Biochemometric Analysis of Fatty Acid Amide Hydrolase Inhibition by Echinacea Root Extracts.

Recent research demonstrates that Echinacea possesses cannabimimetic activity with potential applications beyond common contemporary uses for relief of cold and flu symptoms. In this study, we investigated the in vitro inhibitory effect of root extracts of Echinacea purpurea and Echinacea angustifolia on fatty acid amide hydrolase, the main enzyme that degrades the endocannabinoid anandamide. The objective was to relate variation in bioactivity between commercial Echinacea genotypes to their phytochemical profiles and to identify determinants of activity using biochemometric analysis. Forty root extracts of each of species were tested for inhibition of fatty acid amide hydrolase and analyzed by HPLC-DAD/MS to identify and quantitate alkylamides and caffeic acid derivatives. Fatty acid amide hydrolase inhibition ranged from 34 - 80% among E. angustifolia genotypes and from 33 - 87% among E. purpurea genotypes. Simple linear regression revealed the caffeic acid derivatives caftaric acid and cichoric acid, and the alkylamide dodeca-2E,4Z-diene-8,10-diynioc acid 2-methylbutylamide, as the strongest determinants of inhibition in E. purpurea (r* = 0.53, 0.45, and 0.20, respectively) while in E. angustifolia, only CADs were significantly associated with activity, most notably echinacoside (r* = 0.26). Regression analysis using compound groups generated by hierarchical clustering similarly indicated that caffeic acid derivatives contributed more than alkylamides to in vitro activity. Testing pure compounds identified as determinants of activity revealed cichoric acid (IC50 = 45 ± 4 µM) and dodeca-2E,4E,8Z,10E-tetraenoic acid isobutylamide (IC50 = 54 ± 2 µM) as the most active. The results suggest that several phytochemicals may contribute to Echinacea's cannabimimetic activity and that ample variation in genotypes exists for selection of high-activity germplasm in breeding programs.

Growth environment and organ specific variation in in-vitro cytoprotective activities of Picea mariana in PC12 cells exposed to glucose toxicity: a plant used for treatment of diabetes symptoms by the Cree of Eeyou Istchee (Quebec, Canada).

BACKGROUND: The Cree of Eeyou Istchee (James Bay area of northern Quebec) suffer from a high rate of diabetes and its complications partly due to the introduction of the western lifestyle within their culture. As part of a search for alternative medicine based on traditional practice, this project evaluates the biological activity of Picea mariana (Mill.) Britton, Sterns & Poggenb. needle, bark, and cone, in preventing glucose toxicity to PC12-AC cells in vitro (a diabetic neurophathy model) and whether habitat and growth environment influence this activity. METHODS: Three different organs (needle, bark, and cone) of P. mariana were collected at different geographical locations and ecological conditions and their 80% ethanolic extracts were prepared. Extracts were then tested for their ability to protect PC12-AC cells from hyperglycaemic challenge at physiologically relevant concentrations of 0.25, 0.5, 1.0 and 2.0 µg/mL. Folin-Ciocalteu method was used to determine the total phenolic content of P. mariana extracts. RESULTS: All extracts were well-tolerated in vitro exhibiting LD50 of 25 µg/mL or higher. Extracts from all tested organs showed a cytoprotective concentration-dependent response. Furthermore, the cytoprotective activity was habitat- and growth environment-dependent with plants grown in bog or forest habitats in coastal or inland environments exhibiting different cytoprotective efficacies. These differences in activity correlated with total phenolic content but not with antioxidant activity. In addition, this paper provides the first complete Ultra-Performance Liquid Chromatography-quadrupole time-of-flight (UPLC-QTOF) mass spectrometry analysis of Picea mariana's bark, needles and cones. CONCLUSIONS: Together, these results provide further understanding of the cytoprotective activity of Canadian boreal forest plants identified by the Cree healers of Eeyou Istchee in a cell model of diabetic neuropathy. Their activity is relevant to diabetic peripheral neuropathic complications and shows that their properties can be optimized by harvesting in optimal growth environments.

