Isolating an active and inactive CACTA transposon from lettuce color mutants and characterizing their family.

Dietary flavonoids play an important role in human nutrition and health. Flavonoid biosynthesis genes have recently been identified in lettuce (Lactuca sativa); however, few mutants have been characterized. We now report the causative mutations in Green Super Lettuce (GSL), a natural light green mutant derived from red cultivar NAR; and GSL-Dark Green (GSL-DG), an olive-green natural derivative of GSL. GSL harbors CACTA 1 (LsC1), a 3.9-kb active nonautonomous CACTA superfamily transposon inserted in the 5' untranslated region of anthocyanidin synthase (ANS), a gene coding for a key enzyme in anthocyanin biosynthesis. Both terminal inverted repeats (TIRs) of this transposon were intact, enabling somatic excision of the mobile element, which led to the restoration of ANS expression and the accumulation of red anthocyanins in sectors on otherwise green leaves. GSL-DG harbors CACTA 2 (LsC2), a 1.1-kb truncated copy of LsC1 that lacks one of the TIRs, rendering the transposon inactive. RNA-sequencing and reverse transcription quantitative PCR of NAR, GSL, and GSL-DG indicated the relative expression level of ANS was strongly influenced by the transposon insertions. Analysis of flavonoid content indicated leaf cyanidin levels correlated positively with ANS expression. Bioinformatic analysis of the cv Salinas lettuce reference genome led to the discovery and characterization of an LsC1 transposon family with a putative transposon copy number greater than 1,700. Homologs of tnpA and tnpD, the genes encoding two proteins necessary for activation of transposition of CACTA elements, were also identified in the lettuce genome.

Prenylated Coumaric Acids from Artemisia scoparia Beneficially Modulate Adipogenesis.

Two new diprenylated coumaric acid isomers (1a and 1b) and two known congeners, capillartemisin A (2) and B (3), were isolated from Artemisia scoparia as bioactive markers using bioactivity-guided HPLC fractionation. Their structures were determined by spectroscopic means, including 1D and 2D NMR methods and LC-MS, with their purity assessed by 1D 1H pure shift qNMR spectroscopic analysis. The bioactivity of compounds was evaluated by enhanced accumulation of lipids, as measured using Oil Red O staining, and by increased expression of several adipocyte marker genes, including adiponectin in 3T3-L1 adipocytes relative to untreated negative controls. Compared to the plant's 80% EtOH extract, these purified compounds showed significant but still weaker inhibition of TNFα-induced lipolysis in 3T3-L1 adipocytes. This suggests that additional bioactive substances are responsible for the multiple metabolically favorable effects on adipocytes observed with Artemisia scoparia extract.

Safety and pharmacokinetics of naringenin: A randomized, controlled, single-ascending-dose clinical trial.

AIMS: To evaluate the safety and pharmacokinetics of naringenin in healthy adults consuming whole-orange (Citrus sinensis) extract. METHODS AND METHODS: In a single-ascending-dose randomized crossover trial, 18 adults ingested doses of 150 mg (NAR150), 300 mg (NAR300), 600 mg (NAR600) and 900 mg (NAR900) naringenin or placebo. Each dose or placebo was followed by a wash-out period of at least 1 week. Blood safety markers were evaluated pre-dose and 24 hours post-dose. Adverse events (AEs) were recorded. Serum naringenin concentrations were measured before and over 24 hours following ingestion of placebo, NAR150 and NAR600. Four- and 24-hour serum measurements were obtained after placebo, NAR300 and NAR900 ingestion. Data were analysed using a mixed-effects linear model. RESULTS: There were no relevant AEs or changes in blood safety markers following ingestion of any of the naringenin doses. The pharmacokinetic variables were: maximal concentration: 15.76 ± 7.88 µM (NAR150) and 48.45 ± 7.88 µM (NAR600); time to peak: 3.17 ± 0.74 hours (NAR150) and 2.41 ± 0.74 hours (NAR600); area under the 24-hour concentration-time curve: 67.61 ± 24.36 µM × h (NAR150) and 199.05 ± 24.36 µM × h (NAR600); and apparent oral clearance: 10.21 ± 2.34 L/h (NAR150) and 13.70 ± 2.34 L/h (NAR600). Naringenin half-life was 3.0 hours (NAR150) and 2.65 hours (NAR600). After NAR300 ingestion, serum concentrations were 10.67 ± 5.74 µM (4 hours) and 0.35 ± 0.30 µM (24 hours). After NAR900 ingestion, serum concentrations were 43.11 ± 5.26 µM (4 hours) and 0.24 ± 0.30 µM (24 hours). CONCLUSIONS: Ingestion of 150 to 900 mg doses of naringenin is safe in healthy adults, and serum concentrations are proportional to the dose administered. Since naringenin (8 µM) is effective in primary human adipocytes, ingestion of 300 mg naringenin twice/d will likely elicit a physiological effect.

