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Objective: To discuss the effect of rubusoside (RUB) on the spinal cord injury (SCI) and neuroinflammation in the mice, and to clarify its mechanism. Methods: A total of 48 female Kunming mice were randomly divided into sham operation group, SCI group, SCI+low dose of RUB group, and SCI+high dose of RUB group, and there were 12 mice in each group. Spinal cord injury behavior (BBB) scoring method was used to assess the motor function of the hind limbs of the SCI mice; water content method was used to detect the spinal cord edema of the SCI mice; real-time fluorscence quantitative PCR (RT-qPCR) method was used to detect the expression levels of pro-inflammatory cytokines cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) mRNA of the mice in various groups; ELISA method was used to detect the levels of inflammatory factors in serum of the mice in various groups; HE staining was used to observe the pathomorphology of spinal cord tissue of the mice in various groups；immunofluorescence was used to detect the activation of microglia in spinal cord tissue of the mice in various groups; Western blotting method was used to detect the expression levels of related proteins in spinal cord tissue of the mice in various groups. Results: The BBB score analysis results showed that compared with sham operation group, the score of the mice in SCI group was decreased to 0; compared with SCI group, the BBB scores of the mice in SCI+low dose of RUB group and SCI+high dose of RUB group were gradually increased. The spinal cord tissue water content detection results showed that compared with sham operation group, the water content in spinal cord tissue of the mice in SCI group was significantly increased (P<0. 01) ; compared with SCI group, the water contents in spinal cord tissue of the mice in SCI+ low dose of RUB group and SCI+ high dose of RUB group were significantly decreased (P<0. 01) . The RT-qPCR results showed that compared with sham operation group, the expression levels of COX-2, IL-1β, and TNF-α mRNA in spinal cord tissue of the mice in SCI group were significantly increased (P<0. 001) . Compared with SCI group, the expression levels of COX-2, IL-1, and TNF- α mRNA in spinal cord tissue of the mice in SCI+ low dose of RUB group and SCI+ high dose of RUB group were significantly decreased (P<0. 001). The ELISA results showed that compared with sham operation group, the levels of IL-1β (P<0. 01) and TNF-α (P<0. 001) in serum of the mice in SCI group were increased; compared with SCI group, the levels of IL-1β and TNF-α in spinal cord tissue of the mice in SCI+ low dose of RUB group and SCI+ high dose of RUB group were decreased (P<0. 001). The Western blotting results showed that compared with sham operation group, the expression levels of nuclear factor kappa B (NF-κB) inhibitor protein-α (IκB-α), phosphorylated IκB-α (p-IκB-α), phosphorylated p65 (p-p65), p65, phosphorylated p38 (p-p38), phosphorylated extracellular regulated protein kinase (p-ERK), and phosphorylated c-Jun N-terminal kinase (p-JNK) proteins in spinal cord tissue of the mice in SCI group were significantly increased (P<0. 05 or P<0. 001) ; compared with SCI group, the expression levels of IκB- α, p-IκB- α, p-p65, p65, p-p38, p-ERK, and p-JNK proteins in spinal cord tissue of the mice in SCI+ low dose of RUB group and SCI+ high dose of RUB group were significantly decreased (P<0. 001). The HE staining results showed that compared with sham operation group, the spinal cord tissue of the mice in SCI group had loose organization with the formation of vacuoles, and a large necrotic cavity was present in the center of the spinal cord; compared with SCI group, the central necrotic cavity area of the mice in SCI+ low dose of RUB group and SCI+ high dose of RUB group was significantly decreased (P<0. 05). The immunofluorescence results showed that compared with sham operation group, the number of positive microglia cells in spinal cord tissue of the mice in SCI group was significantly increased (P<0. 001) ; compared with SCI group, the numbers of positive microglia cells in spinal cord tissue of the mice in SCI+ low dose of RUB group and SCI+ high dose of RUB group were significantly decreased (P<0. 001). Conclusion: Rubusoside can improve the motor function impairment in the SCI mice, mitigate the neuroinflammation in spinal cord tissue, and inhibit the activation of microglia. The mechanism may be related to the downregulation of expression of proteins associated with the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways in spinal cord tissue. [ABSTRACT FROM AUTHOR]

Terpenoids are important secondary metabolites in Rubus . Rubusoside is a relatively specific diterpenoid bioactive component in the leaves of Chinese Sweet Tea ( Rubus suavissimus ). However, the terpenoid anabolic pathway of Rubus and the molecular mechanism underlying the specific accumulation of rubusoside in R. suavissimus remain unclear. Here, metabolomics and transcriptomics analyses were performed on differences in terpenoid metabolism levels between R. suavissimus (sweet leaves) and Rubus chingii (bitter leaves). Steviol glycosides and goshonosides primarily accumulated in R. suavissimus and R. chingii , respectively. Three pairs of highly homologous glycosyltransferase genes ( UGT85A57 , UGT75L20 , and UGT75T4 ) associated with rubusoside biosynthesis in the two Rubus species were identified. The three pairs of UGT proteins in both species could glycosylate steviol. Thus, the transcriptional regulation of UGTs in R. suavissimus appears to play a pivotal role in rubusoside accumulation. Our findings provide insights into the differences in terpenoid metabolism between R. suavissimus and R. chingii and reveal the molecular mechanism of rubusoside accumulation in R. suavissimus .

The current study aimed at investigating the therapeutic effects of rubusoside on type 2 diabetes mellitus (T2DM) mice models as an alternative hypoglycemic candidate drug. T2DM mice models were established with a combination of streptozotocin (STZ) intraperitoneal injection and high-fat diet. After 10 weeks of rubusoside intragastric administration (100, 200 mg/kg/day) to the mice, the body weight, fasting blood glucose, glucose tolerance, and blood lipids were measured. The liver protein expression levels of p-AMPK, GLUT2, GLUT4 and total antioxidant capacity were also investigated. After 10 weeks of rubusoside administration, the levels of blood glucose and lipids were decreased in T2DM mice. Compared with the model group, rubusoside administration significantly decreased the liver mass-to-body weight ratio, upregulated p-AMPK and GLUT4, and downregulated GLUT2 expression levels in the liver. Activities of superoxide dismutase (SOD), catalase (CAT), and gluathione peroxidase (GSH-Px) were increased, and the concentration of malondialdehyde (MDA) was decreased to reduce oxidative stress in the liver. Liver hematoxylin and eosin (H&E) pathological analysis also showed that rubusoside had a protective effect on T2DM mice liver. These results demonstrate that rubusoside could be used as an anti-diabetic candidate drug, and that its hypoglycemic mechanismmight be related to the activation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) to modulate the expression of GLUT2 and GLUT4. Finally, rubusoside could also increase total antioxidant capacity to protect the liver from oxidative stress. [ABSTRACT FROM AUTHOR]