Geophagy among East African Chimpanzees: consumed soils provide protection from plant secondary compounds and bioavailable iron.

Geophagy, the intentional consumption of earth materials, has been recorded in humans and other animals. It has been hypothesized that geophagy is an adaptive behavior, and that clay minerals commonly found in eaten soil can provide protection from toxins and/or supplement micronutrients. To test these hypotheses, we monitored chimpanzee geophagy using camera traps in four permanent sites at the Budongo Forest Reserve, Uganda, from October 2015-October 2016. We also collected plants, and soil chimpanzees were observed eating. We analyzed 10 plant and 45 soil samples to characterize geophagic behavior and geophagic soil and determine (1) whether micronutrients are available from the soil under physiological conditions and if iron is bioavailable, (2) the concentration of phenolic compounds in plants, and (3) if consumed soils are able to adsorb these phenolics. Chimpanzees ate soil and drank clay-infused water containing 1:1 and 2:1 clay minerals and > 30% sand. Under physiological conditions, the soils released calcium, iron, and magnesium. In vitro Caco-2 experiments found that five times more iron was bioavailable from three of four soil samples found at the base of trees. Plant samples contained approximately 60 µg/mg gallic acid equivalent. Soil from one site contained 10 times more 2:1 clay minerals, which were better at removing phenolics present in their diet. We suggest that geophagy may provide bioavailable iron and protection from phenolics, which have increased in plants over the last 20 years. In summary, geophagy within the Sonso community is multifunctional and may be an important self-medicative behavior.

Soil eaten by chacma baboons adsorbs polar plant secondary metabolites representative of those found in their diet.

Geophagy, the deliberate consumption of earth materials, is common among humans and animals. However, its etiology and function(s) remain poorly understood. The major hypotheses about its adaptive functions are the supplementation of essential elements and the protection against temporary and chronic gastrointestinal (GI) distress. Because much less work has been done on the protection hypothesis, we investigated whether soil eaten by baboons protected their GI tract from plant secondary metabolites (PSMs) and described best laboratory practices for doing so. We tested a soil that baboons eat/preferred, a soil that baboons never eat/non-preferred, and two clay minerals, montmorillonite a 2:1 clay and kaolinite a 1:1 clay. These were processed using a technique that simulated physiological digestion. The phytochemical concentration of 10 compounds representative of three biosynthetic classes of compounds found in the baboon diet was then assessed with and without earth materials using high-performance liquid chromatography with diode-array detection (HPLC-DAD). The preferred soil was white, contained 1% halite, 45% illite/mica, 14% kaolinite, and 0.8% sand; the non-preferred soil was pink, contained 1% goethite and 1% hematite but no halite, 40% illite/mica, 19% kaolinite, and 3% sand. Polar phenolics and alkaloids were generally adsorbed at levels 10× higher than less polar terpenes. In terms of PSM adsorption, the montmorillonite was more effective than the kaolinite, which was more effective than the non-preferred soil, which was more effective than the preferred soil. Our findings suggest that HPLC-DAD is best practice for the assessment of PSM adsorption of earth materials due to its reproducibility and accuracy. Further, soil selection was not based on adsorption of PSMs, but on other criteria such as color, mouth feel, and taste. However, the consumption of earth containing clay minerals could be an effective strategy for protecting the GI tract from PSMs.

Labrador tea (Rhododendron groenlandicum) attenuates insulin resistance in a diet-induced obesity mouse model.

