Western diet-induced cognitive and metabolic dysfunctions in aged mice are prevented by rosmarinic acid in a sex-dependent fashion.

BACKGROUND & AIMS: Unhealthy lifestyles, such as chronic consumption of a Western Diet (WD), have been associated with increased systemic inflammation and oxidative stress (OS), a condition that may favour cognitive dysfunctions during aging. Polyphenols, such as rosmarinic acid (RA) may buffer low-grade inflammation and OS, characterizing the aging brain that is sustained by WD, promoting healthspan. The aim of this study was to evaluate the ability of RA to prevent cognitive decline in a mouse model of WD-driven unhealthy aging and to gain knowledge on the specific molecular pathways modulated within the brain. METHODS: Aged male and female C57Bl/6N mice were supplemented either with RA or vehicle for 6 weeks. Following 2 weeks on RA they started being administered either with WD or control diet (CD). Successively all mice were tested for cognitive abilities in the Morris water maze (MWM) and emotionality in the elevated plus maze (EPM). Glucose and lipid homeostasis were assessed in trunk blood while the hippocampus was dissected out for RNAseq transcriptomic analysis. RESULTS: RA prevented insulin resistance in males while protecting both males and females from WD-dependent memory impairment. In the hippocampus, RA modulated OS pathways in males and immune- and sex hormones-related signalling cascades (Lhb and Lhcgr genes) in females. Moreover, RA overall resulted in an upregulation of Glp1r, recently identified as a promising target to prevent metabolic derangements. In addition, we also found an RA-dependent enrichment in nuclear transcription factors, such as NF-κB, GR and STAT3, that have been recently suggested to promote healthspan and longevity by modulating inflammatory and cell survival pathways. CONCLUSIONS: Oral RA supplementation may promote brain and metabolic plasticity during aging through antioxidant and immune-modulating properties possibly affecting the post-reproductive hormonal milieu in a sex-dependent fashion. Thus, its supplementation should be considered in the context of precision medicine as a possible strategy to preserve cognitive functions and to counteract metabolic derangements.

Rosmarinic Acid Improves Cognitive Abilities and Glucose Metabolism in Aged C57Bl/6N Mice While Disrupting Lipid Profile in Young Adults in a Sex-Dependent Fashion.

A growing body of evidence suggests that regular consumption of natural products might promote healthy aging; however, their mechanisms of action are still unclear. Rosmarinic acid (RA) is a polyphenol holding anti-inflammatory, antioxidant and neuroprotective properties. The aim of this study was to characterise the efficacy of an oral administration of RA in promoting healthspan in a mouse model of physiological aging. Aged C57Bl/6 male and female (24-month-old) mice were either administered with RA (500 mg/Kg) or a vehicle in drinking bottles for 52 days while 3-month-old mice receiving the same treatment were used as controls. All subjects were assessed for cognitive abilities in the Morris water maze (MWM) and for emotionality in the elevated-plus maze test (EPM). Brain-derived Neurotrophic Factor (BDNF) protein levels were evaluated in the hippocampus. Since the interaction between metabolic signals and cerebral functions plays a pivotal role in the etiopathogenesis of cognitive decline, the glycaemic and lipid profiles of the mice were also assessed. RA enhanced learning and memory in 24-month-old mice, an effect that was associated to improved glucose homeostasis. By contrast, the lipid profile was disrupted in young adults. This effect was associated with worse glycaemic control in males and with reduced BDNF levels in females, suggesting powerful sex-dependent effects and raising a note of caution for RA administration in young healthy adult subjects.

Ion-Pairing Chromatography and Amine Derivatization Provide Complementary Approaches for the Targeted LC-MS Analysis of the Polar Metabolome.