The DESIGNER Approach Helps Decipher the Hypoglycemic Bioactive Principles of Artemisia dracunculus (Russian Tarragon).

Complementing classical drug discovery, phytochemicals act on multiple pharmacological targets, especially in botanical extracts, where they form complex bioactive mixtures. The reductionist approach used in bioactivity-guided fractionation to identify single bioactive phytochemicals is inadequate for capturing the full therapeutic potential of the (bio)chemical interactions present in such complex mixtures. This study used a DESIGNER (Deplete and Enrich Select Ingredients to Generate Normalized Extract Resources) approach to selectively remove the known bioactives, 4'-O-methyldavidigenin (1; 4,2'-dihydroxy-4'-methoxydihydrochalcone, syn. DMC-1) and its isomer 4-O-methyldavidigenin (2; syn. DMC-2), from the mixture of phytochemicals in an ethanol extract from Artemisia dracunculus to determine to what degree the more abundant 2 accounts for the established antidiabetic effect of the A. dracunculus extract. Using an otherwise chemically intact "knock-out extract" depleted in 2 and its regioisomer, 1, in vitro and in vivo outcomes confirmed that 2 (and likely 1) acts as major bioactive(s) that enhance(s) insulin signaling in skeletal muscle, but also revealed that 2 does not account for the breadth of detectable biological activity of the extract. This is the first report of generating, at a sufficiently large preparative scale, a "knock-out extract" used as a pharmacological tool for in vitro and in vivo studies to dissect the biological impact of a designated bioactive in a complex phytochemical mixture.

Phytoecdysteroids and flavonoid glycosides among Chilean and commercial sources of Chenopodium quinoa: variation and correlation to physico-chemical characteristics.

BACKGROUND: Little is known about varietal differences in the content of bioactive phytoecdysteroids (PE) and flavonoid glycosides (FG) from quinoa (Chenopodium quinoa Willd.). The aim of this study was to determine the variation in PE and FG content among 17 distinct quinoa sources and identify correlations to genotypic (highland vs. lowland) and physico-chemical characteristics (seed color, 100-seed weight, protein content, oil content). RESULTS: PE and FG concentrations exhibited over four-fold differences across quinoa sources, ranging from 138 ± 11 µg g(-1) to 570 ± 124 µg g(-1) total PE content and 192 ± 24 µg g(-1) to 804 ± 91 µg g(-1) total FG content. Mean FG content was significantly higher in highland Chilean varieties (583.6 ± 148.9 µg g(-1)) versus lowland varieties (228.2 ± 63.1 µg g(-1)) grown under the same environmental conditions (P = 0.0046; t-test). Meanwhile, PE content was positively and significantly correlated with oil content across all quinoa sources (r = 0.707, P = 0.002; Pearson correlation). CONCLUSION: FG content may be genotypically regulated in quinoa. PE content may be increased via enhancement of oil content. These findings may open new avenues for the improvement and development of quinoa as a functional food.

Leishmanicidal activity of a daucane sesquiterpene isolated from Eryngium foetidum.

