3'sialyllactose and 6'sialyllactose enhance performance in endurance-type exercise through metabolic adaptation.

Human milk oligosaccharides (HMOs) belong to a group of multifunctional glycans that are abundantly present in human breast milk. While health effects of neutral oligosaccharides have been investigated extensively, a lot remains unknown regarding health effects of acidic oligosaccharides, such as the two sialyllactoses (SLs), 3'sialyllactose (3'SL), and 6'sialyllactose (6'SL). We utilized Caenorhabditis elegans (C. elegans) to investigate the effects of SLs on exercise performance. Using swimming as an endurance-type exercise, we found that SLs decrease exhaustion, signifying an increase in endurance that is strongest for 6'SL. Through an unbiased metabolomics approach, we identified changes in energy metabolism that correlated with endurance performance. Further investigation suggested that these metabolic changes were related to adaptations of muscle mitochondria that facilitated a shift from beta oxidation to glycogenolysis during exercise. We found that the effect of SLs on endurance performance required AMPK- (aak-1/aak-2) and adenosine receptor (ador-1) signaling. We propose a model where SLs alter the metabolic status in the gut, causing a signal from the intestine to the nervous system toward muscle cells, where metabolic adaptation increases exercise performance. Together, our results underline the potential of SLs in exercise-associated health and contribute to our understanding of the molecular processes involved in nutritionally-induced health benefits.

Synergistic Inhibiting Effect of Phytochemicals in Rheum palmatum on Tyrosinase Based on Metabolomics and Isobologram Analyses.

Tyrosinase (TYR) plays a key role in the enzymatic reaction that is responsible for a range of unwanted discoloration effects, such as food browning and skin hyperpigmentation. TYR inhibitors could, therefore, be candidates for skin care products that aim to repair pigmentation problems. In this study, we used a metabolomics approach combined with the isobologram analysis to identify anti-TYR compounds within natural resources, and evaluate their possible synergism with each other. Rheum palmatum was determined to be a model plant for observing the effect, of which seven extracts with diverse phytochemicals were prepared by way of pressurized solvent extraction. Each Rheum palmatum extract (RPE) was profiled using nuclear magnetic resonance spectroscopy and its activity of tyrosinase inhibition was evaluated. According to the orthogonal partial least square analysis used to correlate phytochemicals in RPE with the corresponding activity, the goodness of fit of the model (R2 = 0.838) and its predictive ability (Q2 = 0.711) were high. Gallic acid and catechin were identified as the active compounds most relevant to the anti-TYR effect of RPE. Subsequently, the activity of gallic acid and catechin were evaluated individually, and when combined in various ratios by using isobologram analysis. The results showed that gallic acid and catechin in the molar ratios of 9:5 and 9:1 exhibited a synergistic inhibition on TYR, with a combination index lower than 0.77, suggesting that certain combinations of these compounds may prove effective for use in cosmetic, pharmaceutical, and food industries.

C. elegans as a test system to study relevant compounds that contribute to the specific health-related effects of different cannabis varieties.

BACKGROUND: The medicinal effects of cannabis varieties on the market cannot be explained solely by the presence of the major cannabinoids Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Evidence for putative entourage effects caused by other compounds present in cannabis is hard to obtain due to the subjective nature of patient experience data. Caenorhabditis elegans (C. elegans) is an objective test system to identify cannabis compounds involved in claimed health and entourage effects. METHODS: From a medicinal cannabis breeding program by MariPharm BV, the Netherlands a set of 12 varieties were selected both THC rich varieties as well as CBD rich varieties. A consecutive extraction process was applied resulting in a non-polar (cannabinoid-rich) and polar (cannabinoid-poor) extract of each variety. The test model C. elegans was exposed to these extracts in a broad set of bioassays for appetite control, body oscillation, motility, and nervous system function. RESULTS: Exposing C. elegans to extracts with a high concentration of cannabinoids (> 1 µg/mL) reduces the life span of C. elegans dramatically. Exposing the nematodes to the low-cannabinoid (< 0.005 µg/mL) polar extracts, however, resulted in significant effects with respect to appetite control, body oscillation, motility, and nervous system-related functions in a dose-dependent and variety-dependent manner. DISCUSSION: C. elegans is a small, transparent organism with a complete nervous system, behavior and is due to its genetic robustness and short life cycle highly suitable to unravel entourage effects of Cannabis compounds. Although C. elegans lacks an obvious CB1 and CB2 receptor it has orthologs of Serotonin and Vanilloid receptor which are also involved in (endo)cannabinoid signaling. CONCLUSION: By using C. elegans, we were able to objectively distinguish different effects of different varieties despite the cannabinoid content. C. elegans seems a useful test system for studying entourage effects, for targeted medicinal cannabis breeding programs and product development.

