Increased Glycolytic Activity Is Part of Impeded M1(LPS) Macrophage Polarization in the Presence of Urolithin A.

Urolithin A is a gut metabolite of ellagitannins and reported to confer health benefits, e.g., by increased clearance of damaged mitochondria by macroautophagy or curbed inflammation. One targeted cell type are macrophages, which are plastic and able to adopt pro- or anti-inflammatory polarization states, usually assigned as M1 and M2 macrophages, respectively. This flexibility is tightly coupled to characteristic shifts in metabolism, such as increased glycolysis in M1 macrophages, and protein expression upon appropriate stimulation. This study aimed at investigating whether the anti-inflammatory properties of U: rolithin A may be driven by metabolic alterations in cultivated murine M1(lipopolysaccharide) macrophages. Expression and extracellular flux analyses showed that urolithin A led to reduced il1ß, il6, and nos2 expression and boosted glycolytic activity in M1(lipopolysaccharide) macrophages. The pro-glycolytic feature of UROLITHIN A: occurred in order to causally contribute to its anti-inflammatory potential, based on experiments in cells with impeded glycolysis. Mdivi, an inhibitor of mitochondrial fission, blunted increased glycolytic activity and reduced M1 marker expression in M1(lipopolysaccharide/UROLITHIN A: ), indicating that segregation of mitochondria was a prerequisite for both actions of UROLITHIN A: . Overall, we uncovered a so far unappreciated metabolic facet within the anti-inflammatory activity of UROLITHIN A: and call for caution about the simplified notion of increased aerobic glycolysis as an inevitably proinflammatory feature in macrophages upon exposure to natural products.

In Silico and In Vitro Approach to Assess Direct Allosteric AMPK Activators from Nature.

The 5'-adenosine monophosphate-activated protein kinase (AMPK) is an important metabolic regulator. Its allosteric drug and metabolite binding (ADaM) site was identified as an attractive target for direct AMPK activation and holds promise as a novel mechanism for the treatment of metabolic diseases. With the exception of lusianthridin and salicylic acid, no natural product (NP) is reported so far to directly target the ADaM site. For the streamlined assessment of direct AMPK activators from the pool of NPs, an integrated workflow using in silico and in vitro methods was applied. Virtual screening combining a 3D shape-based approach and docking identified 21 NPs and NP-like molecules that could potentially activate AMPK. The compounds were purchased and tested in an in vitro AMPK α 1 ß 1 γ 1 kinase assay. Two NP-like virtual hits were identified, which, at 30 µM concentration, caused a 1.65-fold (± 0.24) and a 1.58-fold (± 0.17) activation of AMPK, respectively. Intriguingly, using two different evaluation methods, we could not confirm the bioactivity of the supposed AMPK activator lusianthridin, which rebuts earlier reports.

Regulation of Nrf2 and NF-κB activities may contribute to the anti-inflammatory mechanism of xylopic acid.

Xylopic acid (XA) is a kaurene diterpene which naturally exists in African plants such as Xylopia aethiopica. It has been established to exhibit acute and chronic anti-inflammatory activities from our earlier studies. This current work sets out to shed light on the potential molecular target(s) of xylopic acid. Selection of investigated targets (NF-κB, Nrf2 and PTP1B) was based on an unbiased approach, using the SPiDER in silico prediction tool, and a candidate approach, examining well-known anti-inflammatory targets. Reporter gene assays were used to test for altered NF-κB and Nrf2 activities in transfected HEK or CHO cells, respectively, and immunoblot and flow cytometric analyses examined protein expression of the Nrf2/NF-kB target genes HO-1 and VCAM-1 in HUVEC. An effect of XA on PTP1B activity assay was studied using an in vitro enzyme assay with recombinant human enzyme and pNPP as substrate as well as by looking at insulin receptor phosphorylation in HepG2 cells. XA at 30 µM significantly (p < 0.001) inhibited the NF-κB-dependent reporter gene expression and enhanced activation of Nrf2 in a concentration-dependent manner when compared to the control. XA also marginally increased HO-1 protein expression levels while expression of VCAM-1 was reduced to 70% in XA-treated endothelial cells. However, XA did not show any sign of inhibition of PTP1B or a related phosphatase. Our findings suggest that the anti-inflammatory mechanism of XA entails the inhibitory effect on NF-κB and an increased activity of Nrf2, accompanied by increased expression of HO-1 and reduced expression of VCAM-1.