CONTEXT: Eryngium foetidum L. (Apiaceae) is a traditional herb that has been used for numerous medicinal applications, including as a treatment for parasitic infections, especially in the Neotropics from where it originates. OBJECTIVE: This study evaluates the in vitro leishmanicidal and cytotoxicity activities of isolated compounds based on a bioassay-guided fractionation approach. MATERIALS AND METHODS: Defatted aerial parts of E. foetidum were subjected to extraction with methanol followed by partitioning with n-hexane, ethyl acetate and 50% methanol. Then, the first two fractions were subsequently fractionated by column chromatography and HPLC. Compound identity was confirmed by mass spectrometry and NMR spectroscopy. Leishmania tarentolae (promastigotes) and L. donovani (amastigotes) were used as testing parasites. L6 rat myoblasts were used for cytotoxicity. All extracts and fractions were tested at 20 µg/mL. RESULTS: The initial methanol extract showed 20% growth inhibition of L. tarentolae. Then, the n-hexane and ethyl acetate fractions were also active showing approximately 40% growth inhibition. From these two fractions, the following compounds were isolated: lasidiol p-methoxybenzoate (1), a daucane sesquiterpene; and 4-hydroxy-1,1,5-trimethyl-2-formyl-cyclohexadien-(2,5)-[α-acetoxymethyl-cis-crotonate] (2), a terpene aldehyde ester derivative. Compound 1 inhibited the growth of both L. tarentolae and L. donovani with IC50 values of 14.33 and 7.84 µM, respectively; and showed no cytotoxicity (IC50 > 50 µM). Compound 2 was inactive in the L. tarentolae assay (IC50 > 50 µM). DISCUSSION AND CONCLUSION: This study presented the bioassay-guided fractionation with the leishmanicidal and cytotoxicity activities of two compounds isolated for the first time from an Eryngium species.

Pregnane glycosides interfere with steroidogenic enzymes to down-regulate corticosteroid production in human adrenocortical H295R cells.

A group of bioactive steroidal glycosides (pregnanes) with anorectic activity in animals was isolated from several genera of milkweeds including Hoodia and Asclepias. In this study, we investigated the effects, structure-activity relationships, and mechanism of action of pregnane glycosides on steroidogenesis in human adrenocortical H295R cells. Administration of pregnane glycosides for 24 h suppressed the basal and forskolin-stimulated release of androstenedione, corticosterone, and cortisone from H295R cells. The conversion of progesterone to 11-deoxycorticosterone and 17-hydroxyprogesterone to either androstenedione or 11-deoxycortisol was most strongly affected, with 12-cinnamoyl-, benzoyl-, and tigloyl-containing pregnanes showing the highest activity. Incubation of pregnane glycosides for 24 h had no effect on mRNA transcripts of CYP11A1, CYP21A1, CYP11B1 cytochrome enzymes and steroidogenic acute regulatory protein (StaR) protein, yet resulted in twofold decrease in HSD3B1 mRNA levels. At the same time, pregnane glycosides had no effect on the CYP1, 2, or 3 drug and steroid metabolism enzymes and showed weak Na(+) /K(+) ATPase and glucocorticoid receptor binding. Taken together, these data suggest that pregnane glycosides specifically suppress steroidogenesis through strong inhibition of 11ß-hydroxylase and steroid 17-alpha-monooxygenase, and weak inhibition of cytochrome P450 side chain cleavage enzyme and 21ß-hydroxylase, but not 3ß-hydroxysteroid dehydrogenase/isomerase.

Phytochemical and Agronomic Characterization of High-Flavonoid Lettuce Lines Grown under Field Conditions.

Flavonoids are antioxidant phytochemicals that confer a beneficial effect on human health. We have previously developed and characterized eight lettuce (Latuca sativa L.) lines that accumulated high levels of diverse flavonoids and their precursors in controlled environment conditions. Three Rutgers Scarlet lettuce (RSL) lines selected in tissue culture for deep-red color (RSL-NAR, RSL-NBR, RSL-NFR) accumulate anthocyanins and quercetin, three lines identified in a chemically mutagenized red lettuce population accumulate kaempferol (KfoA and KfoB) or naringenin chalcone (Nco), and two lines that were spontaneous green mutants derived from the red line RSL-NAR (GSL, GSL-DG) accumulate quercetin. These eight lines were field-grown in the Salinas Valley of California for four years together with seven control accessions of varying colors (light green, dark green, red, and dark red). At market maturity, a substantial variation in plant composition was observed, but the three RSL lines consistently accumulated high levels of cyanidin, GSL and GSL-DG accumulated the highest levels of quercetin, KfoA and KfoB accumulated kaempferol, and Nco amassed naringenin chalcone, confirming that these mutant lines produce high levels of beneficial phytochemicals under field conditions. Mutant lines and control accessions were also assessed for their biomass production (plant weight, height, and width), overall content of pigments (leaf chlorophyll and anthocyanins), resistance to diseases (downy mildew, lettuce drop, and Impatiens necrotic spot virus), postharvest quality of processed tissue (deterioration and enzymatic discoloration), and composition of 23 mineral elements. All but one mutant line had a fresh plant weight at harvest comparable to commercial leaf cultivars; only Nco plants were significantly (p < 0.05) smaller. Therefore, except for Nco, the new, flavonoid hyperaccumulating lines can be considered for field cultivation.