As a natural sweetening and solubilizing agent, rubusoside has great potential in the application of healthy beverages and pharmaceuticals. However, the direct extraction and purification of rubusoside from raw materials is inefficient. In this work, a novel β-glucosidase (Cs BGL) was obtained from Chryseobacterium scophthalmum 1433 through screening of the environmental microorganisms. Cs BGL markedly hydrolyzed sophorese (Glcβ1-2Glc) and laminaribiose (Glcβ1-3Glc), but for steviol glycosides, it only hydrolyzed the C-13/C-19-linked sophorese, instead of the C-13/C-19-linked Glcβ1-2[Glcβ1-3]Glc trisaccharide and Glcβ1-monosaccharide. It efficiently hydrolyzed stevioside (240 g/L) to produce rubusoside (99% yield) at 47.5°C for 70 min. Even when using a crude steviol glycosides extract (500 g/L) containing ∼226 g/L stevioside as the substrate, Cs BGL could also convert stevioside to rubusoside (99% yield) at 47.5°C for 2 h, in which the rubusoside concentration increased from the initial 42 g/L to the final 222 g/L. These results reveal that Cs BGL would be a promising biocatalyst for the industry-scale production of rubusoside from stevioside or/and the crude steviol glycosides extract. As a natural sweetening and solubilizing agent, rubusoside has great potential in the application of healthy beverages and pharmaceuticals. A novel β-glucosidase CsBGL obtained here from Chryseobacterium scophthalmum 1433 through screening of the environmental microorganisms could specifically hydrolyze the C-13- or/and C-19-linked sophorese of steviol glycosides, but not the C-13- or/and C-19-linked Glcβ1-2[Glcβ1-3]Glc trisaccharide and Glcβ1-monosaccharide of steviol glycosides, and thus could be a promising biocatalyst for efficient rubusoside production from stevioside. [ABSTRACT FROM AUTHOR]

As a natural sweetening and solubilizing agent, rubusoside has great potential in the application of healthy beverages and pharmaceuticals. However, the direct extraction and purification of rubusoside from raw materials is inefficient. In this work, a novel β-glucosidase (CsBGL) was obtained from Chryseobacterium scophthalmum 1433 through screening of the environmental microorganisms. CsBGL markedly hydrolyzed sophorese (Glcβ1-2Glc) and laminaribiose (Glcβ1-3Glc), but for steviol glycosides, it only hydrolyzed the C-13/C-19-linked sophorese, instead of the C-13/C-19-linked Glcβ1-2[Glcβ1-3]Glc trisaccharide and Glcβ1-monosaccharide. It efficiently hydrolyzed stevioside (240 g/L) to produce rubusoside (99% yield) at 47.5°C for 70 min. Even when using a crude steviol glycosides extract (500 g/L) containing ∼226 g/L stevioside as the substrate, CsBGL could also convert stevioside to rubusoside (99% yield) at 47.5°C for 2 h, in which the rubusoside concentration increased from the initial 42 g/L to the final 222 g/L. These results reveal that CsBGL would be a promising biocatalyst for the industry-scale production of rubusoside from stevioside or/and the crude steviol glycosides extract.

The anti‐inflammatory and anti‐asthmatic effects of rubusoside (Rbs) were investigated in the ovalbumin (OVA)‐induced asthmatic mice, followed by effective attenuation of Rbs treatment on the airway hyperresponsiveness and reduction of inflammatory cells inside the bronchoalveolar lavage fluid (BALF). The mitigation of inflammatory infiltration as a result of Rbs treatment was histologically observed in these mice lungs. Rbs contributed to the decrease of inflammatory cytokines (TNF‐α, IL‐13, IL‐6, IL‐5, and IL‐4) inside the BALF of mice with asthma. A decline of OVA‐dependent IgE and IgG1 inside the serum was also noticed in these mice. Rbs was proved to enhance the mRNA level of Foxp3 inside the mice lung affected with asthma while decrease that of IL‐17A, IL‐23, and RORγt. NF‐κB pathway activation elicited by OVA was suppressed by Rbs inside the pulmonary tissues. Rbs played significantly in the reduction of airway inflammation induced by OVA which with modulating NF‐κB pathway activation. Practical applications: Simultaneous therapy with medicine and food is strategically significant for disease prevention and treatment in traditional Chinese medicine. Rbs is a diterpene glycoside isolated from Rubus suavissimus. The anti‐inflammatory and anti‐asthmatic mechanism dependent of Rbs need further study clinically. The goal of current investigation is to explore the anti‐inflammatory as well as anti‐asthmatic activity of Rbs in mouse models of OVA‐induced experimental allergic asthma. Results of the present study are scientifically supportive for the use of Rbs as an adjunctive reagent for clinical treatment of allergic asthma. [ABSTRACT FROM AUTHOR]

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Parkinson's disease (PD) is a neurodegenerative condition characterized by the progressive loss of dopaminergic neurons within the substantia nigra. Neuroinflammation plays a pivotal role in the pathogenesis of PD, involving the activation of microglia cells, heightened production of proinflammatory cytokines, and perturbations in the composition of the gut microbiota. Rubusoside (Ru), the principal steviol bisglucoside present in Rubus chingii var. suavissimus (S.K.Lee) L.T.Lu (Rosaceae), has been documented for its anti-inflammatory properties in diverse disease models. Nonetheless, there is an imperative need to comprehensively assess and elucidate the protective and anti-inflammatory attributes of Ru concerning PD, as well as to uncover the underlying mechanism involved. The aim of this study is to evaluate the neuroprotective and anti-inflammatory effects of Ru on PD and investigate its potential mechanisms associated with microbes. We pre-treated mice and cell lines with Ru in order to simulate the progression of PD and the neuroinflammatory state. The mouse model was induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), SN4741 cells were induced by 1-methyl-4-phenylpyridine (mpp+), and BV-2 cells were induced by lipopolysaccharide (LPS). We assessed the impact of Ru on motor function, neuroinflammation, neuron apoptosis, the composition of gut microbes, and their metabolites. Ru treatment reduces the release of pro-inflammatory mediators by inhibiting microglia activation. It also prevents neuronal apoptosis, thereby safeguarding dopaminergic neurons and ameliorating motor dysfunction. Furthermore, it induces alterations in the fecal microbiota composition and metabolites profile in PD mice. In vitro experiments have demonstrated that Ru inhibits neuronal apoptosis in SN4741 cells induced by mpp+, suppresses the production of pro-inflammatory mediators, and activates the c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase (p38 MAPK), and nuclear factor kappa-B (NF-κB) signaling pathways. Ru exhibits inhibitory effects on the MPTP-induced PD model by mitigating neuroinflammation and neuronal apoptosis while also inducing changes in the gut microbiota and metabolite composition. [Display omitted] [ABSTRACT FROM AUTHOR]