Evodiamine Lowers Blood Lipids by Up-Regulating the PPARγ/ABCG1 Pathway in High-Fat-Diet-Fed Mice.

The natural alkaloid evodiamine enhances cholesterol efflux from cultured THP-1-derived macrophages, but whether it has any impact on blood lipids in vivo remains unknown. In this study, the effect of evodiamine on hyperlipidemia induced by a high-fat diet (HFD) was investigated in mice. Intragastric administrations of evodiamine (10 and 20 mg/kg) for 8 weeks resulted in a significant improvement of metabolic lipid profiles by reducing the plasma levels of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C). Evodiamine also significantly decreased hepatic lipid accumulation and hepatic total bile acids (TBA). Mechanistically, evodiamine increased ATP-binding cassette transporter G1 (ABCG1) mRNA and protein expression and up-regulated peroxisome proliferator-activated receptor gamma (PPARγ) expression in the liver. Taken together, the natural product evodiamine lowers blood lipids in HFD-fed mice likely through promoting the PPARγ-ABCG1 signaling pathway.

Fast and Highly Efficient Affinity Enrichment of Azide-A-DSBSO Cross-Linked Peptides.

Cross-linking mass spectrometry is an increasingly used, powerful technique to study protein-protein interactions or to provide structural information. Due to substochiometric reaction efficiencies, cross-linked peptides are usually low abundance. This results in challenging data evaluation and the need for an effective enrichment. Here we describe an improved, easy to implement, one-step method to enrich azide-tagged, acid-cleavable disuccinimidyl bis-sulfoxide (DSBSO) cross-linked peptides using dibenzocyclooctyne (DBCO) coupled Sepharose beads. We probed this method using recombinant Cas9 and E. coli ribosome. For Cas9, the number of detectable cross-links was increased from ∼100 before enrichment to 580 cross-links after enrichment. To mimic a cellular lysate, E. coli ribosome was spiked into a tryptic HEK background at a ratio of 1:2-1:100. The number of detectable unique cross-links was maintained high at ∼100. The estimated enrichment efficiency was improved by a factor of 4-5 (based on XL numbers) compared to enrichment via biotin and streptavidin. We were still able to detect cross-links from 0.25 µg cross-linked E. coli ribosomes in a background of 100 µg tryptic HEK peptides, indicating a high enrichment sensitivity. In contrast to conventional enrichment techniques, like SEC, the time needed for preparation and MS measurement is significantly reduced. This robust, fast, and selective enrichment method for azide-tagged linkers will contribute to mapping protein-protein interactions, investigating protein architectures in more depth, and helping to understand complex biological processes.

Caco-2 Cells for Measuring Intestinal Cholesterol Transport - Possibilities and Limitations.

BACKGROUND: The human Caco-2 cell line is a common in vitro model of the intestinal epithelial barrier. As the intestine is a major interface in cholesterol turnover and represents a non-biliary pathway for cholesterol excretion, Caco-2 cells are also a valuable model for studying cholesterol homeostasis, including cholesterol uptake and efflux. Currently available protocols are, however, either sketchy or not consistent among different laboratories. Our aim was therefore to generate a collection of optimized protocols, considering the different approaches of the different laboratories and to highlight possibilities and limitations of measuring cholesterol transport with this cell line. RESULTS: We developed comprehensive and quality-controlled protocols for the cultivation of Caco-2 cells on filter inserts in a single tight monolayer. A cholesterol uptake as well as a cholesterol efflux assay is described in detail, including suitable positive controls. We further show that Caco-2 cells can be efficiently transfected for luciferase reporter gene assays in order to determine nuclear receptor activation, main transcriptional regulators of cholesterol transporters (ABCA1, ABCB1, ABCG5/8, NPC1L1). Detection of protein and mRNA levels of cholesterol transporters in cells grown on filter inserts can pose challenges for which we highlight essential steps and alternative approaches for consideration. A protocol for viability assays with cells differentiated on filter inserts is provided for the first time. CONCLUSIONS: The Caco-2 cell line is widely used in the scientific community as model for the intestinal epithelium, although with highly divergent protocols. The herein provided information and protocols can be a common basis for researchers intending to use Caco-2 cells in the context of cellular cholesterol homeostasis.

