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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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

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

Synthetic Derivatives of Natural ent-Kaurane Atractyligenin Disclose Anticancer Properties in Colon Cancer Cells, Triggering Apoptotic Cell Demise.

The antitumor activity of different ent-kaurane diterpenes has been extensively studied. Several investigations have demonstrated the excellent antitumor activity of synthetic derivatives of the diterpene atractyligenin. In this research, a series of new synthetic amides and their 15,19-di-oxo analogues obtained from atractyligenin by modifying the C-2, C-15, and C-19 positions were designed in order to dispose of a set of derivatives with different substitutions at the amidic nitrogen. Using different concentrations of the obtained compounds (10-300 µM) a reduction in cell viability of HCT116 colon cancer cells was observed at 48 h of treatment. All the di-oxidized compounds were more effective than their alcoholic precursors. The di-oxidized compounds had already reduced the viability of two colon cancer cells (HCT116 and Caco-2) at 24 h when used at low doses (2.5-15 µM), while they turned out to be poorly effective in differentiated Caco-2 cells, a model of polarized enterocytes. The data reported here provide evidence that di-oxidized compounds induced apoptotic cell death, as demonstrated by the appearance of condensed and fragmented DNA in treated cells, as well as the activation of caspase-3 and fragmentation of its target PARP-1.

Design, Synthesis, and In Vivo Evaluation of Isosteviol Derivatives as New SIRT3 Activators with Highly Potent Cardioprotective Effects.

Cardiovascular diseases (CVDs) persist as the predominant cause of mortality, urging the exploration of innovative pharmaceuticals. Mitochondrial dysfunction stands as a pivotal contributor to CVDs development. Sirtuin 3 (SIRT3), a prominent mitochondrial deacetylase known for its crucial role in protecting mitochondria against damage and dysfunction, has emerged as a promising therapeutic target for CVDs treatment. Utilizing isosteviol, a natural ent-beyerene diterpenoid, 24 derivatives were synthesized and evaluated in vivo using a zebrafish model, establishing a deduced structure-activity relationship. Among these, derivative 5v exhibited significant efficacy in doxorubicin-induced cardiomyopathy in zebrafish and murine models. Subsequent investigations revealed that 5v selectively elevated SIRT3 expression, leading to the upregulation of SOD2 and OPA1 expression, effectively preventing mitochondrial dysfunction, mitigating oxidative stress, and preserving cardiomyocyte viability. As a novel structural class of SIRT3 activators with robust therapeutic effects, 5v emerges as a promising candidate for further drug development.

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

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

Prevalence of youth experiencing homelessness and its association with suicidal thoughts and behaviors: Findings from a population-based study.

Although various studies have examined factors associated with suicidal behaviors among youth, few studies have investigated the association between youth experiencing homelessness (YEH) and suicidal thoughts and behaviors (STBs) using a large nationally representative sample. The objectives of this study were to investigate prevalence of YEH and its association with STBs. Data for this study came from the 2021 Youth Risk Behavior Survey. An analytic sample of 17,033 youth aged 14-18 (51.7 % male) was analyzed using binary logistic regression. Of the 17,033 youth examined, 3 % experienced homelessness during the past 30 days, 21.3 % experienced suicidal ideation, 17.3 % made a suicide plan, and 10.9 % attempted suicide during the past 12 months. Controlling for demographic characteristics and feeling sad or hopeless, YEH was associated with 2.48 times higher odds of experiencing suicidal ideation (AOR=2.48, p<.001), 2.46 times higher odds of making a suicide plan (AOR=2.46, p<.001), and 4.38 times higher odds of making a suicide attempt (AOR=4.38, p<.001). The findings of this study highlight the importance of identifying youth who are at risk of experiencing homelessness to ensure early interventions are put in place to prevent suicidal behaviors.

Effect of steviol glycosides as natural sweeteners on glucose metabolism in adult participants.