PURPOSE: Using a diet-induced obesity (DIO) mouse model, we investigated the antidiabetic effect of Labrador tea [Rhododendron groenlandicum (Oeder) Kron and Judd], a beverage and medicinal tea used by the Cree Nations of northern Quebec. METHODS: C57BL6 mice were divided into five groups and given standard chow (~4 % of lipids) or high-fat diet (~35 % of lipids) for 8 weeks until they became obese and insulin resistant. Treatment began by adding the plant extract at three doses (125, 250 and 500 mg/kg) to the high-fat diet for another 8 weeks. At the end of the study, insulin-sensitive tissues (liver, skeletal muscle, adipose tissue) were collected to investigate the plant's molecular mechanisms. RESULTS: Labrador tea significantly reduced blood glucose (13 %), the response to an oral glucose tolerance test (18.2 %) and plasma insulin (65 %) while preventing hepatic steatosis (42 % reduction in hepatic triglyceride levels) in DIO mice. It stimulated insulin-dependent Akt pathway (55 %) and increased the expression of GLUT4 (53 %) in skeletal muscle. In the liver, Labrador tea stimulated the insulin-dependent Akt and the insulin-independent AMP-activated protein kinase pathways. The improvement in hepatic steatosis observed in DIO-treated mice was associated with a reduction in inflammation (through the IKK α/ß) and a decrease in the hepatic content of SREBP-1 (39 %). CONCLUSIONS: Labrador tea exerts potential antidiabetic action by improving insulin sensitivity and mitigating high-fat diet-induced obesity and hyperglycemia. They validate the safety and efficacy of this plant, a promising candidate for culturally relevant complementary treatment in Cree diabetics.

Antifungal Saponins from the Maya Medicinal Plant Cestrum schlechtendahlii G. Don (Solanaceae).

Bioassay-guided fractionation of the crude extract (80% EtOH) of the leaves of Cestrum schlechtendahlii, a plant used by Q'eqchi' Maya healers for treatment of athlete's foot, resulted in the isolation and identification of two spirostanol saponins (1 and 2). Structure elucidation by MS, 1D-NMR, and 2D-NMR spectroscopic methods identified them to be the known saponin (25R)-1ß,2α-dihydroxy-5α-spirostan-3-ß-yl-O-α-L-rhamnopyranosyl-(1 → 2)-ß-D-galactopyranoside (1) and new saponin (25R)-1ß,2α-dihydroxy-5α-spirostan-3-ß-yl-O-ß-D-galactopyranoside (2). While 2 showed little or no antifungal activity at the highest concentration tested, 1 inhibited growth of Saccharomyces cerevisiae (minimum inhibitory concentration (MIC) of 15-25 µM), Candida albicans, Cryptococcus neoformans, and Fusarium graminearum (MIC of 132-198 µM).

Phytochemical Comparison of the Water and Ethanol Leaf Extracts of the Cree medicinal plant, Sarracenia purpurea L. (Sarraceniaceae).

PURPOSE: The Cree of Eeyou Istchee in Northern Quebec identified Sarracenia purpurea L. as an important plant for the treatment of Type 2 diabetes. Traditionally the plant is used as a decoction (boiling water extract) of the leaf, however, in order to study the extract in a laboratory setting, an 80% ethanol extract was used. In this study, the phytochemistry of both extracts of the leaves was compared and quantified. METHODS: Two S. purpurea leaf extracts were prepared, one a traditional hot water extract and the other an 80% ethanol extract. Using UPLC-ESI-MS, the extracts were phytochemically compared for 2 triterpenes, betulinic acid and ursolic acid, using one gradient method and for 10 additional substances, including the actives quercetin-3-O-galactoside and morroniside, using a different method. RESULTS: The concentrations of the nine phenolic substances present, as well as an active principle, the iridoid glycoside morroniside, were very similar between the two extracts, with generally slightly higher concentrations of phenolics in the ethanol extract as expected. However, two triterpenes, betulinic acid and ursolic acid, were 107 and 93 times more concentrated, respectively, in the ethanol extract compared to the water extract. CONCLUSION: The main phytochemical markers and most importantly the antidiabetic active principles, quercetin-3-O-galactoside and morroniside, were present in similar amounts in the two extracts, which predicts similar bioactivity.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Evaluation by microarray of the potential safety of Sarracenia purpurea L. (Sarraceniaceae) a traditional medicine used by the Cree of Eeyou Istchee.