Extracting relevant physiological information from polar auxin transport data in Panax ginseng.

BACKGROUND: Measuring polar auxin transport (PAT) in plants and drawing conclusions from the observed transport data is only meaningful if these data are being analysed with a mathematical model which describes PAT. In this report we studied the polar auxin transport in Panax ginseng stems of different age and grown on different substrates. METHODS: We measured polar IAA transport in stems using a radio labelled IAA and analysed the transport data with a mathematical model we developed for Arabidopsis. RESULTS: We found that PAT in ginseng stems, as compared to Arabidopsis inflorescence stems, has a 2-fold lower transport velocity and a 3-fold lower steady state auxin flux. CONCLUSION: We were able to pinpoint two physiological parameters that influenced the observed transport characteristics in ginseng which differ from Arabidopsis, namely an increase in immobilization together with a reduced reflux of IAA from the surrounding tissue back to the transporting cells.

Characterization of ginsenoside extracts by delayed luminescence, high-performance liquid chromatography, and bioactivity tests.

Ginsenoside extracts are often used as raw materials for various pharmaceutical, cosmetic and food supplement products. Development of a direct, rapid, cheap, and comprehensive measurement tool for the quality assessment of ginsenoside extracts, and indeed all herbal extracts, is urgently needed. In addition, a bioactivity-based assessment should be linked with quality control. In this report, we try to develop a novel quality control tool using ginsenoside extracts as an example. High-performance liquid chromatography (HPLC) was used to detect nine principal ginsenosides in 11 batches of ginsenoside extracts. Delayed luminescence (DL) was used to analyze the same ginsenoside extract samples. DL measurements showed the same results in terms of differentiating 11 ginsenoside extract samples compared with chemical analysis, and DL properties could be closely linked to index ginsenosides in the quality control of ginsenoside extracts. Next, a zebrafish tail-fin amputation model was used to study differences in anti-inflammatory effect between the ginsenoside extract batches. The results indicate that both chemical analysis and DL measurements could partially reflect biological activity. Thus, DL may serve as a rapid, direct, sensitive, and systemic tool for studying the overall properties of ginsenoside extracts. Our proposal for linking bioactivities as a tool for evaluation of the quality of ginsenoside extracts opens a new direction for quality control.

Broad range chemical profiling of natural deep eutectic solvent extracts using a high performance thin layer chromatography-based method.

Natural deep eutectic solvents (NADES) made mainly with abundant primary metabolites are being increasingly applied in green chemistry. The advantages of NADES as green solvents have led to their use in novel green products for the food, cosmetics and pharma markets. However, one of the main difficulties encountered in the development of novel products and their quality control arises from their low vapour pressure and high viscosity. These features create the need for the development of new analytical methods suited to this type of sample. In this study, such a method was developed and applied to analyse the efficiency of a diverse set of NADES for the extraction of compounds of interest from two model plants, Ginkgo biloba and Panax ginseng. The method uses high-performance thin-layer chromatography (HPTLC) coupled with multivariate data analysis (MVDA). It was successfully applied to the comparative quali- and quantitative analysis of very chemically diverse metabolites (e.g., phenolics, terpenoids, phenolic acids and saponins) that are present in the extracts obtained from the plants using six different NADES. The composition of each NADES was a combination of two or three compounds mixed in defined molar ratios; malic acid-choline chloride (1:1), malic acid-glucose (1:1), choline chloride-glucose (5:2), malic acid-proline (1:1), glucose-fructose-sucrose (1:1:1) and glycerol-proline-sucrose (9:4:1). Of these mixtures, malic acid-choline chloride (1:1) and glycerol-proline-sucrose (1:1:1) for G. biloba leaves, and malic acid-choline chloride (1:1) and malic acid-glucose (1:1) for P. ginseng leaves and stems showed the highest yields of the target compounds. Interestingly, none of the NADES extracted ginkgolic acids as much as the conventional organic solvents. As these compounds are considered to be toxic, the fact that these NADES produce virtually ginkgolic acid-free extracts is extremely useful. The effect of adding different volumes of water to the most efficient NADES was also evaluated and the results revealed that there is a great influence exerted by the water content, with maximum yields of ginkgolides, phenolics and ginsenosides being obtained with approximately 20% water (w/w).