Norway Spruce Balm: Phytochemical Composition and Ability to Enhance Re-epithelialization In Vitro.

The balm of the Norway spruce (Picea abies) is a well-known traditional herbal medicine used to cure wounds. Even though clinical trials have confirmed its empirical use, the active constituents, their mode of action, and the exact composition of this natural product are still unknown. In this study, the balm was subjected to fractionated extraction and further purified employing flash chromatography, HPLC-PDA-ELSD, preparative and analytical TLC. Hydroxycinnamic acids ( 1: - 3: ), the lignan pinoresinol ( 4: ), four hydroxylated derivatives of dehydroabietic acid (DHAA) ( 5:  -  8: ), and dehydroabietic acid ( 9: ) were isolated. Their structures were elucidated by LC-MS, 1D- and 2D-NMR. Four extracts, two commercially available resin acids-pimaric acid ( 10: ) and isopimaric acid ( 11: )-and the isolated compounds were tested for increased re-epithelialization of cell-free areas in a human adult low calcium high temperature keratinocytes monolayer. Lysophosphatidic acid (10 µM) served as positive control and ranged between 100% and 150% rise in cell-covered area related to the vehicle control. Two extracts containing carboxylic acids and non-acidic apolar constituents, respectively, boosted wound closure by 47% and 36% at 10 and 3 µg/mL, respectively. Pinoresinol, DHAA, three of its hydroxylated derivatives, and pimaric and isopimaric acid as well as defined combinations of the hydroxylated DHAA derivatives led to a significantly enhanced wound closure by up to 90% at concentrations between 1 and 10 µM. Overall, lignans and diterpene resin acids, main constituents of Norway spruce balm, are able to increase migration or proliferation of keratinocytes in vitro. The presented data link the phytochemistry of this natural wound healing agent with boosted re-epithelialization.

Narciclasine inhibits angiogenic processes by activation of Rho kinase and by downregulation of the VEGF receptor 2.

The process of angiogenesis is involved in several pathological conditions, such as tumor growth or age-related macular degeneration. Although the available anti-angiogenic drugs have improved the therapy of these diseases, major drawbacks, such as unwanted side effects and resistances, still exist. Consequently, the search for new anti-angiogenic substances is still ongoing. Narciclasine, a plant alkaloid from different members of the Amaryllidaceae family, has extensively been characterized as anti-tumor compound. Beyond the field of cancer, the compound has recently been shown to possess anti-inflammatory properties. Surprisingly, potential actions of narciclasine on endothelial cells in the context of angiogenesis have been neglected so far. Thus, we aimed to analyze the effects of narciclasine on angiogenic processes in vitro and in vivo and to elucidate the underlying mechanism. Narciclasine (100-300 nM) effectively inhibited the proliferation, undirected and directed migration, network formation and angiogenic sprouting of human primary endothelial cells. Moreover, narciclasine (1 mg/kg/day) strongly reduced the VEGF-triggered angiogenesis in vivo (Matrigel plug assay in mice). Narciclasine mediated its anti-angiogenic effects in part by a RhoA-independent activation of the Rho kinase ROCK. Most importantly, however, the compound reduced the de novo protein synthesis in endothelial cells by approx. 50% without exhibiting considerable cytotoxic effects. As a consequence, narciclasine diminished the presence of proteins with a short half-life, such as the VEGF receptor 2, which is the basis for its anti-angiogenic effects. Taken together, our study highlights narciclasine as an interesting anti-angiogenic compound that is worth to be further evaluated in preclinical studies.

Urolithin A gains in antiproliferative capacity by reducing the glycolytic potential via the p53/TIGAR axis in colon cancer cells.