Liquid chromatography coupled to mass spectrometry is a key metabolomics/metabonomics technology. Reversed-phase liquid chromatography (RPLC) is very widely used as a separation step, but typically has poor retention of highly polar metabolites. Here, we evaluated the combination of two alternative methods for improving retention of polar metabolites based on 6-aminoquinoloyl-N-hydroxysuccinidimyl carbamate derivatization for amine groups, and ion-pairing chromatography (IPC) using tributylamine as an ion-pairing agent to retain acids. We compared both of these methods to RPLC and also to each other, for targeted analysis using a triple-quadrupole mass spectrometer, applied to a library of ca. 500 polar metabolites. IPC and derivatization were complementary in terms of their coverage: combined, they improved the proportion of metabolites with good retention to 91%, compared to just 39% for RPLC alone. The combined method was assessed by analyzing a set of liver extracts from aged male and female mice that had been treated with the polyphenol compound ampelopsin. Not only were a number of significantly changed metabolites detected, but also it could be shown that there was a clear interaction between ampelopsin treatment and sex, in that the direction of metabolite change was opposite for males and females.

Novel mannosylerythritol lipid biosurfactant structures from castor oil revealed by advanced structure analysis.

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by fungi of the Ustilaginaceae family in the presence of hydrophobic carbon sources like plant oils. In the present study, we investigated the structural composition of MELs produced from castor oil using seven different microorganisms and compared them to MEL structures resulting from other plant oils. Castor oil is an industrially relevant plant oil that presents as an alternative to currently employed edible plant oils like rapeseed or soybean oil. The main fatty acid in castor oil is the mono-hydroxylated ricinoleic acid, providing the possibility to produce novel MEL structures with interesting features. Analysis of the produced MELs from castor oil by different chromatographic and mass spectrometry techniques revealed that all seven microorganisms were generally able to integrate hydroxylated fatty acids into the MEL molecule, although at varying degrees. These novel MELs containing a hydroxy fatty acid (4-O-[2'-O-alka(e)noyl-3'-O-hydroxyalka(e)noyl-4'/6'-O-acetyl-ß-D-mannopyranosyl]-erythritol) were more hydrophilic than conventional MEL and therefore showed a different elution behavior in chromatography. Large shares of novel hydroxy MELs (around 50% of total MELs) were found for the two MEL-B/C producing species Ustilago siamensis and Ustilago shanxiensis, but also for the MEL-A/B/C producer Moesziomyces aphidis (around 25%). In addition, tri-acylated hydroxylated MELs with a third long-chain fatty acid esterified to the free hydroxyl group of the hydroxy fatty acid were identified for some species. Overall, production of MEL from castor oil with the investigated organisms provided a complex mixture of various novel MEL structures that can be exploited for further research.

Tonantzitlolone is a nanomolar potency activator of transient receptor potential canonical 1/4/5 channels.

BACKGROUND AND PURPOSE: The diterpene ester tonantzitlolone (TZL) is a natural product, which displays cytotoxicity towards certain types of cancer cell such as renal cell carcinoma cells. The effect is similar to that of (-)-englerin A, and so, although it is chemically distinct, we investigated whether TZL also targets transient receptor potential canonical (TRPC) channels of the 1, 4 and 5 type (TRPC1/4/5 channels). EXPERIMENTAL APPROACH: The effects of TZL on renal cell carcinoma A498 cells natively expressing TRPC1 and TRPC4, modified HEK293 cells overexpressing TRPC4, TRPC5, TRPC4-TRPC1 or TRPC5-TRPC1 concatemer, TRPC3 or TRPM2, or CHO cells overexpressing TRPV4 were studied by determining changes in intracellular Ca2+ , or whole-cell or excised membrane patch-clamp electrophysiology. KEY RESULTS: TZL induced an elevation of intracellular Ca2+ in A498 cells, similar to that evoked by englerin A. TZL activated overexpressed channels with EC50 values of 123 nM (TRPC4), 83 nM (TRPC5), 140 nM (TRPC4-TRPC1) and 61 nM (TRPC5-TRPC1). These effects of TZL were reversible on wash-out and potently inhibited by the TRPC1/4/5 inhibitor Pico145. TZL activated TRPC5 channels when bath-applied to excised outside-out but not inside-out patches. TZL failed to activate endogenous store-operated Ca2+ entry or overexpressed TRPC3, TRPV4 or TRPM2 channels in HEK 293 cells. CONCLUSIONS AND IMPLICATIONS: TZL is a novel potent agonist for TRPC1/4/5 channels, which should be useful for testing the functionality of this type of ion channel and understanding how TRPC1/4/5 agonists achieve selective cytotoxicity against certain types of cancer cell.

