasing extracellular matrix (ECM) deposition. Moreover, STE markedly improved oxidative stress (MDA levels were decreased, while SOD and GSH activity were enhanced), the inflammatory response (the levels of TNF-α, IL-1β, IL-6, and NO were reduced), and EMT (the expression of α-SMA and vimentin was downregulated, and the expression of E-cadherin and ZO-1 was upregulated). Further mechanistic analysis revealed that STE could activate the Nrf2 pathway and inhibit the NF-κB and TGF-β1/Smad2/3 pathways. Conclusion: STE may alleviate oxidative stress by activating the Nrf2 pathway, suppress the inflammatory response by downregulating the NF-κB pathway, and inhibit EMT progression by blocking the TGF-β1/Smad2/3 pathway, thereby improving BLM-induced PF.

Long-day photoperiodic requirements for steviol glycosides and gibberellins biosynthesis and bio-sweetener levels optimization in Stevia rebaudiana Bertoni

Stevia rebaudiana, as a short-day perennial, produces steviol glycoside (StGlys) metabolites recognized as healthy natural sweeteners, ca. 300 times sweeter than sucrose, being stevioside (ST) and rebaudioside A (Reb-A), the main StGlys for the natural sweetener demanding industries. Nevertheless, discussions persist regarding the ideal light regimen and respective StGlys biosynthesis regulation in S. rebaudiana aimed at enhancing and optimizing the StGlys large-scale production for industrial purposes. The present work reports on the biosynthesis and the contents of StGlys in leaves and stems in different developmental stages of S. rebaudiana plants submitted to different photoperiods. The final contents resulted from the response of genes in the shared StGlys and gibberellins (GAs) biosynthesis pathway, notably showing differential gene expression patterns in leaves and stems at the different developmental stages. Plants developing under 16/8 h (light/dark) photoperiod increased StGlys contents resulting from the upregulation of S. rebaudiana kaurenoic acid 13-hydroxylase (SrKAH) and uridine diphosphate glycosyltransferase (SrUGTs) genes in association with the downregulation of GAs biosynthesis-related genes in leaves. In contrast, the positive overall regulation of GAs biosynthesis genes in stems appeared as a requirement of GAs for stem elongation and plant growth. Conclusively, the research reports a deeper understanding of transcriptional responses, biosynthesis, and ST and Reb-A yield mechanisms in S. rebaudiana plants under different photoperiodic conditions. Altogether, leading to the potential optimization of commercial production, extraction, and purification while highlighting photoperiodic-responsive StGlys/GAs genes as potential molecular targets for metabolic engineering and future development of superior S. rebaudiana genotypes aiming further large-scale StGlys production and yield to attend the continuous increasing commercial and industrial demand.

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.

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

BackgroundType 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 β-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.MethodsThe 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.ResultsSE 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.ConclusionSE 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.

Stevioside protects primary articular chondrocytes against IL-1β-induced inflammation and catabolism by targeting integrin.

Osteoarthritis (OA) is a common, progressive, and chronic disorder of the joints that is characterized by the inflammation and degradation of articular cartilage and is known to significantly impair quality of daily life. Stevioside (SVS) is a natural diterpenoid glycoside that has anti-inflammatory benefits. Hence, in the current research, it was hypothesized that SVS might exert anti-inflammatory effects on articular chondrocytes and alleviate cartilage degradation in mice with OA. The expression of inflammatory cytokines, like inducible nitric oxide synthase (iNOS), NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), and cyclooxygenase-2 (COX-2) in chondrocytes after interleukin-1β (IL-1β) exposure, was inhibited by the pretreatment of SVS. As well, SVS inhibited the reduction of collagen II and sry-box transcription factor 9 (SOX9) in chondrocytes stimulated by IL-1β and suppressed the expression of MMP3 and MMP13. Further, after treatment with SVS, cell cytometry, autophagy flux, and related protein expression showed diminished cell apoptosis and reduced autophagy impairment. Moreover, SVS blocked the activation of phosphoinositide-3-kinase/Akt/nuclear factor-kappa beta (PI3K/Akt/NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways stimulated by IL-1β. This resulted in decreased cellular inflammation. In vivo experiments with intra-articular injections of SVS in mice with the DMM mouse model demonstrated a decrease in cartilage degradation and an improvement in subchondral bone remodeling. After the integrin αVβ3-related knockdown using siRNA, a reversed effect was observed on the anti-inflammatory, anabolic promoting, catabolic blocking, and NF-κB and MAPK signaling pathway inhibition of SVS on chondrocytes treated with IL-1β. The above findings highlighted that SVS blocked IL-1β, triggered an inflammatory response in mice chondrocytes, and prevented cartilage degradation in vivo through integrin αVβ3. This suggested that SVS might serve as a novel therapeutic option for OA.