Polyphenols have shown promising bioactivity in experimental in vitro and in vivo models for cancer chemoprevention. However, consumed orally, they are often transformed by gut microbes into new active principles with so far incompletely deciphered molecular mechanisms. Here, enterolacton, S-equol and urolithin A as representatives of metabolites of lignans, isoflavones and ellagitannins, respectively, were examined for their impact on HCT116 colon cancer cell growth, cooperativity with oxaliplatin and p53 dependency in vitro. Whereas enterolacton and S-equol (≤60 µM) did not elicit growth inhibition or positive cooperativity with oxaliplatin, urolithin A showed an IC50 value of 19 µM (72 h) and synergism with oxaliplatin. Urolithin A induced p53 stabilization and p53 target gene expression, and absence of p53 significantly dampened the antiproliferative effect of urolithin A (IC50(p53-/-) = 38 µM). P53 was dispensable for the G2/M arrest in HCT116 cells but required for induction of a senescence-like phenotype upon long-term exposure and for the observed synergism with oxaliplatin. Moreover, extracellular flux analyses and knockdown approaches uncovered a reduced glycolytic potential via the p53/TIGAR axis which was linked to the higher susceptibility of wildtype cells to urolithin A. Overall, the p53 status turned out to be an important determinant for the potential benefit of dietary ellagitannins in cancer chemoprevention or use in adjuvant therapy.

Bioactive Molecules and Their Mechanisms of Action.

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An increased autophagic flux contributes to the anti-inflammatory potential of urolithin A in macrophages.

BACKGROUND: An extract of Phyllanthus muellerianus and its constituent geraniin have been reported to exert anti-inflammatory activity in vivo. However, orally consumed geraniin, an ellagitannin, shows low bioavailability and undergoes metabolization to urolithins by gut microbiota. This study aimed at comparing geraniin and urolithin A with respect to inhibition of M1 (LPS) polarization of murine J774.1 macrophages and shedding more light on possible underlying mechanisms. METHODS: Photometric, fluorimetric as well as luminescence-based assays monitored production of reactive oxygen species (ROS) and nitric oxide (NO), cell viability or reporter gene expression. Western blot analyses and confocal microscopy showed abundance and localization of target proteins, respectively. RESULTS: Urolithin A is a stronger inhibitor of M1 (LPS) macrophage polarization (production of NO, ROS and pro-inflammatory proteins) than geraniin. Urolithin A leads to an elevated autophagic flux in macrophages. Inhibition of autophagy in M1 (LPS) macrophages overcomes the suppressed nuclear translocation of p65 (NF-kB; nuclear factor kB), the reduced expression of pro-inflammatory genes as well as the diminished NO production brought about by urolithin A. The increased autophagic flux is furthermore associated with impaired Akt/mTOR (mammalian target of rapamycin) signaling in urolithin A-treated macrophages. CONCLUSIONS AND GENERAL SIGNIFICANCE: Intestinal metabolization may boost the potential health benefit of widely consumed dietary ellagitannins, as suggested by side by side comparison of geraniin and urolithin A in M1(LPS) macrophages. Increased activity of the autophagic cellular recycling machinery aids the anti-inflammatory bioactivity of urolithin A.

Dihydrochalcone Glucosides from the Subaerial Parts of Thonningia sanguinea and Their in Vitro PTP1B Inhibitory Activities.

Six new and four known dihydrochalcone glucoside derivatives (1-10), the phenylpropanoid coniferin (11), and the lignans (+)-pinoresinol (12) and lariciresinol (13) were isolated from the subaerial plant parts of Thonningia sanguinea in the course of a screening campaign for new antidiabetic lead compounds. The structures of the new substances were elucidated by HRESIMS, NMR, GC-MS, and ECD data evaluation. 2'- O-(3-Galloyl-4,6- O- Sa-hexahydroxydiphenoyl-ß-d-glucopyranosyl)-3-hydroxyphloretin (4), 2'- O-(4,6- O- Sa-hexahydroxydiphenoyl-ß-d-glucopyranosyl)phloretin (5), 2'- O-(3- O-galloyl-4,6- O- Sa-hexahydroxydiphenoyl-ß-d-glucopyranosyl)phloretin (6), and thonningianin B (9) showed moderate protein tyrosine phosphatase-1B inhibition in an enzyme assay (IC50 values ranging from 19 to 25 µM), whereas thonningianin A (10) was identified as a more potent inhibitor (IC50 = 4.4 µM). The observed activity differences could be explained by molecular docking experiments. The activity of 10 could further be confirmed in HEPG2 liver carcinoma cells, where the compound was able to increase the level of phosphorylated insulin receptors in a concentration-dependent manner.

