aspalathin and nothofagin

Aspalathin (Aspt) and nothofagin (Not) were reported to have antioxidant activity and are the two major active dihydrochalcones in green rooibos. This study was conducted to determine whether Asp and Not can modulate renal functional damage in a mouse model of sepsis and to elucidate the underlying mechanisms.. The potential of Aspt and Not treatment to reduce renal damage induced by cecal ligation and puncture (CLP) surgery in mice was measured by assessing blood urea nitrogen (BUN), serum creatinine, total urine protein, levels of lactate dehydrogenase (LDH), nitric oxide (NO), tumour necrosis factor (TNF)-α, interleukin (IL)-6, and myeloperoxidase (MPO), lipid peroxidation, total glutathione, glutathione peroxidase activity, catalase activity, and superoxide dismutase activity.. Treatment with Aspt and Not decreased plasma levels of BUN, creatinine, urine protein, and LDH in mice with CLP-induced renal damage. Moreover, Aspt and Not inhibited nuclear factor (NF)-κB activation and reduced the induction of NO synthase and excessive production of nitric acid. Aspt and Not treatment also reduced the plasma levels of NO, TNF-α, IL-6, and MPO and reduced lethality due to CLP-induced sepsis, increased lipid peroxidation, and markedly enhanced the antioxidant defence system by restoring the levels of superoxide dismutase, glutathione peroxidase, and catalase in the kidney tissues.. Our results suggest that Aspt and Not protect mice against sepsis-triggered renal injury.

Topics: Animals; Aspalathus; Chalcones; Disease Models, Animal; Human Umbilical Vein Endothelial Cells; Humans; Kidney; Male; Mice; Mice, Inbred C57BL; Protective Agents; Sepsis

Aspalathin (Asp) and nothofagin (Not) are two major active dihydrochalcones found in green rooibos tea (Aspalathus linearis; family, Fabaceae; tribe, Crotalarieae), which have been reported for their anti-oxidant activity. Here, the anticoagulant activities of Asp and Not were examined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), and the activities of thrombin (Factor IIa, FIIa) and activated factor X (FXa). And, the effects of Asp and Not on expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were evaluated in tumor necrosis factor (TNF)-α activated human umbilical vein endothelial cells (HUVECs). Treatment with Asp and Not resulted in prolonged aPTT and PT and inhibition of the activities of thrombin and FXa, as well as inhibited production of thrombin and FXa in HUVECs. In addition, Asp and Not inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation. Asp and Not also elicited anticoagulant effects in mice. In addition, treatment with Asp and Not resulted in significant reduction of the PAI-1 to t-PA ratio. Collectively, Asp and Not possesses antithrombotic activities and offers a basis for development of a novel anticoagulant.

Topics: Adult; Animals; Blood Coagulation; Cell Survival; Chalcones; Factor Xa; Female; Fibrinolytic Agents; Human Umbilical Vein Endothelial Cells; Humans; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Plasminogen Activator Inhibitor 1; Platelet Aggregation Inhibitors; Prothrombin; Prothrombin Time; Thrombin; Tumor Necrosis Factor-alpha; Young Adult

Vascular inflammation plays a key role in the initiation and progression of atherosclerosis, a major complication of diabetes mellitus. Aspalathin (Asp) and nothofagin (Not) are two major active dihydrochalcones found in green rooibos, which have been reported for their antioxidant activity. In this study, we assessed whether Asp or Not can suppress vascular inflammation induced by high glucose (HG) in human umbilical vein endothelial cells (HUVECs) and mice. We monitored the effects of Asp or Not on HG-induced vascular hyperpermeability, expression of cell adhesion molecules (CAMs), formation of reactive oxygen species (ROS), and activation of nuclear factor (NF)-κB in vitro and in vivo. Our data indicate that HG markedly increased vascular permeability, monocyte adhesion, expression of CAMs, formation of ROS, and activation of NF-κB. Remarkably, treatment of Asp or Not inhibited HG-mediated vascular hyperpermeability, adhesion of monocytes toward HUVECs, and expression of CAMs. In addition, Asp or Not suppressed the formation of ROS and the activation of NF-κB. Since vascular inflammation induced by HG is critical in the development of diabetic complications, our results suggest that Asp or Not may have significant benefits in the treatment of diabetic complications.