Stevioside targets the NF-κB and MAPK pathways for inhibiting inflammation and apoptosis of chondrocytes and ameliorates osteoarthritis in vivo.

Osteoarthritis (OA) is a joint disease that is characterized by articular cartilage degeneration and destruction. Stevioside (SVS) is a diterpenoid glycoside extracted from Stevia rebaudiana Bertoni with some specific effects against inflammatory and apoptotic, whereas it is still unclear what function SVS has in osteoarthritis. This study focuses on the anti-inflammatory and anti-apoptosis functions of SVS on chondrocytes induced by interleukin (IL)-1beta, and the role of SVS in an osteoarthritis model for mice. We can detect the production of inflammatory factors such as nitric oxide (NO) and prostaglandin E2 (PGE2) using real-time quantitative polymerase chain reaction (RT-qPCR), the Griess reaction, and enzyme linked immunosorbent assay (ELISA). On the basis of Western blot, we have observed the protein expressions of cartilage matrix metabolism, inflammatory factors, and apoptosis of chondrocytes. Simultaneously, the pharmacological effects of SVS in mice were evaluated by hematoxylin and eosin (HE), toluidine blue, Safranin O, and immunohistochemical staining. The results show that SVS slows extracellular matrix degradation and chondrocyte apoptosis. In addition, SVS mediates its cellular effect by inhibiting the activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways. Meanwhile, molecular docking studies revealed that SVS has excellent binding capabilities to p65, extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). The study suggests that SVS can be developed as a potential osteoarthritis treatment.

Biological Assessment of Stevioside and Sucralose as Sucrose Substitutes for Diabetics on STZ-Induced Diabetes in Rats.

Numerous food organizations have identified excessive calorie consumption and accompanying ailments as significant health risks associated with high sugar consumption. Administering stevioside (ST), sucralose (SU), and the two synergically (SU+ST) affected normal rats' weight gain. In the current study, SU showed the highest undesired effect. Indeed, administering the three treatments to diabetic rats (DR) did not improve the rats' weight gain. Although, insulin injection synergically with the treatments improved the weight gain, as recorded after three weeks. The best-improving rate was observed in the ST group. After the administration of ST and ST+SU to the DR, the blood glucose level (GL) was positively affected, with SU having no effects on reducing the GL. A considerable reduction in serum insulin (SIL) was noted in the DR+SU group. On the contrary, ST did not negatively affect the SIL, rather an improvement was recorded. In addition, giving SU did not significantly affect the ALT level in the DR or normal rats (NR). A significant improvement in total bilirubin (TBILI) was observed when insulin was injected with ST or SU in DR groups. Further, triglycerides (TG) after administering ST, SU, or ST+SU to NR had no significant difference compared to the control group (NR). Although, the three treatments markedly but not significantly lowered TG in the DR. For total cholesterol (CHO), both DR and NR had no significant effect after the three treatments. No histopathological alterations were recorded in the NR group. Diffuse and severe atrophy of the islands of Langerhans due to depletion of their cells and mild papillary hyperplasia of the pancreatic ducts were represented by a slightly folded ductal basement membrane and newly formed ductules in STZ-DR. Simultaneous atrophy and absence of the cells of islands of Langerhans besides ductal hyperplasia were evident in DR+SU. Hyperplastic ductal epithelium and atrophic Langerhans cells were seen in DR+SU+In. Degeneration and mild atrophy were observed in the islands of Langerhans structures. There was essentially no noticeable change after utilizing ST. A slight shrinkage of the Langerhans' islets was detected in DR+ST. In DR+ST+In, no histopathological alterations in the islands of Langerhans were recorded. Congestion in the stromal blood vessels associated with degenerative and necrotic changes in the cells of the islands of Langerhans in DR+SU+ST was observed. In NR+SU, congestion of the blood vessels associated with mild atrophy in the islands of Langerhans and dilatation in stromal blood vessels was noticed. In conclusion, ST is safe, and SU should be taken cautiously, such as mixing with ST and/or taken at a very low concentration to avoid its drastic effect on the human body.