R. suavissimus (Rubus suavissimus S. Lee), also known as Chinese sweet tea, is a deciduous shrub of the genus Rubus in the family Rosaceae in China. R. suavissimus leaves contain various chemical components with excellent physiological functions, making them both drinkable as tea and medicinal. Moreover, rubusoside, regarded as the main sweet component in R. suavissimus , is an ideal sweetener with the characteristics of high sweetness and low-calorie. However, less attention has been paid to R. suavissimus , which hinders its popularization and application. This paper comprehensively summarizes the research progress on the nutritional components of R. suavissimus leaves, including chemical structure, extraction methods, health benefits, and mechanism of action, as well as expounds on the application status of R. suavissimus. Additionally, the production of rubusoside through biotransformation and biosynthesis is concerned. R. suavissimus leaves are rich in nutrients such as rubusoside, polyphenols, flavonoids, and polysaccharides, which can resist allergies, reduce inflammation, prevent dental caries, regulate glucose and lipid metabolism, and are widely applied in the fields of food, beverages, medicine, and skincare. As the main sweet component, rubusoside is expected to be a natural green substitute for sweeteners such as sucrose. In addition to being extracted from R. suavissimus , rubusoside can also be produced in large quantities through biotransformation and biosynthesis. Overall, R. suavissimus leaves have three major functions of "tea, sugar and medicine", and are a kind of health care product and sweetener with great development value. • The chemical components of R. suavissimus are comprehensively summarized. • The health benefits of R. suavissimus are emphasized. • The application status of R. suavissimus is elaborated. • The production methods of rubusoside through biotransformation and biosynthesis are concerned. [ABSTRACT FROM AUTHOR]

Abstract Many anti-cancer drugs have a common problem of poor solubility. Increasing the solubility of the drugs is very important for its clinical applications. In the present study, we revealed that the solubility of insoluble drugs was significantly enhanced by adding rubusoside (RUB). Further, it was demonstrated that RUB could form micelles, which was well characterized by Langmuir monolayer investigation, transmission electron microscopy, atomic-force microscopy, and cryogenic transmission electron microscopy. The RUB micelles were ellipsoid with the horizontal distance of ~25 nm and vertical distance of ~1.2 nm. Insoluble synergistic anti-cancer drugs including curcumin and resveratrol were loaded in RUB to form anti-cancer micelles RUB/CUR + RES. MTT assay showed that RUB/CUR + RES micelles had more significant toxicity on MCF-7 cells compared to RUB/CUR micelles + RUB/RES micelles. More importantly, it was confirmed that RUB could load other two insoluble drugs together for remarkably enhanced anti-cancer effect compared to that of RUB/one drug + RUB/another drug. Overall, we concluded that RUB-based micelles could efficiently load insoluble drugs for enhanced anti-cancer effect.

Aim: The aim of this study is to improve the water solubility and stability of cannabidiol (CBD) utilizing micelle technology., Materials and Methods: Mixing of rubusoside (RUB) with Poloxamer 407 (P407) was explored as the wall material for the preparation of CBD micelles. In this study, CBD-loaded mixed micelles (CBD-M) composed of P407 and RUB was successfully formed by self-assembly then solid were prepared by solvent evaporation. The saturated solubility of CBD-loaded micelles in water increased to 15.60 mg/mL, 1560-fold compared with its intrinsic solubility (0.01 mg/mL). The average size of CBD-M was 103 ± 2.66 nm and the encapsulation efficiency for CBD was 92.8 + 4.7 %, and 18.6 + 0.94% for drug-loading., Results: The morphology and encapsulation of CBD-M were characterized by TEM, FI-IR, DSC and TG. The CBD-M solution was stable and CBD-M did not precipitate or leak after being diluted and centrifugated. Besides, CBD-M solution was stable for 6 months at 4°C and room temperature. In vitro antioxidant studies found that the antioxidant activity of CBD remained at the same level after micellization., Conclusion: These results suggest that CBD-M may be a promising and competitive formulation for the delivery of CBD, laying a foundation for improving the bioavailability in the future., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Streptococcus mutans plays an important role in the development of dental caries in humans by synthesizing adhesive insoluble glucans from sucrose by mutansucrase activity. To explore the anti-cariogenic characteristics of rubusoside (Ru), a natural sweetener component in Rubus suavissimus S. Lee (Rosaceae), we investigated the inhibitory effect of Ru against the activity of mutansucrase and the growth of Streptococcus mutans. Ru (50 mM) showed 97% inhibitory activity against 0.1 U/mL mutansucrase of S. mutans with 500 mM sucrose. It showed competitive inhibition with a Ki value of 1.1 ± 0.2 mM and IC50 of 2.3 mM. Its inhibition activity was due to hydrophobic and hydrogen bonding interactions based on molecular docking analysis. Ru inhibited the growth of S. mutans as a bacteriostatic agent, with MIC and MBC values of 6 mM and 8 mM, respectively. In addition, Ru showed synergistic anti-bacterial activity when it was combined with curcumin. Therefore, Ru is a natural anti-cariogenic agent with anti-mutansucrase activity and antimicrobial activity against S. mutans. [ABSTRACT FROM AUTHOR]

Rubusoside is a natural sweetener and the active component of Rubus suavissimus. The preventive and therapeutic effect of rubusoside on high-fat diet-induced (HFD) serum metabolite changes in golden hamsters was analyzed by 1H-NMR metabolomics to explore the underlying mechanism of lipid metabolism regulation. 1H-NMR serum metabolomics analyses revealed a disturbed amino acid-, sugar-, fat-, and energy metabolism in HFD animals. Animals supplemented with rubusoside can partly reverse the metabolism disorders induced by high-fat diet and exerted good anti-hypertriglyceridemia effect by intervening in some major metabolic pathways, involving amino acid metabolism, synthesis of ketone bodies, as well as choline and 4-hydroxyphenylacetate metabolism. This study indicates that rubusoside can interfere with and normalize high-fat diet-induced metabolic changes in serum and could provide a theoretical basis to establish rubusoside as a potentially therapeutic tool able to revert or prevent lipid metabolism disorders.