Steviol glycosides (SGs) are recognized as safe natural sweeteners; however, evidence from randomized controlled trials (RCTs) showed an inconclusive effect of SGs on glucose metabolism in adult participants. We aimed to conduct a systematic review and meta-analysis of RCTs to assess the effect of SGs on glucose metabolism. We systematically searched PubMed, Web of Science and EMBASE to include eligible RCTs. Our primary outcomes were differences between SGs and the control group with respect to changes in blood glucose from the baseline to the end of intervention (including fasting blood glucose [FBG], and HbA1c measurements). A random-effects meta-analysis was conducted for data synthesis to calculate the pooled mean difference (MD). There were twelve RCTs included for analyses with a total of 871 participants (48% females). A significant effect of SGs on FBG (MD = -4.10 mg dl-1, 95% CI -6.55 to -1.65) was found, while no significant difference in HbA1c (MD = 0.01%, 95% CI -0.12% to 0.13%) was observed between SGs and controls. The whole quality of evidence was rated as low. Subgroup analyses demonstrated favorable effects of SGs on FBG in participants aged ≤50 years, those without diabetes mellitus (DM) or hypertension at the baseline, and overweight and obese adults. Sensitivity analyses yielded results largely similar to the main findings. To conclude, SGs are found to produce significant improvement in glucose metabolism in adult participants when compared with the control. More evidence is required to further clarify and support the benefit of SGs as a sugar substitute for glucose metabolism.

Acute and two-week effects of neotame, stevia rebaudioside M and sucrose-sweetened biscuits on postprandial appetite and endocrine response in adults with overweight/obesity-a randomised crossover trial from the SWEET consortium.

BACKGROUND: Sweeteners and sweetness enhancers (S&SE) are used to replace energy yielding sugars and maintain sweet taste in a wide range of products, but controversy exists about their effects on appetite and endocrine responses in reduced or no added sugar solid foods. The aim of the current study was to evaluate the acute (1 day) and repeated (two-week daily) ingestive effects of 2 S&SE vs. sucrose formulations of biscuit with fruit filling on appetite and endocrine responses in adults with overweight and obesity. METHODS: In a randomised crossover trial, 53 healthy adults (33 female, 20 male) with overweight/obesity in England and France consumed biscuits with fruit filling containing 1) sucrose, or reformulated with either 2) Stevia Rebaudioside M (StRebM) or 3) Neotame daily during three, two-week intervention periods with a two-week washout. The primary outcome was composite appetite score defined as [desire to eat + hunger + (100 - fullness) + prospective consumption]/4. FINDINGS: Each formulation elicited a similar reduction in appetite sensations (3-h postprandial net iAUC). Postprandial insulin (2-h iAUC) was lower after Neotame (95% CI (0.093, 0.166); p < 0.001; d = -0.71) and StRebM (95% CI (0.133, 0.205); p < 0.001; d = -1.01) compared to sucrose, and glucose was lower after StRebM (95% CI (0.023, 0.171); p < 0.05; d = -0.39) but not after Neotame (95% CI (-0.007, 0.145); p = 0.074; d = -0.25) compared to sucrose. There were no differences between S&SE or sucrose formulations on ghrelin, glucagon-like peptide 1 or pancreatic polypeptide iAUCs. No clinically meaningful differences between acute vs. two-weeks of daily consumption were found. INTERPRETATION: In conclusion, biscuits reformulated to replace sugar using StRebM or Neotame showed no differences in appetite or endocrine responses, acutely or after a two-week exposure, but can reduce postprandial insulin and glucose response in adults with overweight or obesity. FUNDING: The present study was funded by the Horizon 2020 program: Sweeteners and sweetness enhancers: Impact on health, obesity, safety and sustainability (acronym: SWEET, grant no: 774293).

Oridonin inhibited epithelial-mesenchymal transition of laryngeal carcinoma by positively regulating LKB1/AMPK signaling.

Oridonin is the main bioactive component of Rabdosia rubescens, and its anticancer activity has been reported in a variety of cancers. However, the molecular mechanism of oridonin in laryngeal carcinoma remains unclear. In the present study, the cytotoxic effect of oridonin on laryngeal carcinoma Hep-2 and TU212 cell lines were initially detected by modified MTT assay. The results showed that oridonin had a dose-dependent anti-proliferative effect on laryngeal carcinoma Hep-2 and TU212 cells. Next, we found that oridonin significantly inhibited the migration and invasion of human laryngeal carcinoma Hep-2 and TU212 cell lines by wound healing assay and transwell assay. Subsequently, the results of quantitative real-time PCR assay and western blotting assay confirmed that oridonin upregulated the expression of E-cadherin while downregulated the expression of N-cadherin in a concentration-dependent manner at mRNA and protein levels. In addition, phosphorylation levels of liver kinase B1 (p-LKB1) and AMP-activated protein kinase (p-AMPK) were also elevated upon oridonin treatment. To further verify the role of LKB1/AMPK signaling pathway in laryngeal carcinoma, overexpression of LKB1 was constructed by plasmid transfection. The data exhibited that overexpression of LKB1 could further reinforce the increase of E-cadherin level and decrease of N-cadherin level mediated by oridonin. Additionally, AMPK inhibitor compound C could reverse anti-metastatic effect of oridonin on laryngeal carcinoma, and antagonise EMT expression. In contrast, AMPK activator AICAR presented the opposite effect. In conclusion, our study revealed that oridonin could remarkably reverse the epithelial-mesenchymal transition of laryngeal carcinoma by positively regulating LKB1/AMPK signaling pathway, which suggested that oridonin may be a potential candidate for the treatment of laryngeal carcinoma in the future.