Auxin-Producing Bacteria from Duckweeds Have Different Colonization Patterns and Effects on Plant Morphology.

The role of auxin in plant-microbe interaction has primarily been studied using indole-3-acetic acid (IAA)-producing pathogenic or plant-growth-promoting bacteria. However, the IAA biosynthesis pathway in bacteria involves indole-related compounds (IRCs) and intermediates with less known functions. Here, we seek to understand changes in plant response to multiple plant-associated bacteria taxa and strains that differ in their ability to produce IRCs. We had previously studied 47 bacterial strains isolated from several duckweed species and determined that 79% of these strains produced IRCs in culture, such as IAA, indole lactic acid (ILA), and indole. Using Arabidopsis thaliana as our model plant with excellent genetic tools, we performed binary association assays on a subset of these strains to evaluate morphological responses in the plant host and the mode of bacterial colonization. Of the 21 tested strains, only four high-quantity IAA-producing Microbacterium strains caused an auxin root phenotype. Compared to the commonly used colorimetric Salkowski assay, auxin concentration determined by LC-MS was a superior indicator of a bacteria's ability to cause an auxin root phenotype. Studies with the auxin response mutant axr1-3 provided further genetic support for the role of auxin signaling in mediating the root morphology response to IAA-producing bacteria strains. Interestingly, our microscopy results also revealed new evidence for the role of the conserved AXR1 gene in endophytic colonization of IAA-producing Azospirillum baldaniorum Sp245 via the guard cells.

A Whole-Grain Diet Increases Whole-Body Protein Balance Compared with a Macronutrient-Matched Refined-Grain Diet.

BACKGROUND: There are limited data from randomized control trials to support or refute the contention that whole-grains can enhance protein metabolism in humans. OBJECTIVES: To examine: 1) the clinical effects of a whole-grain diet on whole-body protein turnover; 2) the cellular effects of whole-grains on protein synthesis in skeletal muscle cells; and 3) the population effects of whole-grain intake on age-related muscle loss. METHODS: Adults with overweight/obesity (n = 14; age = 40 ± 7 y; BMI = 33 ± 5 kg/m2) were recruited into a crossover, randomized controlled trial (NCT01411540) in which isocaloric, macronutrient-matched whole-grain and refined-grain diets were fully provisioned for two 8-wk periods. Diets differed only in the presence of whole-grains (50 g/1000 kcal). Whole-body protein kinetics were assessed at baseline and after each diet in the fasted-state (13C-leucine) and integrated over 24 h (15N-glycine). In vitro studies using C2C12 cells assessed global protein synthesis by surface sensing of translation and anabolic signaling by Western blot. Complementary epidemiological assessments using the NHANES database assessed the effect of whole-grain intake on muscle function assessed by gait speed in older adults (n = 2783). RESULTS: Integrated 24-h net protein balance was 3-fold higher on a whole-grain diet compared with a refined-grain diet (P = 0.04). A whole-grain wheat extract increased submaximal rates of global protein synthesis (27%, P < 0.05) in vitro. In a large sample of older adults, whole-grain intake was associated with greater muscle function (OR = 0.92; 95% CI: 0.86, 0.98). CONCLUSIONS: Consuming 50 g/1000 kcal whole-grains per day promotes greater protein turnover and enhances net protein balance in adults. Whole-grains impact skeletal muscle at the cellular level, and are associated with greater muscle function in older adults. Collectively, these data point to a new mechanism whereby whole-grain consumption favorably enhances protein turnover and improves health outcomes.This clinical trial is registered on clinicaltrials.gov (identifier: NCT01411540).

Proanthocyanidin-Rich Grape Seed Extract Reduces Inflammation and Oxidative Stress and Restores Tight Junction Barrier Function in Caco-2 Colon Cells.