Stevioside attenuates osteoarthritis via regulating Nrf2/HO-1/NF-κB pathway.

Osteoarthritis (OA) is a chronic disease that may cause articular cartilage degeneration, and synovial inflammation, resulting in considerable pain, poor quality of life, and functional limitations. Previous research has shown that ECM degradation and inflammation are involved in the progression of OA. Stevioside (STE), a naturally diterpenoid glycoside, is isolated from the Stevia rebaudiana (Bertoni), which has been exerted a variety of pharmacological activities, involving anti-inflammatory, anti-oxidative, and neuroprotective effects. However, STE's effects on OA and its mechanism still need further research. In the present study, we examined the anti-inflammatory effects of STE (STE) in both mouse chondrocytes and OA model induced by destabilization of the medial meniscus (DMM). In vitro, the mouse chondrocytes were treated with STE (0, 10, 20, 40​M, 24​h) after stimulated with IL-1β (10​ng/mL, 24​h). The expression of ant-inflammation-relative mediators iNOS and Cox-2 were detected by Western blot and RT-PCR. The catabolic factors (MMP-13, ADAMTS-4) and cartilage matrix constituent (Aggrecan, Collagen II) were measured by Western blot and Immunofluorescence staining. The Nrf2/HO-1/NF-κB signaling molecules were detected by Western blot. In vivo, histological analysis was used to evaluate the severity of mouse OA models. STE remarkably inhibited the IL-1β-induced expression of iNOS and Cox-2, generation of MMP-13, ADAMTS-4 and degradation of Aggrecan and Collagen II. Furthermore, we found that the chondroprotective effect of STE via Nrf2/HO-1/NF-κB signaling pathway. In vivo, the cartilage treated with STE displayed attenuated degeneration, low OARIS scores compared with DMM group. In conclusion, we considered that STE might be a promising therapeutic agent for the treatment of OA in future. Our findings indicated that STE can ameliorate the development of OA via inhibiting the inflammation. The underlying mechanism may be related to the Nrf2/HO-1/NF-κB signaling pathway. Moreover, the treatment of STE significantly relieves the progression in the mouse DMM model. All of the results demonstrated the therapeutic of STE in OA treatment. This study demonstrates a more efficient and safe application of STE in treating osteoarthritis, provide a new concept for the cartilage targeted application of natural compounds.

Stevia and Stevioside Attenuate Liver Steatosis through PPARα-Mediated Lipophagy in db/db Mice Hepatocytes.

Lipophagy, a type of autophagy that breaks down lipid droplets, is essential in the regulation of intracellular lipid accumulation and intracellular free fatty acid levels in numerous organisms and metabolic conditions. We investigated the effects of Stevia rebaudiana Bertoni (S), a low-calorie sweetener, and stevioside (SS) on hepatic steatosis and autophagy in hepatocytes, as well as in db/db mice. S and SS reduced the body and liver weight and levels of serum triglyceride, total cholesterol, and hepatic lipogenic proteins. In addition, S and SS increased the levels of fatty acid oxidase, peroxisome proliferator-activated receptor alpha (PPARα), and microtubule-associated protein light chain 3 B but decreased that of sequestosome 1 (p62) in the liver of db/db mice. Additionally, Beclin 1, lysosomal associated membrane protein 1, and phosphorylated adenosine monophosphate-activated protein kinase protein expression was augmented following S and SS treatment of db/db mice. Furthermore, the knockdown of PPARα blocked lipophagy in response to SS treatment in HepG2 cells. These outcomes indicate that PPARα-dependent lipophagy is involved in hepatic steatosis in the db/db mouse model and that SS, a PPARα agonist, represents a new therapeutic option for managing associated diseases.