PURPOSE: The purpose of this study was to assess safety of the traditional antidiabetic extracts of either S. purpurea or its lead active principle, morroniside at the transcriptional level. The overarching objective was to profile and validate transcriptional changes in the cytochrome P450 family of genes, in response to treatment with S. purpurea ethanolic extract or its lead active, morroniside. METHODS: Transcriptional activity was profiled using a 19K human cDNA microarray in C2BBe1 cells, clone of Caco-2 intestinal cells, which are a model of first-pass metabolism (1, 2). Cells were treated with S. purpurea extract for 4 and 24 hrs, as well as the pure compound morroniside for 4 hrs, to determine their effects. RESULTS: No evidence of cytochrome P450 transcriptome regulation or of transcriptional activation of other diabetes relevant mRNA was detected after rigorous quantitative-PCR validation of microarray results. CONCLUSION: Our data do not support a transcriptional mechanism of action for either S. purpurea extract or its lead active, morroniside. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Ovulation but not milt production is inhibited in fathead minnows (Pimephales promelas) exposed to a reproductively inhibitory pulp mill effluent.

BACKGROUND: A 5-day fathead minnow (FHM) spawning assay is used by industry to monitor pulp mill effluent quality, with some mill effluents capable of completely inhibiting spawning. The purpose of this report is to characterize the effect of an inhibitory effluent on egg and milt production in FHM. METHODS: Eight tanks were treated with an inhibitory effluent while eight were kept with clean water. Each tank contained two males and four females as per the 5-day FHM spawning assay used by industry. Females were stripped of ovulated eggs and males of milt in four effluent-exposed and four control tanks. Eggs oviposited in every tank were also counted and checked for fertilization and data analyzed with 2-way ANOVA. RESULTS: We show that female, but not male, fathead minnow reproductive function is impaired in the 5-day fathead minnow spawning assay used by industry to evaluate pulp mill effluent quality in Canada. Milt production was not changed in the control or exposed males mid-way and at the end of the five day exposure (p > 0.05; n = 8). Total egg production (stripped + oviposited) was impaired (p < 0.05) in fathead minnows exposed to effluent (288 eggs/tank, n = 4 tanks) compared to those in control tanks (753 eggs/tank, n = 4 tanks). CONCLUSIONS: Our results indicate that males are able to detect female signals and prepare appropriately for spawning while in females inhibition of ovulation is occurring somewhere along the hypothalamus-pituitary-gonad reproductive axis. These results suggest female-specific neuroendocrine disruption and provide mechanistic insight into an assay used by industry to assess pulp mill effluent quality.

Inhibition of insect glutathione S-transferase (GST) by conifer extracts.

Insecticide synergists biochemically inhibit insect metabolic enzyme activity and are used both to increase the effectiveness of insecticides and as a diagnostic tool for resistance mechanisms. Considerable attention has been focused on identifying new synergists from phytochemicals with recognized biological activities, specifically enzyme inhibition. Jack pine (Pinus banksiana Lamb.), black spruce (Picea mariana (Mill.) BSP.), balsam fir (Abies balsamea (L.) Mill.), and tamarack larch (Larix laricina (Du Roi) Koch) have been used by native Canadians as traditional medicine, specifically for the anti-inflammatory and antioxidant properties based on enzyme inhibitory activity. To identify the potential allelochemicals with synergistic activity, ethanol crude extracts and methanol/water fractions were separated by Sephadex LH-20 chromatographic column and tested for in vitro glutathione S-transferase (GST) inhibition activity using insecticide-resistant Colorado potato beetle, Leptinotarsa decemlineata (Say) midgut and fat-body homogenate. The fractions showing similar activity were combined and analyzed by ultra pressure liquid chromatography-mass spectrometry. A lignan, (+)-lariciresinol 9'-p-coumarate, was identified from P. mariana cone extracts, and L. laricina and A. balsamea bark extracts. A flavonoid, taxifolin, was identified from P. mariana and P. banksiana cone extracts and L. laricina bark extracts. Both compounds inhibit GST activity with taxifolin showing greater activity compared to (+)-lariciresinol 9'-p-coumarate and the standard GST inhibitor, diethyl maleate. The results suggested that these compounds can be considered as potential new insecticide synergists.