Steviol glucosides (SGs) such as rubusoside (Ru), stevioside (Ste), rebaudioside A (RebA) and stevioside glucosides (SG) are herbal tea sweeteners that enhance the solubility and stability of a number of pharmaceutically important compounds. The complex of epigallocatechin gallate (EGCG) with 10% (w/v) each Ru, Ste, RebA or SG enhanced the water solubility of EGCG over 15 times to 345, 312, 341, or 320 mg/mL, respectively. The 2,2-diphenyl-1-picrylhydrazyl radical scavenging (SC) activities of EGCG, EGCG-Ru, EGCG-Ste, EGCG-RebA, and EGCG-SG in water were 5.88, 6.03, 6.52, 4.89, and 4.23 μg/mL, respectively. EGCGs complexed with different SGs maintained inhibitory activities against human intestinal maltase, human pancreatic α-amylase, and the growth of Streptococcus mutans, Helicobacter pylori, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, and Clostridium difficile. In glucose tolerance test using C57BL/6 mice, plasma glucose levels in mice treated with EGCG or EGCG-Ste complex were decreased by 9.34%, which was 31.08% lower than those treated with maltose. The efficient and cost-effective EGCG-SGs production method might be applicable to produce water soluble bioactive nutraceuticals in large scale. [ABSTRACT FROM AUTHOR]

Many anti-cancer drugs have a common problem of poor solubility. Increasing the solubility of the drugs is very important for its clinical applications. In the present study, we revealed that the solubility of insoluble drugs was significantly enhanced by adding rubusoside (RUB). Further, it was demonstrated that RUB could form micelles, which was well characterized by Langmuir monolayer investigation, transmission electron microscopy, atomic-force microscopy, and cryogenic transmission electron microscopy. The RUB micelles were ellipsoid with the horizontal distance of ~25 nm and vertical distance of ~1.2 nm. Insoluble synergistic anti-cancer drugs including curcumin and resveratrol were loaded in RUB to form anti-cancer micelles RUB/CUR + RES. MTT assay showed that RUB/CUR + RES micelles had more significant toxicity on MCF-7 cells compared to RUB/CUR micelles + RUB/RES micelles. More importantly, it was confirmed that RUB could load other two insoluble drugs together for remarkably enhanced anti-cancer effect compared to that of RUB/one drug + RUB/another drug. Overall, we concluded that RUB-based micelles could efficiently load insoluble drugs for enhanced anti-cancer effect. [ABSTRACT FROM AUTHOR]

• Isolation and characterization of rare α-1,3-glycosylated rubusoside derivatives. • Structural characterization via ESI-IMS-QToF-MS and NMR. • Sensory evaluation and comparison to previously known glycosylated rubusoside. • Investigation of degradation by α-amylase from human saliva. In this work we investigated mixtures from α-glycosylation of rubusoside with cyclodextrin glycosyltransferases. In addition to the previously known α-1,4 glycosylated derivatives, nine new compounds with rare α-1,3-glycosidic bonds were identified based on nuclear magnetic resonance spectroscopy and mass spectrometric analysis. Furthermore, sensory properties of monoglycosylated rubusoside derivatives were investigated and compared to previously described monoglycosylated compounds. Additionally, digestion with α-amylase from human saliva was investigated for different glycosylated rubusoside derivatives. [ABSTRACT FROM AUTHOR]

Stevia rebaudiana is of great importance due to its steviol glycosides (SGs) which are natural sweeteners used by the food industry as well as having medicinal purposes. In the present study, the effect of plant growth regulators (PGRs) and explant types on in vitro propagation and shoot growth of S. rebaudiana were studied, the effect of PGRs on SGs production was determined. For this purpose, nodal explants and shoot tip explants were cultured on woody plant medium (WPM) supplemented with cytokinins [6-benzyladenine (BA), kinetin (Kn), or thidiazuron (TDZ)] or cytokinins + auxins combinations [BA + indoleacetic acid (IAA); BA + naphthaleneacetic acid (NAA); Kn + IAA; Kn + NAA]. Although, the best shoot proliferation was obtained on WPM supplemented with BA + NAA combinations, shoots grown on PGR-containing media produced callus at the base of the shoots and showed chlorosis and necrosis. Additionally, shoots showed at all concentrations of TDZ, and at higher concentrations of BA, morphological changes such as malformed leaves and poor shoot growth. In contrast to PGR-containing media, on the PGR-free control medium, the development of shoots and roots occurred simultaneously and healthy and well-developed plantlets were obtained. Thus, we developed an economical viable means of in vitro propagation by minimizing the micropropagation steps and removing the requirement of PGRs. According to the high-performance liquid chromatography (HPLC) results, PGR-free control medium (WPM) led to considerably higher stevioside content in the leaves compared with the PGR(s)-containing media and the highest stevioside [34 mg·g-1 dry weight (DW)] and rebaudioside A content was only detected on the control medium without PGRs. Steviolbioside, rubusoside, and dulcoside A were detected qualitatively in the leaves of shoots grown on WPM supplemented with 2.27 µM TDZ, 4.54 µM TDZ, 2.22 µM BA + 2.69 µM NAA, 2.22 µM BA + 5.37 µM NAA, 2.32 µM Kn + 5.71 µM IAA, or 2.32 µM Kn + 2.69 µM NAA. [ABSTRACT FROM AUTHOR]

The industrial extraction and purification of steviol glycoside produce huge impure steviol glycosides mixture, commonly knowing as the mother liquor sugar that contains remarkable rebaudioside A (RA, the main steviol glycoside), which is hard to harvest through regular separation. This study presents a strategy to further elevate the harvest of rebaudioside A and to raise the add value of the mother liquor sugar, by raising the recovery rate of RA with enzymatic producing rubusoside directly from the Stevia rebaudiana Bertoni leaves, and obtaining monoglucosyl RA from mother liquor sugar. The produced rubusoside is an edible biosurfactant with superior solubilization ability to drugs, mainly existed in sweet tree plant (Rubus suavissimus S. Lee). Herein, the extraction from Stevia rebaudiana Bertoni leaves in the presence of the β -glucosidase was conducted. The reaction coupling extraction simultaneously produced rubusoside and recovered other steviol glycosides, in which the recovery rate of RA was significantly increased to 184.59% of that obtained from extraction without enzyme. No eutectic crystallization obviously happened during the recrystallization of the reaction-coupling-extracted steviol glycosides. The monoglucosyl RA was obtained from pure steviol glycosides and the extract excluded polyphenols and proteins, instead of producing in the reaction coupling extraction. Moreover, although the mother liquor sugar contains many impurities (total steviol glycosides of 71.8%), the β -glucosidase still can result the production of Ru and monoglucosyl RA simultaneously. The underneath mechanisms and issues in this reaction coupling extraction were investigated in detail. • The reaction coupling extraction raises recover rate of rebaudioside A by 184.59%. • Enzymatic production of rubusoside (Ru) directly from stevia leaves. • Simultaneously harvesting rebaudioside A, Ru and adding value to mother liquor sugar. • Simultaneous production of Ru and RAG1 in mother liquor sugar with β -glucosidase. [ABSTRACT FROM AUTHOR]