Grape polyphenols have previously been shown to improve gut health and attenuate the symptoms of metabolic syndrome; however, the mechanism of these beneficial effects is still debated. In this study, we investigated the protective effect of proanthocyanidin-rich grape seed extract (GSE) on bacterial lipopolysaccharide (LPS)-induced oxidative stress, inflammation, and barrier integrity of human Caco-2 colon cells. GSE significantly reduced the LPS-induced intracellular reactive oxygen species (ROS) production and mitochondrial superoxide production, and upregulated the expression of antioxidant enzyme genes. GSE also restored the LPS-damaged mitochondrial function by increasing mitochondrial membrane potential. In addition, GSE increased the expression of tight junction proteins in the LPS-treated Caco-2 cells, increased the expression of anti-inflammatory cytokines, and decreased pro-inflammatory cytokine gene expression. Our findings suggest that GSE exerts its beneficial effects on metabolic syndrome by scavenging intestinal ROS, thus reducing oxidative stress, increasing epithelial barrier integrity, and decreasing intestinal inflammation.

Metabolomic differences between invasive alien plants from native and invaded habitats.

Globalization facilitated the spread of invasive alien species (IAS), undermining the stability of the world's ecosystems. We investigated the metabolomic profiles of three IAS species: Chromolaena odorata (Asteraceae) Datura stramonium (Solanaceae), and Xanthium strumarium (Asteraceae), comparing metabolites of individual plants in their native habitats (USA), to their invasive counterparts growing in and around Kruger National Park (South Africa, ZA). Metabolomic samples were collected using RApid Metabolome Extraction and Storage (RAMES) technology, which immobilizes phytochemicals on glass fiber disks, reducing compound degradation, allowing long-term, storage and simplifying biochemical analysis. Metabolomic differences were analyzed using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) of samples eluted from RAMES disks. Partial Least Squares-Discriminant Analysis (PLS-DA) of metabolomes of individual plants allowed statistical separation of species, native and invasive populations of each species, and some populations on the same continent. Invasive populations of all species were more phytochemically diverse than their native counterparts, and their metabolomic profiles were statistically distinguishable from their native relatives. These data may elucidate the mechanisms of successful invasion and rapid adaptive evolution of IAS. Moreover, RAMES technology combined with PLS-DA statistical analysis may allow taxonomic identification of species and, possibly, populations within each species.

Tricin levels and expression of flavonoid biosynthetic genes in developing grains of purple and brown pericarp rice.

The methylated flavone tricin has been associated with numerous health benefits, including reductions in intestinal and colon cancers in animal models. Tricin is found in a wide range of plant species and in many different tissues. However, whole cereal grains, such as rice, barley, oats, and wheat, are the only food sources of tricin, which is located in the bran portion of the grain. Variation in tricin levels was found in bran from rice genotypes with light brown, brown, red, and purple pericarp color, with the purple pericarp genotypes having the highest levels of tricin. Here, we analyzed tricin and tricin derivative levels in developing pericarp and embryo samples of a purple pericarp genotype, IAC600, that had high tricin and tricin derivative levels in the bran, and a light brown pericarp genotype, Cocodrie, that had no detectable tricin or tricin derivatives in the bran. Tricin and tricin derivatives were detected in both the pericarp and embryo of IAC600 but only in the embryo of Cocodrie. The purple pericarp rice had higher total levels of free tricin plus tricin derivatives than the light brown pericarp rice. When expressed on a per grain basis, most of the tricin component of IAC600 was in the pericarp. In contrast, Cocodrie had no detectable tricin in the pericarp samples but did have detectable chrysoeriol, a precursor of tricin, in the pericarp samples. We also used RNA-Seq analysis of developing pericarp and embryo samples of the two cultivars to compare the expression of genes involved in the flavonoid biosynthetic pathway. The results presented here suggest that understanding the basis of tricin accumulation in rice pericarp may lead to an approach to increasing tricin levels in whole grain rice. From analysis of gene expression levels in the pericarp samples it appears that regulation of the flavone specific genes is independent of regulation of the anthocyanin biosynthetic genes. It therefore may be feasible to develop brown pericarp rice cultivars that accumulate tricin in the pericarp.