Glaucocalyxin A delays the progression of OA by inhibiting NF-κB and MAPK signaling pathways.

BACKGROUND: Osteoarthritis (OA) is a common degenerative joint condition marked by inflammation and cartilage breakdown. Currently, there is a dearth of treatment medications that can clearly slow the course of OA. Glaucocalyxin A (GLA) is a diterpene chemical identified and extracted from Rabdosia japonica with antithrombotic, anticoagulant, anti-tumor, anti-inflammatory, anti-oxidant, and other pharmacological properties. Previous research has linked inflammation to abnormalities in the homeostasis of the extracellular matrix (ECM). Although GLA has been shown to have anti-inflammatory qualities, its effects on the progression of OA are unknown. As a result, the goal of this study was to see if GLA could slow the course of OA. METHODS: ATDC5 cells were stimulated by IL-1ß to create an inflammatory chondrocyte damage model. Quantitative polymerase chain reaction, Western Blot, high-density culture, and immunofluorescence were used to detect the expression levels of associated gene phenotypes. We also created a mouse model of OA induced by destabilization of the medial meniscus (DMM) instability, and GLA was administered intraperitoneally once every two days for eight weeks. Mice knee specimens were stained with hematoxylin-eosin, Safranin O/fast green, and immunohistochemical, and the Osteoarthritis Research Society International grade system and Mankin's score were used to assess the protective effect of GLA on cartilage. RESULTS: In vitro and in vivo, we explored the effects and molecular processes of GLA as a therapy for OA. The findings demonstrated that GLA might reduce the expression of associated inflammatory mediators and protect the ECM by inhibiting the NF-κB and MAPK signaling pathways. Animal research revealed that GLA could protect against the DMM-induced OA model mice by stabilizing ECM. CONCLUSION: Taken together, our findings show that GLA has a protective impact on cartilage throughout OA progression, implying that GLA could be employed as a possible therapeutic agent for OA, thus giving a new therapeutic method for the treatment of OA.

Heterologous Biosynthesis of Kauralexin A1 in Saccharomyces cerevisiae through Metabolic and Enzyme Engineering.

Kauralexin A1 (KA1) is a key intermediate of the kauralexin A series metabolites of maize phytoalexins. However, their application is severely limited by their low abundance in maize. In this study, an efficient biosynthetic pathway was constructed to produce KA1 in Saccharomyces cerevisiae. Also, metabolic and enzyme engineering strategies were applied to construct the high-titer strains, such as chassis modification, screening synthases, the colocalization of enzymes, and multiple genomic integrations. First, the KA1 precursor ent-kaurene was synthesized using the efficient diterpene synthase GfCPS/KS from Fusarium fujikuroi, and optimized to reach 244.36 mg/L in shake flasks, which displayed a 200-fold increase compared to the initial strain. Then, the KA1 was produced under the catalysis of ZmCYP71Z18 from Zea mays and SmCPR1 from Salvia miltiorrhiza, and the titer was further improved by integrating the fusion protein into the genome. Finally, an ent-kaurene titer of 763.23 mg/L and a KA1 titer of 42.22 mg/L were achieved through a single-stage fed-batch fermentation in a 5 L bioreactor. This is the first report of the heterologous biosynthesis of maize diterpene phytoalexins in S. cerevisiae, which lays a foundation for further pathway reconstruction and biosynthesis of the kauralexin A series maize phytoalexins.