Antimicrobial and P450 inhibitory properties of common functional foods.

PURPOSE: To study the effect of functional foods on human cytochrome P450 (CYP) and the gut bacterial microflora that may potentially affect drug metabolism and ultimately affect human health and wellness. METHODS: This study examined a variety of food plants from the Apiaceae, Fabaceae, and Lamiaceae families for their inhibitory potential on cytochrome 2D6-, 3A4-, 3A5-, and 3A7-mediated metabolism. The antimicrobial effects of these samples were also investigated with 7 selected bacterial surrogate species to determine potential effects on the gut microflora. RESULTS: The highest CYP inhibitory activities, based upon visual examination, were observed from extracts of celery seed, cumin, fennel seed, basil, oregano, and rosemary belonging to the Apiaceae and Lamiaceae families, respectively. Likewise, the strongest antimicrobial activities were also observed in the Apiaceae and Lamiaceae. No significant antimicrobial and CYP inhibition was observed in the Fabaceae extracts. CONCLUSION: Results demonstrated the possible risk of food-drug interactions from spice and herb plants may affect drug disposition and safety.

Souroubea sympetala (Marcgraviaceae): a medicinal plant that exerts anxiolysis through interaction with the GABAA benzodiazepine receptor.

The mode of action of the anxiolytic medicinal plant Souroubea sympetala was investigated to test the hypothesis that extracts and the active principle act at the pharmacologically important GABAA-benzodiazepine (GABAA-BZD) receptor. Leaf extracts prepared by ethyl acetate extraction or supercritical extraction, previously determined to have 5.54 mg/g and 6.78 mg/g of the active principle, betulinic acid, respectively, reduced behavioural parameters associated with anxiety in a rat model. When animals were pretreated with the GABAA-BZD receptor antagonist flumazenil, followed by the plant extracts, or a more soluble derivative of the active principle, the methyl ester of betulinic acid (MeBA), flumazenil eliminated the anxiety-reducing effect of plant extracts and MeBA, demonstrating that S. sympetala acts via an agonist action on the GABAA-BZD receptor. An in vitro GABAA-BZD competitive receptor binding assay also demonstrated that S. sympetala extracts have an affinity for the GABAA-BZD receptor, with an EC50 value of 123 µg/mL (EtOAc leaf extract) and 154 µg/mL (supercritical CO2 extract). These experiments indicate that S. sympetala acts at the GABAA-BZD receptor to elicit anxiolysis.

Inhibition of bacterial quorum sensing and biofilm formation by extracts of neotropical rainforest plants.

Bacterial biofilms are responsible for many persistent infections by many clinically relevant pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa. Biofilms are much more resistant to conventional antibiotics than their planktonic counterparts. Quorum sensing, an intercellular communication system, controls pathogenesis and biofilm formation in most bacterial species. Quorum sensing provides an important pharmacological target since its inhibition does not provide a selective pressure for resistance. In this study, we investigated the quorum sensing and biofilm inhibitory activities of 126 plant extracts from 71 species collected from neotropical rainforests in Costa Rica. Quorum sensing and biofilm interference were assessed using a modified disc diffusion bioassay with Chromobacterium violaceum ATCC 12,472 and a spectrophotometric bioassay with Pseudomonas aeruginosa PA14, respectively. Species with significant anti-quorum sensing and/or anti-biofilm activities belonged to the Meliaceae, Melastomataceae, Lepidobotryaceae, Sapindaceae, and Simaroubaceae families. IC50 values ranged from 45 to 266 µg/mL. Extracts of these active species could lead to future development of botanical treatments for biofilm-associated infections.