Eurycomalactone Inhibits Expression of Endothelial Adhesion Molecules at a Post-Transcriptional Level.

The C-19 quassinoid eurycomalactone (1) has recently been shown to be a potent (IC50 = 0.5 µM) NF-κB inhibitor in a luciferase reporter model. In this study, we show that 1 with similar potency inhibited the expression of the NF-κB-dependent target genes ICAM-1, VCAM-1, and E-selectin in TNFα-activated human endothelial cells (HUVECtert) by flow cytometry experiments. Surprisingly, 1 (2 µM) did not inhibit TNFα-induced IKKα/ß or IκBα phosphorylation significantly. Also, the TNFα-induced degradation of IκBα remained unchanged in response to 1 (2 µM). In addition, pretreatment of HUVECtert with 1 (2 µM) had no statistically significant effect on TNFα-mediated nuclear translocation of the NF-κB subunit p65 (RelA). Quantitative RT-PCR revealed that 1 (0.5-5 µM) exhibited diverse effects on the TNFα-induced transcription of ICAM-1, VCAM-1, and SELE genes since the mRNA level either remained unchanged (ICAM-1, E-selectin, and VCAM-1 at 0.5 µM 1), was reduced (VCAM-1 at 5 µM 1), or even increased (E-selectin at 5 µM 1). Finally, the time-dependent depletion of a short-lived protein (cyclin D1) as well as the measurement of de novo protein synthesis in the presence of 1 (2-5 µM) suggested that 1 might act as a protein synthesis inhibitor rather than an inhibitor of early NF-κB signaling.

Xanthohumol Blocks Proliferation and Migration of Vascular Smooth Muscle Cells in Vitro and Reduces Neointima Formation in Vivo.

Xanthohumol (1) is a principal prenylated chalcone found in hops. The aim of this study was to examine its influence on platelet-derived growth factor (PDGF)-BB-triggered vascular smooth muscle cell (VSMC) proliferation and migration in vitro and on experimentally induced neointima formation in vivo. Quantification of resazurin conversion indicated that 1 can inhibit PDGF-BB-induced VSMC proliferation concentration-dependently (IC50 = 3.49 µM). Furthermore, in a wound-healing assay 1 potently suppresses PDGF-BB-induced VSMC migration at 15 µM. Tested in a mouse femoral artery cuff model, 1 significantly reduces neointima formation. Taken together, we show that 1 represses PDGF-BB-induced VSMC proliferation and migration in vitro as well as neointima formation in vivo. This novel activity suggests 1 as an interesting candidate for further studies addressing a possible therapeutic application to counteract vascular proliferative disease.

Activation of the Nrf2-ARE pathway by the Alternaria alternata mycotoxins altertoxin I and II.

The mycotoxins altertoxin I and II (ATX I and II) are secondary metabolites produced by Alternaria alternata fungi and may occur as food and feed contaminants, especially after long storage periods. Although the toxic potential of altertoxins has been previously investigated, little is known about the pathways that play a role in their intracellular metabolism. In order to identify potential targets of ATX I and ATX II, the two toxins were tested for interaction with the nuclear factor erythroid-derived 2-like 2/antioxidant response element (Nrf2/ARE) pathway in mammalian cells. This pathway can be activated by various stressors resulting in the expression of enzymes important for metabolism and detoxification. In the present study, only ATX II triggered a concentration-dependent increase in Nrf2-ARE-dependent luciferase expression. Consistently, confocal microscopy revealed an ATX II-induced increase in Nrf2 signal in HT29 intestinal cells. In agreement with these data, ATX II induced the transcription of γ-glutamate cysteine ligase, the key enzyme in catalyzing GSH synthesis of the cells and which is regulated by Nrf2. Further investigations demonstrated that ATX II induced a concentration-dependent depletion of the cellular GSH levels after short incubation time (3 h) and an increase after longer incubation time (24 h). In conclusion, it was demonstrated that ATX II can interact at several levels of the Nrf2-ARE pathway in mammalian cells and that ATX I does not share the same mechanism of action.