Plant-derived food ingredients for stimulation of energy expenditure.

The development of obesity is related to the regulation of energy intake, energy expenditure, and energy storage in the body. Increasing energy expenditure by inducing lipolysis followed by fat oxidation is one of the alternatives which could help to reverse this increasingly widespread condition. Currently, there is no approved drug targeting on stimulation of energy expenditure available. The use of herbal medicines has become a preferred alternative, supported by the classical consensus on the innocuity of herbal medicine vs synthetic drugs, something that often lacks a scientific basis (ban on Ephedra, for example). The inclusion of functional food in the daily diet has also been promoted although its efficacy requires further investigation. This review summarizes the results of recent work focused on the investigation of edible plant materials targeted at various important pathways related to stimulation of energy expenditure. The aim is to evaluate a number of plants that may be of interest for further studies because of their potential to provide novel lead compounds or functional foods which may be used to combat obesity, but require further studies to evaluate their antiobesity activity in humans.

Red wines attenuate TNFα production in human histiocytic lymphoma cell line: an NMR spectroscopy and chemometrics based study.

Nuclear magnetic resonance (NMR) spectroscopy and multivariate data analyses methods are applied to the metabolic profiling of different red wines from Portugal. The water, methanol-water (1:1), and methanol fractions from solid phase extraction (SPE) with C18 resin were subjected to in vitro TNFα activity assay. Principal component analysis allowed the clear separation among the different SPE fractions according to the activity. Various supervised data reduction algorithms were tested and compared to highlight the TNFα inhibition by SPE fractions of wines. Partial least squares-discriminant analysis and orthogonal bidirectional PLS-DA were found most effective in discriminating the samples with different activities. By calculating variable importance in the projection, the active ingredients in the high activity samples have been identified as caftaric acid, quercetin, and catechin. Among the different vintages, samples from 2010 vintage were found with maximum anti-TNFα activity. The effectiveness of NMR spectroscopy in combination with chemometrics to identify the possible bioactivity in the several crude extracts is highlighted.

NMR spectroscopy and chemometrics as a tool for anti-TNFα activity screening in crude extracts of grapes and other berries.

The identification of active ingredients in crude plant extracts offers great advantages. In this study, nuclear magnetic resonance and chemometrics were used for the screening of in vitro anti-TNFα activity in different berry types. Solid phase extraction was applied and the resulting water, methanol-water (1:1), and methanol fractions were tested for the activity. The methanol-water fraction contained most of the phenolics and showed significantly higher activity than the other two fractions. In the second phase of this study, grapes from 'Trincadeira', 'Touriga Nacional', and 'Aragonês', at four developmental stages were metabolically classified and tested for the TNFα inhibition. The initial stages of grape development, green and veraison, were found more active against TNFα production as compared to the later ripe and harvest stages. Among the cultivars, 'Touriga Nacional' was found to be the most potent inhibitor. Different multivariate data analyses algorithms based on projections to latent structures were applied to correlate the NMR and TNFα inhibition data. The variable importance in the projections plot showed that phenolics like quercetin, myricetin, (+)-catechin, (-)-epicatechin, caftarate, and coutarate, were positively correlated with high activity. This work demonstrates the great potential of NMR spectroscopy in combination with chemometrics for the screening of large set of crude extracts, to study the effects of different variables on the activity, and identifying active compounds in complex mixtures like plant extracts. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-012-0406-8) contains supplementary material, which is available to authorized users.

Lipidomics reveals multiple pathway effects of a multi-components preparation on lipid biochemistry in ApoE*3Leiden.CETP mice.