Bridging drug discovery through hierarchical subtractive genomics against asd, trpG, and secY of pneumonia causing MDR Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is an opportunistic gram-positive, non-motile, and non-sporulating bacteria that induces pneumonia, a provocative lung infection affecting mainly the terminal bronchioles and the small air sacs known as alveoli. Recently, it has developed antibiotic resistance to the available consortium as per the WHO reports; thereby, novel remedial targets and resilient medications to forestall and cure this illness are desperately needed. Here, using pan-genomics, a total of 1,387 core proteins were identified. Subtractive proteome analyses further identified 12 proteins that are vital for bacteria. One membrane protein (secY) and two cytoplasmic proteins (asd and trpG) were chosen as possible therapeutic targets concerning minimum % host identity, essentiality, and other cutoff values, such as high resistance in the MDR S. aureus. The UniProt AA sequences of the selected targets were modelled and docked against 3 drug-like chemical libraries. The top-ranked compounds i.e., ZINC82049692, ZINC85492658 and 3a of Isosteviol derivative for Aspartate-semialdehyde dehydrogenase (asd); ZINC38222743, ZINC70455378, and 5 m Isosteviol derivative for Anthranilate synthase component II (trpG); and finally, ZINC72292296, ZINC85632684, and 7 m Isosteviol derivative for Protein translocase subunit secY (secY), were further subjected to molecular dynamics studies for thermodynamic stability and energy calculation. Our study proposes new therapeutic targets in S. aureus, some of which have previously been reported in other pathogenic microorganisms. Owing to further experimental validation, we anticipate that the adapted methodology and the predicted results in this work could make major contributions towards novel drug discovery and their targets in S. aureus caused pneumonia.

Oridonin loaded peptide nanovesicles alleviate nonalcoholic fatty liver disease in mice.

This study was to construct a nanovesicle delivery system to improve the loading efficiency and stability of ORI for the treatment of nonalcoholic fatty liver disease (NAFLD). This nanovesicles (NVs) exerted a narrow size distribution (195.6 ± 11.49 nm) and high entrapment efficiency (84.46 ± 1.34%). In vitro cell studies demonstrated that the NVs treatment enhanced the cellular uptake of ORI and reduced lipid over-accumulation and total cholesterol levels in NAFLD cell model. At the same time, in vivo study proved that, compared with the normal group, the model group mice showed a decrease in body weight, a significant increase in liver index (6.71 ± 0.62, p < 0.01), and symptoms of liver lipid accumulation, lipid vesicles, and liver tissue fibrosis. Compared with the model group, after high-dose ORI NVs intervention, mice gained weight, decreased liver index (4.69 ± 0.55, p < 0.01), reduced hepatic lipid droplet vacuoles, reduced lipid accumulation (reduced oil red area, p < 0.001), and alleviated the degree of liver fibrosis (reduced blue collagen area, p < 0.001). In conclusion, ORI/HP-ß-CD/H9-HePC NVs showed specific liver accumulation and improved therapeutic effects, the nano drug loading system provides a promising strategy for the encapsulation of ORI to effectively alleviate the process of NAFLD.

A sustainable strategy for biosynthesis of Rebaudioside D using a novel glycosyltransferase of Solanum tuberosum.

Bioconversion of Rebaudioside D faces high-cost obstacles. Herein, a novel glycosyltransferase StUGT converting Rebaudioside A to Rebaudioside D was screened and characterized, which exhibits stronger affinity and substrate specificity for Rebaudioside A than previously reported enzymes. A whole-cell catalytic system was thus developed using the StUGT strain. The production of Rebaudioside D was enhanced significantly by enhancing cell permeability, and the maximum production of 6.12 g/L and the highest yield of 98.08% by cell catalyst was obtained by statistical-based optimization. A new cascade process utilizing this recombinant strain and E. coli expressing sucrose synthase was further established to reduce cost through replacing expensive UDPG with sucrose. A StUGT-GsSUS1 system exhibited high catalytic capability, and 5.27 g L-1 Rebaudioside D was achieved finally without UDPG addition by systematic optimization. This is the best performance reported in cell-cascaded biosynthesis, which paves a new cost-effective strategy for sustainable synthesis of scarce premium sweeteners from biomass.

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

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

Comparison of a Daily Steviol Glycoside Beverage compared with a Sucrose Beverage for Four Weeks on Gut Microbiome in Healthy Adults.