Natural 6-hydroxy-chromanols and -chromenols: structural diversity, biosynthetic pathways and health implications.

We present the first comprehensive and systematic review on the structurally diverse toco-chromanols and -chromenols found in photosynthetic organisms, including marine organisms, and as metabolic intermediates in animals. The focus of this work is on the structural diversity of chromanols and chromenols that result from various side chain modifications. We describe more than 230 structures that derive from a 6-hydroxy-chromanol- and 6-hydroxy-chromenol core, respectively, and comprise di-, sesqui-, mono- and hemiterpenes. We assort the compounds into a structure-activity relationship with special emphasis on anti-inflammatory and anti-carcinogenic activities of the congeners. This review covers the literature published from 1970 to 2017.

Flavonol Glycosides and Cytotoxic Steroidal Saponins from Furcraea tuberosa (Agavaceae).

Phytochemical analysis of the mature fruits of Furcraea tuberosa (Agavaceae) resulted in the isolation of a new bisdesmosidic spirostanol saponin (1), along with eight known steroidal glycosides (2-9), one known phenolic carboxylic acid ester (10) and three known flavonol glycosides (11-13). The structures of these compounds were assigned using a combination of ID and 2D NMR techniques including ¹H, ¹³C, COSY, TOCSY, HSQC and HMBC NMR, and confirmed by mass spectrometry. Thus the new saponin was elucidated as (25R)-6α-(ß-D-glucopyranosyloxy)-5α-spirostane-3ß-Ο-[(6-Ο-hexadecanoyl)-ß-D- glucopyranoside]. The literature survey revealed that most of the steroidal saponins isolated have shown potent cytotoxic effects against various human cancer cell lines and the results are herein reviewed.

Sesquiterpene Lactone Composition and Cellular Nrf2 Induction of Taraxacum officinale Leaves and Roots and Taraxinic Acid ß-d-Glucopyranosyl Ester.

Taraxacum officinale, the common dandelion, is a plant of the Asteraceae family, which is used as a food and medical herb. Various secondary plant metabolites such as sesquiterpene lactones, triterpenoids, flavonoids, phenolic acids, coumarins, and steroids have been described to be present in T. officinale. Dandelion may exhibit various health benefits, including antioxidant, anti-inflammatory, and anticarcinogenic properties. We analyzed the leaves and roots of the common dandelion (T. officinale) using high-performance liquid chromatography/mass spectrometry to determine its sesquiterpene lactone composition. The main compound of the leaf extract taraxinic acid ß-d-glucopyranosyl ester (1), a sesquiterpene lactone, was isolated and the structure elucidation was conducted by nuclear magnetic resonance spectrometry. The leaf extract and its main compound 1 activated the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in human hepatocytes more significantly than the root extract. Furthermore, the leaf extract induced the Nrf2 target gene heme oxygenase 1. Overall, present data suggest that compound 1 may be one of the active principles of T. officinale.

Long-Lasting WNT-TCF Response Blocking and Epigenetic Modifying Activities of Withanolide F in Human Cancer Cells.

The WNT-TCF signaling pathway participates in adult tissue homeostasis and repair, and is hyperactive in a number of human diseases including cancers of the colon. Whereas to date there are no antagonists approved for patient use, a potential problem for their sustained use is the blockade of WNT signaling in healthy tissues, thus provoking potentially serious co-lateral damage. Here we have screened a library of plant and microorganism small molecules for novel WNT signaling antagonists and describe withanolide F as a potent WNT-TCF response blocker. This steroidal lactone inhibits TCF-dependent colon cancer xenograft growth and mimics the effects of genetic blockade of TCF and of ivermectin, a previously reported WNT-TCF blocker. However, withanolide F is unique in that it imposes a long-lasting repression of tumor growth, WNT-TCF targets and cancer stem cell clonogenicity after drug treatment. These findings are paralleled by its modulation of chromatin regulators and its alteration of overall H3K4me1 levels. Our results open up the possibility to permanently repress essential signaling responses in cancer cells through limited treatments with small molecules.

Synthesis and antiproliferative activity of new tonantzitlolone-derived diterpene derivatives.

The synthesis of the diterpene (+)-tonantzitlolone A and a series of derivatives is reported. The study includes the determination of their antiproliferative activities against selected cancer cell lines.