Steviol Glycoside, L-Arginine, and Chromium(III) Supplementation Attenuates Abnormalities in Glucose Metabolism in Streptozotocin-Induced Mildly Diabetic Rats Fed a High-Fat Diet.

Stevia rebaudiana Bertoni and its glycosides are believed to exhibit several health-promoting properties. Recently, the mechanisms of the anti-diabetic effects of steviol glycosides (SG) have been the subject of intense research. The following study aims to evaluate the results of SG (stevioside (ST) and rebaudioside A (RA)) combined with L-arginine (L-Arg) and chromium(III) (CrIII) supplementation in streptozotocin- (STZ) induced mild type 2 diabetic rats fed a high-fat diet (HFD), with particular emphasis on carbohydrate and lipid metabolisms. The experiment was carried out on 110 male Wistar rats, 100 of which were fed an HFD to induce insulin resistance, followed by an intraperitoneal injection of streptozotocin to induce mild type 2 diabetes. After confirmation of hyperglycemia, the rats were divided into groups. Three groups served as controls: diabetic untreated, diabetic treated with metformin (300 mg/kg BW), and healthy group. Eight groups were fed an HFD enriched with stevioside or rebaudioside A (2500 mg/kg BW) combined with L-arginine (2000 or 4000 mg/kg BW) and Cr(III) (1 or 5 mg/kg BW) for six weeks. The results showed that supplementation with SG (ST and RA) combined with L-arg and Cr(III) could improve blood glucose levels in rats with mild type 2 diabetes. Furthermore, ST was more effective in improving blood glucose levels, insulin resistance indices, and very low-density lipoprotein cholesterol (VLDL-C) concentrations than RA. Although L-arg and Cr(III) supplementation did not independently affect most blood carbohydrate and lipid indices, it further improved some biomarkers when combined, particularly with ST. Notably, the beneficial impact of ST on the homeostatic model assessment–insulin resistance (HOMA-IR) and on the quantitative insulin-sensitivity check index (QUICKI) was strengthened when mixed with a high dose of L-arg, while its impact on antioxidant status was improved when combined with a high dose of Cr(III) in rats with mild type 2 diabetes. In conclusion, these results suggest that supplementary stevioside combined with L-arginine and Cr(III) has therapeutic potential for mild type 2 diabetes. However, further studies are warranted to confirm these effects in other experimental models and humans.

End-point determination of the extraction processes for Stevia rebaudiana Bertoni leaves by near-infrared spectroscopy

Rebaudioside A (RA) and Stevioside (STV) are abundant steviol glycosides (SGs) contained in Stevia rebaudiana Bertoni leaves, which exhibit good stability and various pharmacological activities that have been widely developed in the food and pharmaceutical industries. However, the stevia industry still suffers from high consumption, low efficiency, and long-term dependence on the operational experience of workers. The extraction process of Stevia rebaudiana Bertoni leaves one of the fundamental units for the production of SGs, which is crucial for the homogeneous stability of the final product. The applications of near-infrared (NIR) spectroscopy combined with chemometrics in determining the end-point of the extraction process were proposed in this study. Firstly, the quantitative models were established by partial least squares regression (PLSR) and back-propagation artificial neural network (BP-ANN) to rapidly detect the changes of content RA and STV, which have good prediction results. Secondly, the qualitative analysis methods were established by moving block of standard deviation (MBSD), absolute distance of standard deviation (ADSD) and principal component analysis (PCA) to rapidly determine the extraction end-point, which MBSD method was consistent with the high-performance liquid chromatography (HPLC) method. Finally, the variation of the extraction process was revealed by Aquaphotomic to provide a microscopic perspective of water molecules for end-point determination. The HPLC method requires 30 min to determine the content, whereas NIR spectroscopy requires only 18 s to obtain a spectrum. These results indicate that the extraction end-point of Stevia rebaudiana Bertoni leaves can be determined rapidly and accurately using NIR spectroscopy, which provides a significant reference for other food, medicinal plants, and agricultural products production processes.