The metabolic burden caused by terpenoid accumulation limits the development of highly efficient microbial cell factories, which can be circumvented using exporter-mediated product secretion. Although our previous study showed that the pleiotropic drug resistance exporter (PDR11) mediates the export of rubusoside in Saccharomyces cerevisiae , the underlying mechanism is still unclear. Herein, we used GROMACS software to simulate PDR11-mediated rubusoside recruitment and found six residues (D116, D167, Y168, P521, R663, and L1146) on PDR11 that are critical for this process. We also explored the exportation potential of PDR11 for 39 terpenoids by calculating their binding affinity using batch molecular docking. Then, we verified the accuracy of the predicted results by conducting experiments with squalene, lycopene, and β-carotene as examples. We found that PDR11 can efficiently secrete terpenoids with binding affinities lower than -9.0 kcal/mol. Combining the computer-based prediction and experimental verification, we proved that binding affinity is a reliable parameter to screen exporter substrates and might potentially enable rapid screening of exporters for natural products in microbial cell factories.

Background: Production of wheat bran (WB) for human consumption is estimated to be about 90 million tons per year. WB contains an abundant source of dietary fiber, minerals, vitamins, and bioactive compounds. WB is a by-product of milling and contains an abundant source of carbohydrate (60%), protein (12%), fat (0.5%), minerals (2%), and bioactive compounds such as phenolic acids, arabinoxylans, flavonoids, caroteinoids alkylresorcinol and phytosterols. These are known for health promoting properties such as controlling glycemic index, reducing plasma cholesterol level, antioxidant, anti-inflammatory, and anticarcinogenic activities. Several terpene glycosides such as mogroside V, paenoiflorin, geniposide, rubusoside (Ru), stevioside (Ste), rebaudioside A (RebA), steviol monoside, and stevioside glucoside have been discovered to enhance the solubility of a number of pharmaceutically and medically important compounds that normally show poor solubility in water. Context and purpose of this study: In this study, in order to increase soluble extraction of polyphenol compounds of WB using Ru, the expression of β-galactosidase from Thermus thermophilus (T. thermophilus) was optimized using different E. coli hosts and a different concentration of lactose inducer rather than of isopropyl-1-thio-β-D-galactopyranoside (IPTG) for industrial production. Additionally, the effect of different steviol glucosides (Ru, Ste, RebA, and SG) on the enhancement of polyphenol compounds extraction from wheat bran was studied. Results: β-galactosidase from T. thermophilus was used for the specific conversion of stevioside (Ste) to rubusoside (Ru) with 92% productivity. The enzyme was optimized to be expressed in E. coli. With 7 mM lactose, the β-galactosidase activity expressed was 34.3, 14.2, or 34.4 ± 0.5 U/mL in E. coli BL21(DE3)pLysS, Rosetta(DE3)pLysS, or BL21(DE3) at 37°C, and 9.8 ± 0.2, 7.0 ± 0.5, or 7.4 ± 0.2 U/mL at 28°C respectively. The expression of β-galactosidase was dependent on the lactose concentration and the highest activity was obtained with the conditions of 5 mM lactose in E. coli Rosetta(DE3)pLysS, 53.3 ± 1.5 U/mL. 78% of the mesophilic proteins was eliminated by heating at 70°C for 15 min with 89% β-galactosidase activity recovery. The total polyphenol content of WB extracted by water, Ru, Ste, rebaudioside A (RebA), and steviol glucosides (SG) were 533.8 ± 9.6 μg/mL, 633.3 ± 1.25 μg/mL, 604.4 ± 10.1 μg/mL, 654.8 ± 26.5 μg/mL, and 601.2 ± 33.4 μg/mL, respectively. The DPPH radical scavenging activity prepared by water, Ru, Ste, RebA, and SG extraction were 8.76 ± 0.3 mg/mL, 4.87 ± 0.3 mg/mL, 5.34 ± 0.22 mg/mL, 7.27 ± 0.1 mg/mL, and 7.82 ± 0.02 mg/mL respectively. Conclusions: To increase soluble extraction of polyphenol compounds of WB using Ru, the expression of β-galactosidase from T. thermophilus was optimized using different E. coli hosts and a different concentration of lactose inducer rather than isopropyl-1-thio-β-D-galactopyranoside (IPTG) for industrial production of the enzyme. The highest antioxidant activity was shown in WB extracted by Ru. The number of glucosyl units attached to steviol can possibly affect the efficiency of antioxidant activity of WB extracted by steviol glucosides. [ABSTRACT FROM AUTHOR]

Background: One promising way of reducing caries is by using sucrose substitutes in food. rubusoside is a prototype sweet substance isolated from the leaves of the plant Rubrus suavissimus S. Lee. (Rosaceae), and is rated sweeter than sucrose. The purpose of this study was to investigate the effects of rubusoside on Streptococcus mutans growth, acidogenicity, and adherence to glass in vitro. Methods: The effects of rubusoside on the growth and glass surface adhering of Streptococcus mutans were investigated by measuring the optical density of the culture at 540 nm with a spectrophotometer. Rubusoside influence on Streptococcus mutans acidogenicity was determined by measuring the pH of the culture. Sucrose, glucose, maltose, fructose and xylitol were designed to compare with rubusoside. Results: S. mutans growth in the rubusoside-treated group was significantly lower than that in the sucrose, glucose, maltose and fructose groups (p < 0.05) except for xylitol group (p > 0.05). Sucrose-treated S. mutans exhibited the highest adherence to glass, and rubusoside-treated S. mutans exhibited the lowest. S. mutans adherence to a glass surface and acidogenicity with sucrose were significantly reduced by rubusoside. Conclusions: Rubusoside may have some potential as a non-cariogenic, non-caloric sweetener. [ABSTRACT FROM AUTHOR]