Amorfrutin C Induces Apoptosis and Inhibits Proliferation in Colon Cancer Cells through Targeting Mitochondria.

A known (1) and a structurally related new natural product (2), both belonging to the amorfrutin benzoic acid class, were isolated from the roots of Glycyrrhiza foetida. Compound 1 (amorfrutin B) is an efficient agonist of the nuclear peroxisome proliferator activated receptor (PPAR) gamma and of other PPAR subtypes. Compound 2 (amorfrutin C) showed comparably lower PPAR activation potential. Amorfrutin C exhibited striking antiproliferative effects for human colorectal cancer cells (HT-29 and T84), prostate cancer (PC-3), and breast cancer (MCF7) cells (IC50 values ranging from 8 to 16 µM in these cancer cell lines). Notably, amorfrutin C (2) showed less potent antiproliferative effects in primary colon cells. For HT-29 cells, compound 2 induced G0/G1 cell cycle arrest and modulated protein expression of key cell cycle modulators. Amorfrutin C further induced apoptotic events in HT-29 cells, including caspase activation, DNA fragmentation, PARP cleavage, phosphatidylserine externalization, and formation of reactive oxygen species. Mechanistic studies revealed that 2 disrupts the mitochondrial integrity by depolarization of the mitochondrial membrane (IC50 0.6 µM) and permanent opening of the mitochondrial permeability transition pore, leading to increased mitochondrial oxygen consumption and extracellular acidification. Structure-activity-relationship experiments revealed the carboxylic acid and the hydroxy group residues of 2 as fundamental structural requirements for inducing these apoptotic effects. Synergy analyses demonstrated stimulation of the death receptor signaling pathway. Taken together, amorfrutin C (2) represents a promising lead for the development of anticancer drugs.

Tonantzitlolone cytotoxicity toward renal cancer cells is PKCθ- and HSF1-dependent.

Elucidating the targets and mechanism of action of natural products is strategically important prior to drug development and assessment of potential clinical applications. In this report, we elucidated the main targets and mechanism of action of the natural product tonantzitlolone (TZL) in clear cell renal cell carcinoma (CCRCC). We identified TZL as a dual PKCα and PKCθ activator in vitro, although in CCRCC cells its activity was mostly PKCθ-dependent. Through activation of PKCθ, TZL induced an insulin resistant phenotype by inhibiting IRS1 and the PI3K/Akt pathway. Simultaneously, TZL activated the heat shock factor 1 (HSF1) transcription factor driving glucose dependency. Thus, similar to the selective PKCθ activator englerin A, TZL induces a metabolic catastrophe in CCRCC, starving cells of glucose while simultaneously increasing their glycolytic dependency.

Cytotoxic saponins from the seeds of Pittosporum angustifolium.

Three new acylated R1-barrigenol triterpene glycosides, 1-3, were isolated from the seeds of Pittosporum angustifolium Lodd. together with four known glycosides, 4-7, containing R1- and A1-barrigenol backbones. On the basis of spectroscopic, spectrometric, and chemical analyses the novel compounds were named pittangretosides N-P and established as 21beta-acetoxy-22alpha-angeloyloxy- (1), 21beta-acetoxy-22alpha-(2-acetoxy-2-methylbutyroyloxy)- (2), and 21beta-(2-methylbutyroyloxy)-22alpha-acetoxy-3beta-[beta-D-glucopyranosyl- (1 --> 2)]-[alpha-L-arabinopyranosyl-(1 --> 3)]-[alpha-L-arabinofuranosyl-(1 --> 4)]-beta-D-glucuronopyranosyloxyolean-12-ene-15alpha, 6alpha, 28-triol (3). Evaluation of the in vitro cytotoxicity against three tumour cell lines and one non-tumourigenic cell line revealed antiproliferative effects with IC50 values in a range of 1.74-34.1 microM.

Protein kinase and HDAC inhibitors from the endophytic fungus Epicoccum nigrum.