Impact of phase I metabolism on uptake, oxidative stress and genotoxicity of the emerging mycotoxin alternariol and its monomethyl ether in esophageal cells.

Studies on the genotoxicity of Alternaria mycotoxins focus primarily on the native compounds. Alternariol (AOH) and its methyl ether (AME) have been reported to represent substrates for cytochrome P450 enzymes, generating hydroxylated metabolites. The impact of these phase I metabolites on genotoxicity remains unknown. In the present study, the synthesis and the toxicological effects of the metabolites 4-hydroxy alternariol (4-OH-AOH) and 4-hydroxy alternariol monomethyl ether (4-OH-AME) are presented and compared to the effects of the parent molecules. Although the two phase I metabolites contain a catecholic structure, which is expected to be involved in redox cycling, only 4-OH-AOH increased reactive oxygen species (ROS) in human esophageal cells (KYSE510), 4 times more pronounced than AOH. No ROS induction was observed for 4-OH-AME, although the parent compound showed some minor impact. Under cell-free conditions, both metabolites inhibited topoisomerase II activity comparable to their parent compounds. In KYSE510 cells, both metabolites were found to enhance the level of transient DNA-topoisomerase complexes in the ICE assay. Although the level of ROS was significantly increased by 4-OH-AOH, neither DNA strand breaks nor enhanced levels of formamidopyrimidine-DNA-glycosylase (FPG)-sensitive sites were observed. In contrast, AOH induced significant DNA damage in KYSE510 cells. Less pronounced or even absent effects of hydroxylated metabolites compared to the parent compounds might at least partly be explained by their poor cellular uptake. Glucuronidation as well as sulfation appear to have only a minor influence. Instead, methylation of 4-OH-AOH seems to be the preferred way of metabolism in KYSE510 cells, whereby the toxicological relevance of the methylation product remains to be clarified.

Bupleurum chinense Roots: a Bioactivity-Guided Approach toward Saponin-Type NF-κB Inhibitors.

The roots of Bupleurum chinense have a long history in traditional medicine to treat infectious diseases and inflammatory disorders. Two major compounds, saikosaponins A and D, were reported to exert potent anti-inflammatory activity by inhibiting NF-κB. In the present study, we isolated new saikosaponin analogues from the roots of B. chinese interfering with NF-κB activity in vitro. The methanol-soluble fraction of the dichloromethane extract of Radix Bupleuri was subjected to activity-guided isolation yielding 18 compounds, including triterpenoids and polyacetylenes. Their structures were determined by spectroscopic methods as saikogenin D (1), prosaikogenin D (2), saikosaponins B2 (3), W (4), B1 (5), Y (6), D (7), A (8), E (9), B4 (10), B3 (11), and T (12), saikodiyne A (13), D (14), E (15) and F (16), falcarindiol (17), and 1-linoleoyl-sn-glycero-3-phosphorylcholine (18). Among them, 4, 15, and 16 are new compounds, whereas 6, previously described as a semi-synthetic compound, is isolated from a natural source for the first time, and 13-17 are the first reports of polyacetylenes from this plant. Nine saponins/triterpenoids were tested for inhibition of NF-κB signaling in a cell-based NF-κB-dependent luciferase reporter gene model in vitro. Five of them (1, 2, 4, 6, and 8) showed strong (> 50%, at 30 µM) NF-κB inhibition, but also varying degrees of cytotoxicity, with compounds 1 and 4 (showing no significant cytotoxicity) presenting IC50 values of 14.0 µM and 14.1 µM in the cell-based assay, respectively.

Leoligin, the Major Lignan from Edelweiss (Leontopodium nivale subsp. alpinum), Promotes Cholesterol Efflux from THP-1 Macrophages.