Background: Quercetin, a flavonol contained in various vegetables and fruits, has numerous biological activities which include anticancer, antiviral, anti-diabetic, and anti-oxidative properties. However, quercetin also has low oral bioavailability, due to its insolubility in water. Thus, the bioavailability of quercetin administered to human beings in a capsule form was reported to be less than 1%, with only a small percentage of ingested quercetin getting absorbed in the blood. This leads to certain difficulties in creating highly effective medicines. Methods: Quercetin-rubusoside and quercetin-rebaudioside were prepared. The antioxidant activities of quercetin and Q-rubusoside were evaluated by DPPH radical scavenging method. Inhibition activities of quercetin and Quercetin-rubusoside were determined by measuring the remaining activity of 3CLpro with 200 µM inhibitor. The inhibition activity of quercetin, rubusoside and quercetin-rubusoside were determined by measuring the activity of human maltase, which remains at 100 µM rubusoside or quercetin-rubusoside. The mushroom tyrosinase inhibition was assayed with the reaction mixture containing 3.3 mM L-DOPA in 50 mM potassium phosphate buffer (pH 6.8), and 10 U mushroom tyrosinase/ml with or without quercetin or quercetin-rubusoside. Results: With 10% rubusoside treatment, quercetin showed solubility of 7.7 mg/ml in water, and its solubility increased as the concentration of rubusoside increased; the quercetin solubility in water increased to 0.83 mg/ml as rubusoside concentration increased to 1 mg/ml. Quercetin solubilized in rubusoside solution showed DPPH radical-scavenging activity and mushroom tyrosinase inhibition activity, similar to that of quercetin solubilized in dimethyl-sulfoxide. Quercetin-rubusoside also demonstrated 1.2 and 1.9 folds higher inhibition activity against 3CLpro of SARS and human intestinal maltase, respectively, than those of quercetin in DMSO. Conclusions: Quercetin can be solubilized in water with rebaudioside or rubusoside treatment. As Ru concentration increases, the solubility of quercetin in water increases. The solubilization of quercetin in Ru solution did not reduce its biological functions such as the DPPH radicalscavenging and mushroom tyrosinase activity. Additionally, quercetin-rubusoside increased the inhibition activity against the 3CLpro of SARS and human intestinal maltase, when compared with the activity of quercetin in DMSO. Therefore, rubusoside and rebaudioside are promising compounds which enhance the solubility of poorly water soluble compounds. [ABSTRACT FROM AUTHOR]

In order to produce rubusoside, enzymes with preferential specificity for the saccharide sophorose were tested for ability to produce rubusoside from stevioside. We identified BGL1, a β-glucosidase from Streptomyces sp. GXT6, as an enzyme for rubusoside production. Out of several saccharide substrates, BGL1 showed the most affinity to sophorose. This enzyme only hydrolyzes the glucose moiety of the sophoroside at C-13 in stevioside. Production of rubusoside was determined by H and C nuclear magnetic resonance (NMR). Thus, rubusoside was produced from stevioside and the stevioside conversion rate was 98.2 %. The production yield of rubusoside was 78.8 % in 6 h. [ABSTRACT FROM AUTHOR]

Five plant-derived uridine diphosphate glycosyltransferases (UGTs) that catalyzed the glucosylation of stevia glycosides (SGs) were uncovered as the result of sequence mining considering the catalytic residues and conserved motifs of the known UGTs. Thereinto, Lb UGT from Lycium barbarum with high activity toward rubusoside has been enzymatically characterized. The recombinant Lb UGT was demonstrated to catalyze the β-1,6-glucosylation at C19 of rubusoside, producing a monoglucosyl derivative 13-[( O -β-d-glucopyranosyl) oxy] ent-kaur-16-en-19-oic acid-[(6- O -β-d-glucopyranosyl-β-d-glucopyranosyl) ester], which was then submitted to a β-1,2-glucosylation by Lb UGT, resulting in a diglucosyl derivative 13-[( O -β-d-glucopyranosyl) oxy] ent-kaur-16-en-19-oic acid-[(2- O -β-d-glucopyranosyl-6- O -β-d-glucopyranosyl-β-d-glucopyranosyl) ester]. The di-glycosylated product of rubusoside showed an obvious increase in sweetness intensity (134 times sweeter than 5% sucrose) and almost eliminated the unpleasant bitter taste. This work will provide a reference for the taste improvement of SGs.

Solubility is an important factor for achieving the desired plasma level of drug for pharmacological response. About 40% of drugs are not soluble in water in practice and therefore are slowly absorbed, which results in insufficient and uneven bioavailability and GI toxicity. Rubusoside (Ru) is a sweetener component in herbal tea and was discovered to enhance the solubility of a number of pharmaceutically and medicinally important compounds, including anticancer compounds. In this study, thirty-one hydrolyzing enzymes were screened for the conversion of stevioside (Ste) to Ru. Recombinant lactase from Thermus thermophiles which was expressed in Escherichia coli converted stevioside to rubusoside as a main product. Immobilized lactase was prepared and used for the production of rubusoside; twelve reaction cycles were repeated with 95.4% of Ste hydrolysis and 49gL−1 of Ru was produced. The optimum rubusoside synthesis yield was 86% at 200gL−1, 1200U lactase. The purified 10% rubusoside solution showed increased water solubility of liquiritin from 0.98mgmL−1 to 4.70±0.12mgmL−1 and 0mgmL−1 to 3.42±0.11mgmL−1 in the case of teniposide. [ABSTRACT FROM AUTHOR]

Abstract: Rubusoside is a precious bioactive sweetener which mainly exists in Chinese sweet tea plant (Rubus suavissimus S. Lee), while stevioside is an abundant natural sweetener with bitter aftertaste. In this work, a β-galactosidase from Aspergillus sp. presented specific hydrolytic activity on β-1,2 glucosidic linkage of stevioside, and converting stevioside to rubusoside. The hydrolytic activity was weak on the natural stevioside analogs, which makes the process a promising approach to produce rubusoside and utilize stevioside. The highest steviosides conversion was 98.3%, accompanying with a rubusoside yield of 91.4%. [Copyright &y& Elsevier]

Abstract: Etoposide (ETO), a widely used anti-cancer drug, is constrained by its low aqueous solubility and by side effects from both the drug and its solubilizing excipients. In this study, a recently discovered natural solubilizer rubusoside (RUB) was used to achieve the solubilization of ETO. Dynamic light scattering and freeze-fracture transmission electron microscopy studies showed that ETO and RUB formed ETO–RUB nanoparticles (∼6nm in diameter). The powder of ETO–RUB nanoparticles was completely reconstitutable in water and remained stable in this solution at 25 and 37°C for at least 24h. Under other physiologic conditions, ETO solution was clear and free of precipitation at 25°C, but underwent various structural transformations. In PBS and simulated intestinal fluid, RUB-solubilized ETO underwent epimerization and equilibrated to cis-ETO. In simulated gastric fluid, RUB-solubilized ETO degraded to 4′-demethylepipodophyllotoxin-beta-d-glucoside and 4′-demethylepipodophyllotoxin. Higher temperatures favored epimerization or degradation. Furthermore, a side-by-side comparison with DMSO-solubilized ETO confirmed that the RUB-solubilized ETO showed no significant differences in cytotoxicity in colon, breast and prostate cancer cell lines. RUB effectively solubilized and stabilized etoposide, which sets the stage for further toxicology, bioavailability, and efficacy investigations. [Copyright &y& Elsevier]