Topics: Animals; Aspalathus; Cell Survival; Chalcones; Dose-Response Relationship, Drug; Glucose; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Male; Mice; Mice, Inbred C57BL; Plant Extracts

Aspalathin (Asp) and nothofagin (Not) are two major active dihydrochalcones found in green rooibos, which have been reported for their anti-oxidant activity. Increasing evidence has demonstrated that beyond its role in the activation of protein C, endothelial cell protein C receptor (EPCR) is also involved in vascular inflammation. EPCR activity is markedly changed by ectodomain cleavage and its release as the soluble EPCR. EPCR can be shed from the cell surface, which is mediated by tumor necrosis factor-α converting enzyme (TACE). However, little is known about the effects of Asp and Not on EPCR shedding. Our results demonstrated that Asp and Not induced potent inhibition of phorbol-12-myristate 13-acetate (PMA)-, tumor necrosis factor (TNF)-α-, interleukin (IL)-1β, and cecal ligation and puncture (CLP)-induced EPCR shedding. Asp and Not also inhibited the expression and activity of PMA-induced TACE in endothelial cells. Asp and Not also suppressed CLP-induced protein C decrease in mice and thrombin generation in HUVECs. In addition, treatment with Asp and Not resulted in reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinase (ERK) 1/2, and c-Jun N-terminal kinase (JNK). These results demonstrate the potential of Asp and Not as an anti-sEPCR shedding reagent against PMA and CLP-mediated EPCR shedding.

Topics: ADAM Proteins; ADAM17 Protein; Animals; Aspalathus; Blood Coagulation Factors; Chalcones; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-1beta; JNK Mitogen-Activated Protein Kinases; Male; Mice, Inbred C57BL; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Receptors, Cell Surface; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Aspalathin and nothofagin, the major dihydrochalcones in rooibos (Aspalathus linearis), are valuable bioactive compounds, but their bioactivity has not been fully elucidated. Isolation of these compounds using high-performance countercurrent chromatography (HPCCC), a gentle, support-free, up-scalable technique, offers an alternative to synthesis for obtaining sufficient amounts. An HPLC-DAD method was adapted to allow rapid (16 min from injection to injection) quantification of the four major compounds (aspalathin, nothofagin, isoorientin, orientin) during development of the isolation protocol. The traditional shake-flask method, used to determine distribution constants (K(D)) for target compounds, was also adapted to obtain higher repeatability. Green rooibos leaves with a high aspalathin and nothofagin content were selected as source material. Sample loading of the polyphenol-enriched extract was limited due to constituents with emulsifying properties, but could be increased by removing ethanol-insoluble matter. Furthermore, problems with degradation of aspalathin during HPCCC separation and further processing could be limited by acidifying the HPCCC solvent system. Aspalathin was shown to be fairly stable at pH 3 (91% remaining after 29 h) compared to pH 7 (45% remaining after 29 h). Aspalathin and nothofagin with high purities (99% and 100%, respectively) were obtained from HPCCC fractions after semi-preparative HPLC.

Topics: Aspalathus; Chalcones; Chromatography, High Pressure Liquid; Countercurrent Distribution; Flavonoids; Glucosides; Luteolin; Plant Extracts; Plant Leaves; Polyphenols; Spectrometry, Mass, Electrospray Ionization