BACKGROUND: Causes and consequences of the complex changes in lipids occurring in the metabolic syndrome are only partly understood. Several interconnected processes are deteriorating, which implies that multi-target approaches might be more successful than strategies based on a limited number of surrogate markers. Preparations from Chinese Medicine (CM) systems have been handed down with documented clinical features similar as metabolic syndrome, which might help developing new intervention for metabolic syndrome. The progress in systems biology and specific animal models created possibilities to assess the effects of such preparations. Here we report the plasma and liver lipidomics results of the intervention effects of a preparation SUB885C in apolipoprotein E3 Leiden cholesteryl ester transfer protein (ApoE*3Leiden.CETP) mice. SUB885C was developed according to the principles of CM for treatment of metabolic syndrome. The cannabinoid receptor type 1 blocker rimonabant was included as a general control for the evaluation of weight and metabolic responses. METHODOLOGY/PRINCIPAL FINDINGS: ApoE*3Leiden.CETP mice with mild hypercholesterolemia were divided into SUB885C-, rimonabant- and non-treated control groups. SUB885C caused no weight loss, but significantly reduced plasma cholesterol (-49%, p<0.001), CETP levels (-31%, p<0.001), CETP activity (-74%, p<0.001) and increased HDL-C (39%, p<0.05). It influenced lipidomics classes of cholesterol esters and triglycerides the most. Rimonabant induced a weight loss (-9%, p<0.05), but only a moderate improvement of lipid profiles. In vitro, SUB885C extract caused adipolysis stimulation and adipogenesis inhibition in 3T3-L1 cells. CONCLUSIONS: SUB885C, a multi-components preparation, is able to produce anti-atherogenic changes in lipids of the ApoE*3Leiden.CETP mice, which are comparable to those obtained with compounds belonging to known drugs (e.g. rimonabant, atorvastatin, niacin). This study successfully illustrated the power of lipidomics in unraveling intervention effects and to help finding new targets or ingredients for lifestyle-related metabolic abnormality.

Screening of selected Asian spices for anti obesity-related bioactivities.

The potential health effects of 30 spices, commonly used for daily consumption, were submitted to bioactivity screening with several anti-obesity related bioassays: adenosine A1 receptor binding, cannabinoid CB1 receptor binding, TNF-α and 3T3-L1 adipocytes differentiation induction. Sesame seed and red chilli exhibited high binding activity to the adenosine A1 receptor and nutmeg, mace, black pepper and turmeric to the cannabinoid CB1 receptor, while piment and turmeric showed high inhibition of TNF-α accumulation. Black onion seed proved to be the only spice with high 3T3-L1 adipocyte differentiation induction activity. Several well known major compounds found in these active spices were tested with the respective bioassays but did not show activity. Thus, it appears that other minor compounds or the synergistic effects of different constituents are responsible for the observed activity.

Unheated Cannabis sativa extracts and its major compound THC-acid have potential immuno-modulating properties not mediated by CB1 and CB2 receptor coupled pathways.

There is a great interest in the pharmacological properties of cannabinoid like compounds that are not linked to the adverse effects of Delta(9)-tetrahydrocannabinol (THC), e.g. psychoactive properties. The present paper describes the potential immuno-modulating activity of unheated Cannabis sativa extracts and its main non-psychoactive constituent Delta(9)-tetrahydrocanabinoid acid (THCa). By heating Cannabis extracts, THCa was shown to be converted into THC. Unheated Cannabis extract and THCa were able to inhibit the tumor necrosis factor alpha (TNF-alpha) levels in culture supernatants from U937 macrophages and peripheral blood macrophages after stimulation with LPS in a dose-dependent manner. This inhibition persisted over a longer period of time, whereas after prolonged exposure time THC and heated Cannabis extract tend to induce the TNF-alpha level. Furthermore we demonstrated that THCa and THC show distinct effects on phosphatidylcholine specific phospholipase C (PC-PLC) activity. Unheated Cannabis extract and THCa inhibit the PC-PLC activity in a dose-dependent manner, while THC induced PC-PLC activity at high concentrations. These results suggest that THCa and THC exert their immuno-modulating effects via different metabolic pathways.

The RETINOBLASTOMA-RELATED gene regulates stem cell maintenance in Arabidopsis roots.

The maintenance of stem cells in defined locations is crucial for all multicellular organisms. Although intrinsic factors and signals for stem cell fate have been identified in several species, it has remained unclear how these connect to the ability to reenter the cell cycle that is one of the defining properties of stem cells. We show that local reduction of expression of the RETINOBLASTOMA-RELATED (RBR) gene in Arabidopsis roots increases the amount of stem cells without affecting cell cycle duration in mitotically active cells. Conversely, induced RBR overexpression dissipates stem cells prior to arresting other mitotic cells. Overexpression of D cyclins, KIP-related proteins, and E2F factors also affects root stem cell pool size, and genetic interactions suggest that these factors function in a canonical RBR pathway to regulate somatic stem cells. Expression analysis and genetic interactions position RBR-mediated regulation of the stem cell state downstream of the patterning gene SCARECROW.