Leoligin is a natural lignan found in Edelweiss (Leontopodium nivale ssp. alpinum). The aim of this study was to examine its influence on cholesterol efflux and to address the underlying mechanism of action. Leoligin increases apo A1- as well as 1% human plasma-mediated cholesterol efflux in THP-1 macrophages without affecting cell viability as determined by resazurin conversion. Western blot analysis revealed that the protein levels of the cholesterol efflux transporters ABCA1 and ABCG1 were upregulated, whereas the SR-B1 protein level remained unchanged upon treatment with leoligin (10 µM, 24 h). Quantitative reverse transcription PCR further uncovered that leoligin also increased ABCA1 and ABCG1 mRNA levels without affecting the half-life of the two mRNAs in the presence of actinomycin D, a transcription inhibitor. Proteome analysis revealed the modulation of protein expression fingerprint in the presence of leoligin. Taken together, these results suggest that leoligin induces cholesterol efflux in THP-1-derived macrophages by upregulating ABCA1 and ABCG1 expression. This novel activity suggests leoligin as a promising candidate for further studies addressing a possible preventive or therapeutic application in the context of atherosclerosis.

Quantitation of phenylpropanoids and iridoids in insulin-sensitising extracts of Leonurus sibiricus L. (Lamiaceae).

INTRODUCTION: Leonurus sibiricus L. is regularly used in traditional Mongolian medicine including for the treatment of symptoms of diabetes mellitus. OBJECTIVES: To provide a validated quantitation method for the quality control of Leonurus sibiricus and to prove in vitro insulin-sensitisation, thereby supporting the traditional use of Leonurus sibiricus. METHODOLOGY: Pulverised Leonurus sibiricus material was either extracted with methanol or methanol:water (25:75, v/v). HPLC-CAD (charged aerosol detector) separations were performed on a Luna Phenyl-Hexyl column with water and acetonitrile (both modified with 0.1% formic acid) as mobile phase. Gradient elution was employed using theophylline as internal standard. Tentative peak identification was facilitated by HPLC-MS. Validation was carried out according to ICH (International Conference on Harmonisation) guidelines. Potential insulin-sensitisation of accordant extracts was assessed in glucose uptake experiments in C2C12 myocytes and protein tyrosine phosphatase 1B (PTP1B) enzyme assays. RESULTS: Thirty-six compounds were tentatively identified based on their retention times, UV spectra, MS fragments and data from literature. They comprise phenolcarboxylic acids, flavonoids, iridoid glycosides, and phenylpropanoids, among which acetylharpagide, ajugoside, lavandulifolioside, and verbascoside were selected for quantitation. The methanol extract contained 0.42% combined iridoids, and 1.58% combined phenylpropanoids. Validation showed good accuracy, intermediate precision and robustness. The methanol extract of Leonurus sibiricus led to a 1.5 fold increase in insulin-stimulated cellular glucose uptake and inhibition of PTP1B by 40% at a concentration of 10 µg/mL. CONCLUSION: HPLC-CAD analysis allowed sensitive quantitation of the selected marker compounds in Leonurus sibiricus, thereby providing a reliable tool for its quality control.

Natural Products to Counteract the Epidemic of Cardiovascular and Metabolic Disorders.

Natural products have always been exploited to promote health and served as a valuable source for the discovery of new drugs. In this review, the great potential of natural compounds and medicinal plants for the treatment or prevention of cardiovascular and metabolic disorders, global health problems with rising prevalence, is addressed. Special emphasis is laid on natural products for which efficacy and safety have already been proven and which are in clinical trials, as well as on plants used in traditional medicine. Potential benefits from certain dietary habits and dietary constituents, as well as common molecular targets of natural products, are also briefly discussed. A glimpse at the history of statins and biguanides, two prominent representatives of natural products (or their derivatives) in the fight against metabolic disease, is also included. The present review aims to serve as an "opening" of this special issue of Molecules, presenting key historical developments, recent advances, and future perspectives outlining the potential of natural products for prevention or therapy of cardiovascular and metabolic disease.