BACKGROUND: Recent studies suggest that some nonnutritive sweeteners (NNS) have deleterious effects on the human gut microbiome (HGM). The effect of steviol glycosides on the HGM has not been well studied. OBJECTIVE: We aimed to evaluate the effects of stevia- compared with sucrose-sweetened beverages on the HGM and fecal short-chain fatty acid (SCFA) profiles. METHODS: Using a randomized, double-blinded, parallel-design study, n = 59 healthy adults [female/male, n = 36/23, aged 31±9 y, body mass index (BMI): 22.6±1.7 kg/m2] consumed 16 oz of a beverage containing either 25% of the acceptable daily intake (ADI) of stevia or 30 g of sucrose daily for 4 weeks followed by a 4-week washout. At weeks 0 (baseline), 4, and 8, the HGM was characterized via shotgun sequencing, fecal SCFA concentrations were measured using ultra-high performance liquid chromatography-tandem mass spectrometry and anthropometric measurements, fasting serum glucose, insulin and lipids, blood pressure, pulse, and 3-d diet records were obtained. RESULTS: There were no significant differences in the HGM or fecal SCFA between the stevia and sucrose groups at baseline (P > 0.05). At week 4 (after intervention), there were no significant differences in the HGM at the phylum, family, genus, or species level between the stevia and sucrose groups and no significant differences in fecal SCFA. At week 4, BMI had increased by 0.3 kg/m2 (P = 0.013) in sucrose compared with stevia, but all other anthropometric and cardiometabolic measures and food intake did not differ significantly (P > 0.05). At week 8 (after washout), there were no significant differences in the HGM, fecal SFCA, or any anthropometric or cardiometabolic measure between the stevia and sucrose groups (P > 0.05). CONCLUSIONS: Daily consumption of a beverage sweetened with 25% of the ADI of stevia for 4 weeks had no significant effects on the HGM, fecal SCFA, or fasting cardiometabolic measures, compared with daily consumption of a beverage sweetened with 30 g of sucrose. TRIAL REGISTRATION: clinicaltrials.gov as NCT05264636.

Oridonin-induced ferroptosis and apoptosis: a dual approach to suppress the growth of osteosarcoma cells.

BACKGROUND: Osteosarcoma (OS) is one of the most common aggressive bone malignancy tumors in adolescents. With the application of new chemotherapy regimens, finding new and effective anti-OS drugs to coordinate program implementation is urgent for the patients of OS. Oridonin had been proved to mediate anti-tumor effect on OS cells, but its mechanism has not been fully elucidated. METHODS: The effects of oridonin on the viability, clonal formation and migration of 143B and U2OS cells were detected by CCK-8, colony formation assays and wound-healing test. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to explore the mechanism of oridonin on OS. Western blot (WB), real-time quantitative PCR (qRT-PCR) were used to detect the expression levels of apoptosis and ferroptosis-relative proteins and genes. Annexin V-FITC apoptosis detection kit and flow cytometry examination were used to detect the level of apoptosis. Iron assay kit was used to evaluate the relative Fe2+ content. The levels of mitochondrial membrane potential and lipid peroxidation production was determined by mitochondrial membrane potential detection kit and ROS assay kit. RESULTS: Oridonin could effectively inhibit the survival, clonal formation and metastasis of OS cells. The KEGG results indicated that oridonin is associated with the malignant phenotypic signaling pathways of proliferation, migration, and drug resistance in OS. Oridonin was capable of inhibiting expressions of BAX, cl-caspase3, SLC7A11, GPX4 and FTH1 proteins and mRNA, while promoting the expressions of Bcl-2 and ACSL4 in 143B and U2OS cells. Additionally, we found that oridonin could promote the accumulation of reactive oxygen species (ROS) and Fe2+ in OS cells, as well as reduce mitochondrial membrane potential, and these effects could be significantly reversed by the ferroptosis inhibitor ferrostatin-1 (Fer-1). CONCLUSION: Oridonin can trigger apoptosis and ferroptosis collaboratively in OS cells, making it a promising and effective agent for OS therapy.

Angelica Sinensis Polysaccharide-Based Nanoparticles for Liver-Targeted Delivery of Oridonin.