Metabolomics in the context of systems biology: bridging traditional Chinese medicine and molecular pharmacology.

The introduction of the concept of systems biology, enabling the study of living systems from a holistic perspective based on the profiling of a multitude of biochemical components, opens up a unique and novel opportunity to reinvestigate natural products. In the study of their bioactivity, the necessary reductionistic approach on single active components has been successful in the discovery of new medicines, but at the same time the synergetic effects of components were lost. Systems biology, and especially metabolomics, is the ultimate phenotyping. It opens up the possibility of studying the effect of complex mixtures, such as those used in Traditional Chinese Medicine, in complex biological systems; abridging it with molecular pharmacology. This approach is considered to have the potential to revolutionize natural product research and to advance the development of scientific based herbal medicine.

Programmed cell death during the transition from multicellular structures to globular embryos in barley androgenesis.

Androgenesis represents one of the most fascinating examples of cell differentiation in plants. In barley, the conversion of stressed uninucleate microspores into embryo-like structures is highly efficient. One of the bottlenecks in this process is the successful release of embryo-like structures out of the exine wall of microspores. In the present work, morphological and biochemical studies were performed during the transition from multicellular structures to globular embryos. Exine wall rupture and subsequent globular embryo formation were observed only in microspores that divided asymmetrically. Independent divisions of the generative and the vegetative nuclei gave rise to heterogeneous multicellular structures, which were composed of two different cellular domains: small cells with condensed chromatin structure and large cells with normal chromatin structure. During exine wall rupture, the small cells died and their death marked the site of exine wall rupture. Cell death in the small cell domain showed typical features of plant programmed cell death. Chromatin condensation and DNA degradation preceded cell detachment and cytoplasm dismantling, a process that was characterized by the formation of vesicles and vacuoles that contained cytoplasmic material. This morphotype of programmed cell death was accompanied by an increase in the activity of caspase-3-like proteases. The orchestration of such a death program culminated in the elimination of the small generative domain, and further embryogenesis was carried out by the large vegetative domain. To date, this is the first report to show evidence that programmed cell death takes part in the development of microspore-derived embryos.

A tormentor in the quest for plant p53-like proteins.

Over the past few years the presence of p53-like proteins in plants was frequently reported, by using the monoclonal antibody Pab240. By means of protein purification and screening a cDNA library, a Pab240 cross-reacting protein and a cDNA clone were isolated from barley. Peptide- and DNA-sequence analysis identified one and the same protein: 2-oxoglutarate dehydrogenase. Sequence analysis of 2-oxoglutarate dehydrogenase revealed that the protein contains a perfect Pab240 epitope. In barley, the 110 kDa oxoglutarate dehydrogenase was degraded during isolation to a 53 kDa Pab240 cross-reacting polypeptide, thereby mimicking curiously p53-like properties.

14-3-3 proteins and plant development.

The 14-3-3 proteins are a family of ubiquitous regulatory molecules which have been found in virtually every eukaryotic organism and tissue. Discovered 34 years ago, 14-3-3 proteins have first been studied in mammalian nervous tissues, but in the past decade their indispensable role in various plant regulatory and metabolic pathways has been increasingly established. We now know that 14-3-3 members regulate fundamental processes of nitrogen assimilation and carbon assimilation, play an auxiliary role in regulation of starch synthesis, ATP production, peroxide detoxification, and participate in modulation of several other important biochemical pathways. Plant development and seed germination appear also to be under control of factors whose interaction with 14-3-3 molecules is crucial for their activation. Located within the nucleus, 14-3-3 isoforms are constituents of transcription factor complexes and interact with components of abscisic acid (ABA)-induced gene expression machinery. In addition, in animal cells they participate in nucleo-cytoplasmic trafficking and molecular sequestration. Cytoplasmic 14-3-3 members form a guidance complex with chloroplast destined preproteins and facilitate their import into these photosynthetic organelles. Recently, several 14-3-3s have been identified within chloroplasts where they could be involved in targeting and insertion of thylakoid proteins. The identification of 14-3-3 isoform specificity, and in particular the elucidation of the signal transduction mechanisms connecting 14-3-3 members with physiological responses, are central and developing topics of current research in this field.