A chemical investigation of the endophytic fungus Epicoccum nigrum isolated from leaves of Mentha suaveolens collected in Morocco resulted in the isolation of five new polyketides, epicocconigrones A and B (1 and 2), 3-methoxyepicoccone B (3), 3-methoxyepicoccone (4), and 2,3,4-trihydroxy-6-(methoxymethyl)-5-methylbenzaldehyde (5), together with five known compounds (6-10). The structures of the new compounds were unambiguously determined by extensive analysis of the 1D and 2D NMR and mass spectroscopic data. Compounds 1 and 10 showed potent inhibition of at least 15 protein kinases with IC50 values ranging from 0.07 to 9.00 µM. Moreover, compounds 1 and 10 inhibited histone deacetylase (HDAC) activities with IC50 values of 9.8 and 14.2 µM, respectively. A preliminary structure-activity relationship is discussed. Interestingly, compounds 1 and 10 exert mainly cytostatic effects in human lymphoma RAJI and U-937 cell lines.

Triterpene glycosides from the leaves of Pittosporum angustifolium.

Phytochemical investigation of the leaves of Pittosporum angustifolium resulted in the isolation and structural elucidation of nine new triterpene saponins, named pittangretosides A-I (1-9), together with a known compound (10). Mainly by NMR and HRESIMS experiments, eight compounds were identified as A1-barrigenol glycosides (1-7, 10), whereas two compounds exhibited an unusual 17,22-seco-backbone of oleanolic acid (8, 9). All compounds were evaluated for their in vitro cytotoxicities against human urinary bladder carcinoma cells (5637). Only compounds with an angeloyl-residue at C-22 of the aglycone (1-4 and 10) showed antiproliferative effects with IC50 values of 4.1, 5.2, 2.1, 17.9, and 2.4 µM, respectively.

Structure-activity relationships of saponins enhancing the cytotoxicity of ribosome-inactivating proteins type I (RIP-I).

Saponins are amphiphilic secondary plant compounds able to interfere with surfaces and permeabilize membranes. In addition to antimicrobial and anti-inflammatory features, anti-neoplastic activities are described which base on various mechanisms. A very promising anti-cancer strategy is the synergistic enhancement of the cytotoxicity of ribosome-inactivating proteins type I (RIP-I) by suitable saponins. The cytotoxicity of the naturally low cytotoxic activity showing RIP-I can be augmented up to 100,000-fold, if they are applied in combination with appropriate saponins. These saponins have to hold certain structural features. We analyzed 56 different saponins for their non-synergistic, as well as their synergistic cytotoxicity with the RIP-I saporin from Saponaria officinalis L. (Caryophyllaceae). The saponins were assorted into categories, according to their structural features and their cytotoxic behavior, to build up structure-activity relationships. A non-synergistic cytotoxicity as low as possible and a synergistic cytotoxicity as high as possible were desired. The investigations suggest that ideal saponins consist of an oleanane-type scaffold, preferably gypsogenin or quillaic acid, three branched sugars at the C-3-OH-group including glucuronic acid, galactose, xylose and four branched sugars at the C-28-OH-group, including deoxy-sugars and acetyl-residues.

Characterisation of Nox4 inhibitors from edible plants.

NADPH oxidases transport electrons from cytosolic NADPH through biological membranes to generate reactive oxygen species. NADPH oxidase 4, broadly expressed in humans, is an interesting pharmacological target, since its activity is deregulated in several diseases, including pulmonary fibrosis, diabetic nephropathy, and cardiac hypertrophy. Whereas several candidate NADPH oxidase 4 inhibitors were recently described, most of these compounds are either unspecific or toxic. Here we set out to identify new NADPH oxidase 4 inhibitors from edible plants, in an attempt to decrease the number of hits with toxic side effects. We screened a compound library prepared from edible plants for new bioactives with the ability to inhibit the activity of NADPH oxidase 4. Using both cell-based and cell-free assays, we identified several compounds with significant inhibitory activity towards NADPH oxidase 4. For selected compounds, the activity profile towards NADPH oxidase 2 and NADPH oxidase 5 was established, and controls were carried out to exclude general reactive oxygen species scavengers. A number of promising NADPH oxidase 4 inhibitors from edible plants was identified and characterised. Several new chemical entities are disclosed which act as NADPH oxidase 4 inhibitors, and the efficacies of our best hits, in particular several diarylheptanoids and lignans, are comparable to the best available pharmacological NADPH oxidase 4 inhibitors. These findings will provide valuable tools to study mechanisms of NADPH oxidase inhibition.