A Hybrid RSM-ANN-GA Approach on Optimization of Ultrasound-Assisted Extraction Conditions for Bioactive Component-Rich Stevia rebaudiana (Bertoni) Leaves Extract.

Stevia rebaudiana (Bertoni) leaves consist of dietetically important diterpene steviol glycosides (SGs): stevioside (ST) and rebaudioside-A (Reb-A). ST and Reb-A are key sweetening compounds exhibiting a sweetening potential of 100 to 300 times more intense than that of table sucrose. Ultrasound-assisted extraction (UAE) of SGs was optimized by effective process optimization techniques, such as response surface methodology (RSM) and artificial neural network (ANN) modeling coupled with genetic algorithm (GA) as a function of ethanol concentration (X1: 0–100%), sonication time (X2: 10–54 min), and leaf–solvent ratio (X3: 0.148–0.313 g·mL−1). The maximum target responses were obtained at optimum UAE conditions of 75% (X1), 43 min (X2), and 0.28 g·mL−1 (X3). ANN-GA as a potential alternative indicated superiority to RSM. UAE as a green technology proved superior to conventional maceration extraction (CME) with reduced resource consumption. Moreover, UAE resulted in a higher total extract yield (TEY) and SGs including Reb-A and ST yields as compared to those that were obtained by CME with a marked reduction in resource consumption and CO2 emission. The findings of the present study evidenced the significance of UAE as an ecofriendly extraction method for extracting SGs, and UAE scale-up could be employed for effectiveness on an industrial scale. These findings evidenced that the UAE is a high-efficiency extraction method with an improved statistical approach.

Directional bioconversion and optimization of stevioside into rubusoside by Lelliottia sp. LST-1.

The present study aimed to specifically transform stevioside (ST) into rubusoside (RS) through bioconversion with high efficiency, seeking to endow steviol glycosides (SGs) with subtle flavours for commercial acceptability. An endophytic bacterium named Lelliottia LST-1 was screened and confirmed to specifically convert ST into RS, reaching a conversion rate of 75.4% after response surface optimization. Phylogenetic analysis combined with complete genome sequencing demonstrated that LST-1 was also presumed to be a new species. To further explore the principle and process of biological transformation, the potential beta-glucosidases GH3-1, GH3-2, GH3-3 and GH3-4 were expressed, purified and reacted with SGs. High-performance liquid chromatography revealed that all enzymes hydrolysed ST and generated RS, but substrate specificity analysis indicated that GH3-2had the highest substrate specificity towards STs and the highest enzyme activity. The potential β-glucosidase GH3-2 in Lelliottia sp. LST-1 was found to specifically and efficiently convert ST to RS. The efficient biotransformation of ST into RS will be beneficial to its large-scale production and extensive application in the food and pharmaceutical industries.

The Preparation of High Purity of Rebaudioside A and Stevioside and the Enrichment of Rebaudioside C by Subsequent Twice Crystallizations from Natural Stevia Resources

A comprehensive utilization of rebaudioside A (RA), stevioside (ST), and rebaudioside C (RC) from natural stevia resources was proposed. The influence of the solvent, solvent concentration, solid-liquid ratio, temperature, and time on the purity and recovery were investigated with response surface methodology. A 99% purity and 81% recovery of the RA were achieved by one crystallization of crude stevia under optimized conditions. Around a 95% purity and 80% recovery of the ST were easily achieved by the recrystallization of less value of mother liquor sugar (MLS) with a certain concentration of isopropyl alcohol–methanol aqueous solution. During the crystallization of the ST, the enriched RC in the liquid phase was more than three times higher than that in the crude stevia, which can provide cheaper RC raw material for the subsequent preparation of very expensive and high-purity RC.

Influence of calcium and magnesium elimination on plant biomass and secondary metabolites of Stevia rebaudiana Bertoni.

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