Aspalathin (Asp) and nothofagin (Not) are two major active dihydrochalcones found in green rooibos, which have been reported for their anti-oxidant activity. Here, we investigated the anti-inflammatory effects and underlying mechanisms of Asp and Not against lipopolysaccharide (LPS)-mediated vascular inflammatory responses. The anti-inflammatory activities of Asp and Not were determined by measuring permeability, monocytes adhesion and migration, and activation of pro-inflammatory proteins in LPS-activated human umbilical vein endothelial cells (HUVECs) and mice. We found that each compound inhibited LPS-induced barrier disruption, expression of cell adhesion molecules (CAMs), and adhesion/transendothelial migration of neutrophils to human endothelial cells. Asp and Not also suppressed LPS-induced hyperpermeability and leukocyte migration in vivo. Furthermore, each compound suppressed the production of tumor necrosis factor-α (TNF-α) or interleukin (IL)-6 and the activation of nuclear factor-κB (NF-κB) or extracellular regulated kinases (ERK) 1/2 by LPS. Moreover, treatment with each compound resulted in reduced LPS-induced lethal endotoxemia. These results suggest that Asp and Not posses anti-inflammatory functions by inhibiting hyperpermeability, expression of CAMs, and adhesion and migration of leukocytes, thereby endorsing its usefulness as a therapy for vascular inflammatory diseases.

Topics: Animals; Anti-Inflammatory Agents; Cell Movement; Chalcones; Dose-Response Relationship, Drug; Human Umbilical Vein Endothelial Cells; Humans; Inflammation Mediators; Male; Mice; Mice, Inbred C57BL; Neutrophils

The ubiquitous nuclear protein, high mobility group box 1 (HMGB1), is released by activated macrophages and human umbilical vein endothelial cells (HUVECs) and functions as a late mediator of experimental sepsis. Aspalathin (Asp) and nothofagin (Not), which have been reported to have anti-oxidant activity, are the two major active dihydrochalcones found in green rooibos. In this study, we investigated the antiseptic effects and underlying mechanisms of Asp and Not against HMGB1-mediated septic responses in HUVECs and mice. The anti-inflammatory activities of Asp and Not were determined by measuring permeability, monocyte adhesion and migration, and activation of proinflammatory proteins in HMGB1-activated HUVECs and mice. According to the results, Asp and Not effectively inhibited lipopolysaccharide (LPS)-induced release of HMGB1, and suppressed HMGB1-mediated septic responses, such as hyperpermeability, adhesion and migration of leukocytes, and expression of cell adhesion molecules. In addition, Asp and Not suppressed the production of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), the activation of nuclear factor-κB (NF-κB) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) by HMGB1. Collectively, these results indicate that Asp and Not could be potential therapeutic agents for the treatment of various severe vascular inflammatory diseases via the inhibition of the HMGB1 signaling pathway.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Aspalathus; Chalcones; Disease Models, Animal; HMGB1 Protein; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-6; Male; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; Phytotherapy; Sepsis; Signal Transduction; Tumor Necrosis Factor-alpha

The anti-peroxyl radical quality of two aqueous rooibos infusions and solutions of their most abundant glycosylated polyphenols was evaluated using pyrogallol red and fluorescein-based oxygen radical absorbance ratios. It was observed that the artificial infusions, prepared using only the most abundant polyphenols present in rooibos and at concentrations similar to those found in the natural infusions, showed greater antioxidant quality than the latter infusions, reaching values close to those reported for tea infusions. Additionally, the antimicrobial activity of the natural and artificial infusions was assessed against three species of bacteria: Gram (+) Staphylococus epidermidis and Staphylococcus aureus and Gram (-) Escherichia coli. When compared to the natural infusions the artificial beverages did not demonstrate any bacterostatic/cidal activity, suggesting that the antibacterial activity of rooibos is related to compounds other than the glycosylated polyphenols employed in our study.