Rubusoside, which is used as a natural sweetener or a solubilizing agent for water-insoluble functional materials, is currently expensive to produce owing to the high cost of the membrane-based technologies needed for its extraction and purification from the sweet tea plant (Rubus suavissimus S. Lee). Therefore, this study was carried out to screen for lactic acid bacteria that possess enzymes capable of bio-transforming stevioside into rubusoside. Subsequently, one such rubusoside-producing enzyme was isolated from Lactobacillus plantarum GS100. Located on the bacterial cell surface, this enzyme was stable at pH 4.5–6.5 and 30–40 °C, and it produced rubusoside as a major product through its stevioside-hydrolyzing activity. Importantly, the enzyme showed higher β-glucosidase activity toward the β-linked glucosidic bond of stevioside than toward other β-linked glucobioses. Under optimal conditions, 70 U/L of the rubusoside-producing enzyme could produce 69.03 mM rubusoside from 190 mM stevioside. The β-glucosidase activity on the cell surface was high at 35 h of culture. This is the first report detailing the production of rubusoside from stevioside by an enzyme derived from a food-grade lactic acid bacterium. The application of this β-glucosidase could greatly reduce the cost of rubusoside production, hence benefiting all industries that use this natural product. Biotransformation of stevioside into rubusoside by Lactobacillus plantarum GS100 [Display omitted] • A rare natural sweetener rubusoside was synthesized from abundant stevioside. • The β -glucosidase presented specific hydrolytic activity on stevioside. • The enzyme has optimum activity at pH 6.0 and 40 °C. • The β -glucosidase from Lactobacillus platarum GS100 located on cell surface. • The optimal conditions for rubusoside production (63.09 mM, 60% yield) were 190 mM stevioside and 70 U/L enzyme. [ABSTRACT FROM AUTHOR]

Rubusoside (Rub) is a highly sweet diterpene glycoside mainly isolated from the leaves of Rubus suavissimus (Rosaceae). It has been used as a low-calorie natural sweetener for decades and was recently found to be a potential drug lead. In this study, we designed a whole-cell biocatalyst to achieve the glycosylation of steviol to Rub in Saccharomyces cerevisiae . The sucrose synthases were applied to construct a uridine diphosphate glucose regeneration system, which were coupled with optimal combinations of different uridine diphosphate (UDP) glycosyltransferases from multiple plant species. After optimization of reaction conditions, the residues in SrUGT74G1 probably influencing glycosylation efficiency were subjected to site-directed mutagenesis. Double mutations of S84A/E87A reduced the accumulation of intermediates, finally glucosylating 1.27 g/L steviol to 0.45 ± 0.06 g/L steviolmonoside (conversion rate = 23.3%) and 1.92 ± 0.17 g/L Rub (conversion rate = 74.9%). A high efficiency of Rub biosynthesis could be achieved without supply of additional UDPG. This work provided the first example of multi-step glycosylation reactions in whole-cell biocatalysis, which laid a foundation of scalable production of the value-added diterpene sweetener in the future.

The reaction pathway of Ru production and its solubilization on vitamin. • Rubusoside is produced from 55 wt.% of stevioside with a conversion of 98.8 %. • Reaction pathway of rubusoside production from stevioside with or without rebaudioside A. • 1% rubusoside promoted the solubility of vitamin E and D 2 at 25℃ by 105 and 111 times, respectively. Rubusoside is a natural botanic sweetener with capacity of micelle solubilization. This study revealed the reaction pathway of enzymatically producing rubusoside from a high concentration of stevioside in the absence or presence of rebaudioside A, the identification of the byproduct, as well as the performance of rubusoside in micelle solublization of vitamins. Rebaudioside A initially stimulated rubusoside yield by taking glucosyl from stevioside to form monoglucosyl rebaudioside A, although the β -glucosidase administered was absolutely specific towards stevioside. A trace byproduct was identified as steviol glucosyl ester, yielded from breaking the 13-(β -D-glucopyranosyloxy) linkage of stevioside instead of that of rubusoside. 1% Rubusoside promoted the solubility of vitamin E (VE) and vitamin D 2 at 25℃ by 105 and 111 times, respectively. The driving force for the solubilizing is the synergic effect of VE and rubusoside on the surface activity of rubusoside solution, presented as the erasing of the γ-lgC plateau by VE with increasing concentration, which dramatically increased the concentration for micelle formation. The results are beneficial for the use of concentrated stevia (Stevia rebaudiana Bertoni) extracts to produce rare natural steviol glycoside, yet extend its application in addition to its function as a sweetener. [ABSTRACT FROM AUTHOR]

Monoglucosylation of rubusoside not only could increase its structural diversity but may also improve its taste. To biosynthesize the monoglucosyl rubusosides, a series of glycosyltransferases and glycosynthases were screened to identify the enzymes capable of specifically glycosylating the hydroxyl groups of the 13-O-β-d-glucosyl and 19-COO-β-d-glucosyl moieties. After structural characterization, the effect of structure on sweetness and taste was established based on these rubusoside-derived analogues, including two first characterized compounds. β-Monoglucosylation of two 2-hydroxyl groups, as well as α-monoglucosylations of the 4- and 6-hydroxyl groups of the 13-glucosyl moiety, could significantly increase the relative sweetness of rubusoside to 140 while maintaining or improving the taste quality. In contrast, monoglucosylations of other hydroxyl groups in our study usually decreased the taste quality of the rubusoside. Additionally, the possibility of a negative influence of these monoglucosylated derivatives on the function of islets was preliminarily excluded, which should facilitate the development of rubusoside-derived sweeteners.