Genetic and Phytochemical Characterization of Lettuce Flavonoid Biosynthesis Mutants.

We previously developed red lettuce (Lactuca sativa L.) cultivars with high flavonoid and phenolic acid content and demonstrated their anti-diabetic effect. Here we report on developing three fertile and true-breeding lettuce lines enriched with flavonoids with reported beneficial health effects. These lines were identified in a segregating population of EMS-mutagenized red lettuce and characterized biochemically and genetically. Change in red coloration was used as a visual indicator of a mutation in a flavonoid pathway gene, leading to accumulation of flavonoid precursors of red anthocyanins. Pink-green kaempferol overproducing kfoA and kfoB mutants accumulated kaempferol to 0.6-1% of their dry weight, higher than in any vegetable reported. The yellow-green naringenin chalcone overproducing mutant (nco) accumulated naringenin chalcone, not previously reported in lettuce, to 1% dry weight, a level only observed in tomato peel. Kfo plants carried a mutation in the FLAVONOID-3' HYDROXYLASE (F3'H) gene, nco in CHALCONE ISOMERASE (CHI). This work demonstrates how non-GMO approaches can transform a common crop plant into a functional food with possible health benefits.

Distinct Fractions of an Artemisia scoparia Extract Contain Compounds With Novel Adipogenic Bioactivity.

Adipocytes are important players in metabolic health and disease, and disruption of adipocyte development or function contributes to metabolic dysregulation. Hence, adipocytes are significant targets for therapeutic intervention in obesity and metabolic syndrome. Plants have long been sources for bioactive compounds and drugs. In previous studies, we screened botanical extracts for effects on adipogenesis in vitro and discovered that an ethanolic extract of Artemisia scoparia (SCO) could promote adipocyte differentiation. To follow up on these studies, we have used various separation methods to identify the compound(s) responsible for SCO's adipogenic properties. Fractions and subfractions of SCO were tested for effects on lipid accumulation and adipogenic gene expression in differentiating 3T3-L1 adipocytes. Fractions were also analyzed by Ultra Performance Liquid Chromatography- Mass Spectrometry (UPLC-MS), and resulting peaks were putatively identified through high resolution, high mass accuracy mass spectrometry, literature data, and available natural products databases. The inactive fractions contained mostly quercetin derivatives and chlorogenates, including chlorogenic acid and 3,5-dicaffeoylquinic acid, which had no effects on adipogenesis when tested individually, thus ruling them out as pro-adipogenic bioactives in SCO. Based on these studies we have putatively identified the principal constituents in SCO fractions and subfractions that promoted adipocyte development and fat cell gene expression as prenylated coumaric acids, coumarin monoterpene ethers, 6-demethoxycapillarisin and two polymethoxyflavones.

Evaluation of botanicals for improving human health.

Botanical preparations have been used medicinally for thousands of years. Many commercially available botanical products are being marketed in the United States with little or no publicly available scientific validation of efficacy or consistency. For botanicals to be reliable for research purposes and consumer products, they must be standardized with sufficient quality controls to ensure consistent composition, safety, and potency. This includes uniform cultivation of source plants with controls to monitor for contamination from other species, pesticides, and environmental toxins. The active components of botanicals must be identified by activity-guided fractionation with the use of in vitro assays that require little test material followed by validation in vivo. Concentrations of active compounds within the botanicals can then be accurately measured to ensure the delivery of a dependable dose in the final product. The use of bioenhancing agents may be considered for compounds with poor bioavailability. Standardization of botanical therapeutics can only be achieved when the active compounds are identified and biological activity is confirmed, thus ensuring a consistent product.

A natural history of botanical therapeutics.

Plants have been used as a source of medicine throughout history and continue to serve as the basis for many pharmaceuticals used today. Although the modern pharmaceutical industry was born from botanical medicine, synthetic approaches to drug discovery have become standard. However, this modern approach has led to a decline in new drug development in recent years and a growing market for botanical therapeutics that are currently available as dietary supplements, drugs, or botanical drugs. Most botanical therapeutics are derived from medicinal plants that have been cultivated for increased yields of bioactive components. The phytochemical composition of many plants has changed over time, with domestication of agricultural crops resulting in the enhanced content of some bioactive compounds and diminished content of others. Plants continue to serve as a valuable source of therapeutic compounds because of their vast biosynthetic capacity. A primary advantage of botanicals is their complex composition consisting of collections of related compounds having multiple activities that interact for a greater total activity.