Topics: Anti-Bacterial Agents; Apigenin; Aspalathus; Beverages; Chalcones; Escherichia coli; Flavonoids; Free Radical Scavengers; Glucosides; Microbial Sensitivity Tests; Peroxides; Plant Extracts; Polyphenols; Quercetin; Rutin; Staphylococcus aureus; Staphylococcus epidermidis

The steroid hormone output of the adrenal gland is crucial in the maintenance of hormonal homeostasis, with hormonal imbalances being associated with numerous clinical conditions which include, amongst others, hypertension, metabolic syndrome, cardiovascular disease, insulin resistance and type 2 diabetes. Aspalathus linearis (Rooibos), which has been reported to aid stress-related symptoms linked to metabolic diseases, contains a wide spectrum of bioactive phenolic compounds of which aspalathin is unique. In this study the inhibitory effects of Rooibos and the dihydrochalcones, aspalathin and nothofagin, were investigated on adrenal steroidogenesis. The activities of both cytochrome P450 17α-hydroxylase/17,20 lyase and cytochrome P450 21-hydroxylase were significantly inhibited in COS-1 cells. In order to study the effect of these compounds in H295R cells, a human adrenal carcinoma cell line, a novel UPLC-MS/MS method was developed for the detection and quantification of twenty-one steroid metabolites using a single chromatographic separation. Under both basal and forskolin-stimulated conditions, the total amount of steroids produced in H295R cells significantly decreased in the presence of Rooibos, aspalathin and nothofagin. Under stimulated conditions, Rooibos decreased the total steroid output 4-fold and resulted in a significant reduction of aldosterone and cortisol precursors. Dehydroepiandrosterone-sulfate levels were unchanged, while the levels of androstenedione (A4) and 11β-hydroxyandrostenedione (11βOH-A4) were inhibited 5.5 and 2.3-fold, respectively. Quantification of 11βOH-A4 showed this metabolite to be a major product of steroidogenesis in H295R cells and we confirm, for the first time, that this steroid metabolite is the product of the hydroxylation of A4 by human cytochrome P450 11β-hydroxylase. Taken together our results demonstrate that Rooibos, aspalathin and nothofagin influence steroid hormone biosynthesis and the flux through the mineralocorticoid, glucocorticoid and androgen pathways, thus possibly contributing to the alleviation of negative effects arising from elevated glucocorticoid levels.

Topics: Adenylyl Cyclase Inhibitors; Adrenal Glands; Animals; Aspalathus; Cell Line; Chalcones; Chlorocebus aethiops; Colforsin; COS Cells; Enzyme Inhibitors; Humans; Hydroxylation; Molecular Structure; Papio; Plant Extracts; Recombinant Proteins; Steroid 17-alpha-Hydroxylase; Steroid 21-Hydroxylase; Steroids

Syntheses of the C-glycosyl flavone natural products aspalathin and nothofagin have been accomplished in eight steps from tribenzyl glucal, tribenzylphloroglucinol, and either 4-(benzyloxy)phenylacetylene or 3,4-bis(benzyloxy)phenylacetylene. The key step of the syntheses involves a highly stereoselective Lewis acid promoted coupling of 1,2-di-O-acyl-3,4,6-tribenzylglucose with tribenzylphloroglucinol, which gives rise to the corresponding beta-C-aryl glycoside in 30-65% yields.

Topics: Biological Products; Chalcones; Molecular Structure; Stereoisomerism

Aspalathin and nothofagin are typical ingredients of unfermented rooibos (Krafczyk, N.; Glomb, M. A. J. Agric. Food Chem. 2008, 56, 3368). During oxidation these dihydrochalcones were degraded to higher molecular weight browning products under aerated and nonenzymatic conditions. In the early stages of browning reactions aspalathin formed two dimers. These two compounds were unequivocally established as atropisomers stemming from oxidative A to B ring coupling. Multilayer countercurrent chromatography (MLCCC) and preparative high-performance liquid chromatography (HPLC) were applied to obtain pure substances. The purity and identity of isolated dimers were confirmed by different NMR experiments, HPLC-DAD-MS, and HR-MS. In parallel to the formation of chromophores during the fermentation of black tea, the formation of aspalathin dimers implies an important mechanistic channel for the generation of color during the processing of rooibos.

Topics: Aspalathus; Chalcones; Dimerization; Oxidation-Reduction; Plant Extracts