Dental caries is a biofilm-mediated disease in which Streptococcus mutans is the main pathogenic microorganism, and its incidence is closely related to sucrose. Rubusoside is a natural nonnutritive sweetener isolated from Rubus suavissimus S. Lee. This study was designed to determine the effect of this sucrose substitute on the cariogenic properties and virulence gene expression of S. mutans biofilms. S. mutans was exposed to brain heart infusion (BHI) medium (as a control), 1% sucrose-supplemented medium, 1% rubusoside-supplemented medium, and 1% xylitol-supplemented medium. The growth curve of the biofilm was monitored by crystal violet staining, and the pH was measured every 24 h. After 5 days, the biofilms formed on the glass coverslips were recovered to determine the biomass (dry weight and total amount of soluble proteins), numbers of CFU, and amounts of intra- and extracellular polysaccharides. Biofilm structural imaging was performed using a scanning electron microscope (SEM). Virulence gene expression ( gtfB , gtfC , gtfD , ftf , spaP , gbpB , ldh , atpF , vicR , and comD ) was determined by reverse transcription-quantitative PCR. Growth in rubusoside resulted in lower levels of acid production than observed during growth in sucrose, xylitol, and the control, while it also reduced the level of biofilm accumulation and bacterial viability and even reduced the level of production of extracellular polysaccharides. By SEM, the levels of biofilm formation and extracellular matrix during growth in rubusoside were lower than these levels during growth in sucrose and xylitol. From the perspective of virulence genes, growth in rubusoside and xylitol significantly inhibited the expression of virulence genes compared with their levels of expression after growth in sucrose. Among these genes, gtfB , gtfC , gbpB , ldh , and comD downregulation was found with growth in rubusoside compared with their expression with growth in xylitol. Therefore, rubusoside appears to be less potentially cariogenic than sucrose and xylitol and may become an effective sucrose substitute for caries prevention. Further studies are needed to deepen these findings. IMPORTANCE Dental caries is a major public health challenge and places heavy biological, social, and financial burdens on individuals and health care systems. To palliate the deleterious effect of sucrose on the virulence factors of S. mutans , massive commercial efforts have been oriented toward developing products that may act as sucrose substitutes. Rubusoside, a natural sucrose substitute, is a plant extract with a high level of sweetness. Although some studies have shown that rubusoside does not produce acids or inhibit the growth of S. mutans , little attention has been paid to its effect on dental biofilm and the underlying mechanisms. Our study focuses on the effect of rubusoside on the formation and structure of biofilms and the expression of virulence genes. The results confirm that rubusoside can inhibit accumulation, bacterial viability, polysaccharide production by the biofilm, and related gene expression. These results provide further insight into the cariogenicity of S. mutans biofilms and demonstrate a new perspective for studying the impact of sucrose substitutes on caries., (Copyright © 2020 American Society for Microbiology.)

The demand for steviol glycosides, non-caloric sweet components of Stevia rebaudiana Bertoni (stevia) leaves, has increased considerably as a benefit to enhance human health. However, the supply has remained challenging due to limited production, with the lack of a specific steviol glycoside hydrolyzing enzyme. In this study, a novel β-glucosidase (EcBgl) from Enterococcus casseliflavus was cloned and expressed in Escherichia coli. An EcBgl consists of 721 amino acids corresponding to a molecular mass of 79.37 kDa. The EcBgl was purified to homogeneity, followed by enzyme characterization. The enzyme showed optimum pH and temperature at 6.0 and 37 °C, and exhibited the kinetic constants k cat /K m for p NPG and k cat /K m for stevioside of 8583 mM−1s−1 and 95.41 mM−1s−1, respectively. When compared to the stevioside hydrolyzing β-glycosidases previously reported, EcBgl was found to be the most efficient enzyme. EcBgl also rendered hydrolysis of the stevioside to produce rubusoside, a rare steviol glycoside with a pharmaceutical solubilizing property, by cleaving at the glucose moiety. In addition, the enzyme demonstrated substantial resistance against amygdalin, so it served as a potential enzyme in agricultural and pharmaceutical applications. Image 1 [ABSTRACT FROM AUTHOR]

Almost all functions of cells or organs rely on the activities of cellular enzymes. Indeed, the in-vivo activities that directly represent the cellular effects of enzymes in live organs are critical importance to appreciate the roles enzymes play in modulating physiological or pathological processes, although assessments of such in-vivo enzyme activity are more difficult than typical test-tube assays. Recently, we, for the first time, developed a direct and easy-handling method for HPLC analyzing the in-vivo activity of glucosylceramide synthase (GCS). GCS that converts ceramide into glucosylceramide is a limiting-enzyme in the syntheses of glycosphingolipids and is one cause of cancer drug resistance. In our method developed, rubusoside nanomicelles delivers fluorescence N -[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoyl]-d- erythro -sphingosine (NBD C 6 -ceramide) into mice, tissues uptake the cell-permeable substrate, and GCS converts it into NBD C 6 -glucosylceramide in all organs simultaneously. Further, HPLC analyzes the extracted NBD C 6 -glucosylceramide to assess alterations of the in-vivo GCS activities in tissues. This method can be broadly used to assess the in-vivo GCS activities in any kind of animal models to appreciate either the role GCS plays in diseases or the therapeutic efficacies of GCS inhibitors., Competing Interests: Competing interestsThe authors declare no competing financial interests., (Copyright © 2019 The Authors; exclusive licensee Bio-protocol LLC.)

Background: One promising way of reducing caries is by using sucrose substitutes in food. rubusoside is a prototype sweet substance isolated from the leaves of the plant Rubrus suavissimus S. Lee. (Rosaceae), and is rated sweeter than sucrose. The purpose of this study was to investigate the effects of rubusoside on Streptococcus mutans growth, acidogenicity, and adherence to glass in vitro., Methods: The effects of rubusoside on the growth and glass surface adhering of Streptococcus mutans were investigated by measuring the optical density of the culture at 540 nm with a spectrophotometer. Rubusoside influence on Streptococcus mutans acidogenicity was determined by measuring the pH of the culture. Sucrose, glucose, maltose, fructose and xylitol were designed to compare with rubusoside., Results: S. mutans growth in the rubusoside-treated group was significantly lower than that in the sucrose, glucose, maltose and fructose groups (p < 0.05) except for xylitol group (p > 0.05). Sucrose-treated S. mutans exhibited the highest adherence to glass, and rubusoside-treated S. mutans exhibited the lowest. S. mutans adherence to a glass surface and acidogenicity with sucrose were significantly reduced by rubusoside., Conclusions: Rubusoside may have some potential as a non-cariogenic, non-caloric sweetener.

Glucosyl rubusosides were synthesized by two dextransucrases. LcDexT was obtained from Leuconosotoc citreum, that LlDexT was obtained from Leuconostoc lactis. LcDexT and LlDexT regioselectively transferred a glucosyl residue to the 13-O-glucosyl moiety of rubusoside with high yield of 59-66% as analyzed by TLC and HPLC. Evaluation of the sweetness of these glucosyl rubusosides showed that their quality of taste, in particular, was superior to that of rubusoside. These results indicate that transglucosylation at the 13-O-glucosyl moiety of rubusoside by different regioselective dextransucrases can be applicable for increasing its sweetness and quality of taste.