Quercetin transiently increases energy expenditure but persistently decreases circulating markers of inflammation in C57BL/6J mice fed a high-fat diet.

Quercetin, a polyphenolic compound and a major bioflavonoid in the human diet, has anti-inflammatory properties and has been postulated to enhance energy expenditure (EE). We sought to determine whether quercetin alters body weight, body composition, EE, and circulating markers of inflammation. At 6 weeks (W) of age, 2 cohorts of C57BL/6J mice (N = 80) were placed on one of 2 diets for 3W or 8W: (1) high fat (HF) (45% kcal fat) or (2) high fat + quercetin (HF + Q) (45% kcal fat + 0.8% quercetin). Quercetin concentrations in the diet and plasma were evaluated using mass spectrometry. Body weight, composition (nuclear magnetic resonance), and food consumption were measured weekly. Energy expenditure was measured by indirect calorimetry at 3 and 8W, and inflammatory markers were measured in plasma obtained at 8W. The presence of quercetin in the HF diet did not alter food consumption over time in the HF + Q group and did not differ from the HF group at any time point. However, circulating plasma quercetin concentrations declined between 3 and 8W. At 3W, EE was higher during both day and night phases (P < .0001) in the HF + Q group compared with the HF group; but this difference was not detected at 8W and did not translate into significant differences between the HF + Q and HF groups with respect to body weight or body composition. During the night phase, concentrations of the inflammatory markers (interferon-gamma, interleukin-1alpha, and interleukin-4) were significantly lower when compared with HF treatment group (P < .05). Dietary supplementation with quercetin produces transient (3W) increases in EE that are not detected after 8W on the diet. A corresponding decrease in circulating quercetin between 3 and 8W suggests that metabolic adaptation may have diminished the impact of quercetin's early effect on EE and diminished its overall effect on nutrient partitioning and adiposity. However, quercetin at the levels provided was effective in reducing circulating markers of inflammation observed in animals on an HF diet at 8W.

Phytoecdysteroids increase protein synthesis in skeletal muscle cells.

Phytoecdysteroids, which are structurally similar or identical to insect molting hormones, produce a range of effects in mammals, including increasing growth and physical performance. To study the mechanism of action of phytoecdysteroids in mammalian tissue, an in vitro cellular assay of protein synthesis was developed. In C2C12 murine myotubes and human primary myotubes, phytoecdysteroids increased protein synthesis by up to 20%. In vivo, ecdysteroids increased rat grip strength. Ecdysteroid-containing plant extracts produced similar results. The effect was inhibited by a phosphoinositide kinase-3 inhibitor, which suggests a PI3K-mediated mechanism.

Determination of tripdiolide in root extracts of Tripterygium wilfordii by solid-phase extraction and reversed-phase high-performance liquid chromatography.

Extracts of Tripterygium wilfordii Hook F. have been widely used in China to treat a variety of autoimmune and inflammatory diseases. The diterpenoids triptolide and tripdiolide are two major active components in the T. wilfordii ethyl acetate extract. An efficient solid-phase extraction and high-performance liquid chromatography (SPE-HPLC) method to measure triptolide content in the extract has been previously reported. However, a suitable means of tripdiolide quantification is not available because of interfering compounds in the extract that co-elute with tripdiolide. Therefore, this paper describes a method wherein tripdiolide content can be measured from a small amount of the extract. The extract solution (600 microL) was applied into an aminopropyl SPE tube. Triptolide was eluted with dichloromethane:methanol (1 mL, 49:1 v/v), followed by tripdiolide elution with dichloromethane:methanol (3 mL, 17:3 v/v). The tripdiolide eluate was analysed by HPLC using an isocratic solvent system and was quantified by measuring the peak area at 219 nm. The contents of triptolide and tripdiolide in the extract were determined to be 807.32 +/- 51.94 and 366.13 +/- 17.21 microg/g of extract, respectively. Since tripdiolide is biologically active and makes up a considerable portion of the extract, for extract quality control and standardisation purposes, it should be measured along with triptolide using the proposed SPE-HPLC method.