The present work aimed to study the feasibility of Angelica sinensis polysaccharide (ASP) as an instinctive liver targeting drug delivery carrier for oridonin (ORI) in the treatment of hepatocellular carcinoma (HCC). ASP was reacted with deoxycholic acid (DOCA) via an esterification reaction to form an ASP-DOCA conjugate. ORI-loaded ASP-DOCA nanoparticles (ORI/ASP-DOCA NPs) were prepared by the thin-film water method, and their size was about 195 nm in aqueous solution. ORI/ASP-DOCA NPs had a drug loading capacity of up to 9.2%. The release of ORI in ORI/ASP-DOCA NPs was pH-dependent, resulting in rapid decomposition and accelerated drug release at acidic pH. ORI/ASP-DOCA NPs significantly enhanced the accumulation of ORI in liver tumors through ASGPR-mediated endocytosis. In vitro results showed that ORI/ASP-DOCA NPs increased cell uptake and apoptosis in HepG2 cells, and in vivo results showed that ORI/ASP-DOCA NPs caused effective tumor suppression in H22 tumor-bearing mice compared with free ORI. In short, ORI/ASP-DOCA NPs might be a simple, feasible, safe and effective ORI nano-drug delivery system that could be used for the targeted delivery and treatment of liver tumors.

Fine-Scale Characterization of Plant Diterpene Glycosides Using Energy-Resolved Untargeted LC-MS/MS Metabolomics Analysis.

Plant diterpene glycosides are essential for diverse physiological processes. Comprehensive structural characterization proved to be a challenge due to variations in glycosylation patterns, diverse aglycone structures, and the absence of comprehensive reference databases. In this study, a method for fine-scale characterization was proposed based on energy-resolved (ER) untargeted LC-MS/MS metabolomics analysis using steviol glycosides as a demonstration. Energy-dependent fragmentation patterns were unveiled by a series of model compounds. Distinct glycosylation sites were discerned by leveraging varying fragmentation energies for the precursor ions. The sugar moiety linkage at C19OOH (R1) exhibited facile and intact cleavage at low collision energies, while the sugar moiety at C13-OH (R2) demonstrated consecutive cleavage with increasing energy. Aglycone ions exhibited a higher relative intensity at NCE 50, with relative intensities ranging from 95% to 100%. Subsequently, aglycone candidates, R1 sugar composition, and R2 sugar sequence were deduced through ER-MS/MS analysis. The developed method was applied to Stevia rebaudiana leaves. A total of 91 diterpene glycosides were unambiguously identified, including 16 steviol glycosides with novel acetylglycosylation patterns. This method offers a rapid alternative for glycan analysis and the structural differentiation of isomers. The developed method enhances the understanding of diterpene glycosides in plants, providing a reliable tool for the in-depth characterization of complex metabolite profiles.

Rebaudioside D decreases adiposity and hepatic lipid accumulation in a mouse model of obesity.

Overconsumption of added sugars has been pointed out as a major culprit in the increasing rates of obesity worldwide, contributing to the rising popularity of non-caloric sweeteners. In order to satisfy the growing demand, industrial efforts have been made to purify the sweet-tasting molecules found in the natural sweetener stevia, which are characterized by a sweet taste free of unpleasant aftertaste. Although the use of artificial sweeteners has raised many concerns regarding metabolic health, the impact of purified stevia components on the latter remains poorly studied. The objective of this project was to evaluate the impact of two purified sweet-tasting components of stevia, rebaudioside A and D (RebA and RebD), on the development of obesity, insulin resistance, hepatic health, bile acid profile, and gut microbiota in a mouse model of diet-induced obesity. Male C57BL/6 J mice were fed an obesogenic high-fat/high-sucrose (HFHS) diet and orally treated with 50 mg/kg of RebA, RebD or vehicle (water) for 12 weeks. An additional group of chow-fed mice treated with the vehicle was included as a healthy reference. At weeks 10 and 12, insulin and oral glucose tolerance tests were performed. Liver lipids content was analyzed. Whole-genome shotgun sequencing was performed to profile the gut microbiota. Bile acids were measured in the feces, plasma, and liver. Liver lipid content and gene expression were analyzed. As compared to the HFHS-vehicle treatment group, mice administered RebD showed a reduced weight gain, as evidenced by decreased visceral adipose tissue weight. Liver triglycerides and cholesterol from RebD-treated mice were lower and lipid peroxidation was decreased. Interestingly, administration of RebD was associated with a significant enrichment of Faecalibaculum rodentium in the gut microbiota and an increased secondary bile acid metabolism. Moreover, RebD decreased the level of lipopolysaccharide-binding protein (LBP). Neither RebA nor RebD treatments were found to impact glucose homeostasis. The daily consumption of two stevia components has no detrimental effects on metabolic health. In contrast, RebD treatment was found to reduce adiposity, alleviate hepatic steatosis and lipid peroxidation, and decrease LBP, a marker of metabolic endotoxemia in a mouse model of diet-induced obesity.