Major constituents and cytotoxic effects of Ajuga chamaecistus ssp. tomentella.

The n-butanolic fraction of a methanolic extract (80%) from aerial parts of Ajuga chamaecistus ssp. tomentella was analysed using different chromatographic methods. Column (CC) and high-performance liquid chromatography (HPLC) were used for isolation and purification. 13C, H NMR, H-H COSY, HSQC, HMBC, and ESI-MS were employed for identification of the compounds isolated from this fraction. The structures of the compounds were determined to be cis-melilotoside (1), trans-melilotoside (2), lavandulifolioside (3), 20-hydroxyecdysone (4), leonoside B (5), martynoside (6), ajugalactone (7), makisterone A (8), and 24-dehydroprecyasterone (9). This is the first report on the presence of cis- and trans-melilotoside in Ajuga species. Cytotoxic evaluation of the n-butanolic fraction, cis- and trans-melilotoside against cancer (T47D, HT-29, and Caco-2) and normal (NIH 3T3) cell lines by the mitochondrial tetrazolium test (MTT) showed no cytotoxic effects up to 400 microg/mL. The results of this study suggest that melilotoside, phenylethyl glycosides, and phytoecdysteroids are the main constituents of the n-butanolic fraction of Ajuga chamaecistus ssp. tomentella.

Revisiting old drugs as novel agents for retinoblastoma: in vitro and in vivo antitumor activity of cardenolides.

PURPOSE: Intra-arterial delivery of chemotherapeutic agents offers a new and exciting opportunity for the treatment of advanced intraocular retinoblastoma. It allows local delivery of relatively high doses of chemotherapy agents while bypassing general blood circulation. For this reason, this study was undertaken to revisit some of the FDA-approved drugs for the treatment of retinoblastoma. METHODS: High-throughput screening (HTS) of 2640 approved drugs and bioactive compounds resulted in the identification of cytotoxic agents with potent activity toward both the Y79 and RB355 human retinoblastoma cell lines. Subsequent profiling of the drug candidates was performed in a panel of ocular cancer cell lines. Induction of apoptosis in Y79 cells was assessed by immunofluorescence detection of activated caspase-3. Therapeutic effect was evaluated in a xenograft model of retinoblastoma. RESULTS: Several FDA-approved drugs were identified that showed potent cytotoxic activity toward retinoblastoma cell lines in vitro. Among them were several cardiac glycosides, a class of cardenolides historically associated with the prevention and treatment of congestive heart failure. Caspase-3 activation studies provided an insight into the mechanism of action of cardenolides in retinoblastoma cells. When tested in a xenograft model of retinoblastoma, the cardenolide ouabain induced complete tumor regression in the treated mice. CONCLUSIONS: Cardenolides were identified as a new class of antitumor agents for the treatment of retinoblastoma. Members of this class of cardiotonic drugs could be repositioned for retinoblastoma if administered locally via direct intra-arterial infusion.

Phenylethanoid glycosides from Stachytarpheta cayennensis (Rich. ) Vahl, Verbenaceae, a traditional antimalarial medicinal plant / Glicosídeos feniletanóides de Stachytarpheta cayennensis (Rich. ) Vahl, Verbenaceae, uma planta medicinal tradicional anti-malárica

The boiled juice of Stachytarpheta cayennensis, Verbenaceae, whole plant material is used ethnomedicinally in different countries of Latin-America to treat symptoms of malaria. In the ethyl acetate extract, five phenylethanoid glycosides could be identified. Three of these phenylethanoid glycosides, leucosceptoside A, martynoside and jionoside D were detected for the first time in the genus Stachytarpheta.

O suco fervido de Stachytarpheta cayennensis, Verbenaceae, do material vegetal inteiro é utilizado etnomedicinalmente em diferentes países da América Latina para tratar os sintomas da malária. No extrato acetato de etila, cinco glicosídeos feniletanóides puderam ser identificados. Três destes glicosídeos feniletanóides, leucosceptosídeo A, martinosídeo e jionosídeo D foram detectados pela primeira vez no gênero Stachytarpheta.