Distinguishing Vaccinium species by chemical fingerprinting based on NMR spectra, validated with spectra collected in different laboratories.

A method was developed to distinguish Vaccinium species based on leaf extracts using nuclear magnetic resonance spectroscopy. Reference spectra were measured on leaf extracts from several species, including lowbush blueberry (Vaccinium angustifolium), oval leaf huckleberry (Vaccinium ovalifolium), and cranberry (Vaccinium macrocarpon). Using principal component analysis, these leaf extracts were resolved in the scores plot. Analysis of variance statistical tests demonstrated that the three groups differ significantly on PC2, establishing that the three species can be distinguished by nuclear magnetic resonance. Soft independent modeling of class analogies models for each species also showed discrimination between species. To demonstrate the robustness of nuclear magnetic resonance spectroscopy for botanical identification, spectra of a sample of lowbush blueberry leaf extract were measured at five different sites, with different field strengths (600 versus 700 MHz), different probe types (cryogenic versus room temperature probes), different sample diameters (1.7 mm versus 5 mm), and different consoles (Avance I versus Avance III). Each laboratory independently demonstrated the linearity of their NMR measurements by acquiring a standard curve for chlorogenic acid (R(2) = 0.9782 to 0.9998). Spectra acquired on different spectrometers at different sites classifed into the expected group for the Vaccinium spp., confirming the utility of the method to distinguish Vaccinium species and demonstrating nuclear magnetic resonance fingerprinting for material validation of a natural health product.

Investigating wild berries as a dietary approach to reducing the formation of advanced glycation endproducts: chemical correlates of in vitro antiglycation activity.

Evidence supports the health promoting benefits of berries, particularly with regard to the prevention and management of chronic diseases such cardio- and cerebrovascular disease, diabetes and Alzheimer's disease. Two related pathophysiological features common to many of these conditions are oxidative stress and the accumulation of advanced glycation endproducts (AGEs). Whereas antioxidant properties are well-established in several species of berries and are believed central to their protective mechanisms, few studies have investigated the effects of berries on AGE formation. Here, employing a series of complementary in vitro assays, we evaluated a collection of wild berry extracts for 1) inhibitory effects on fluorescent-AGE and Nε- (carboxymethyl)lysine-albumin adduct formation, 2) radical scavenging activity and 3) total phenolic and anthocyanin content. All samples reduced AGE formation in a concentration-dependent manner that correlated positively with each extract's total phenolic content and, to a lesser degree, total anthocyanin content. Inhibition of AGE formation was similarly related to radical scavenging activities. Adding antiglycation activity to the list of established functional properties ascribed to berries and their phenolic metabolites, our data provide further insight into the active compounds and protective mechanisms through which berry consumption may aid in the prevention and treatment of chronic diseases associated with AGE accumulation and toxicity. As widely available, safe and nutritious foods, berries represent a promising dietary intervention worthy of further investigation.

Distinguishing Ontario ginseng landraces and ginseng species using NMR-based metabolomics.

The use of (1)H-NMR-based metabolomics to distinguish and identify unique markers of five Ontario ginseng (Panax quinquefolius L.) landraces and two ginseng species (P. quinquefolius and P. ginseng) was evaluated. Three landraces (2, 3, and 5) were distinguished from one another in the principal component analysis (PCA) scores plot. Further analysis was conducted and specific discriminating metabolites from the PCA loadings were determined. Landraces 3 and 5 were distinguishable on the basis of a decreased NMR intensity in the methyl ginsenoside region, indicating decreased overall ginsenoside levels. In addition, landrace 5 was separated by an increased amount of sucrose relative to the rest of the landraces. Landrace 2 was separated from the rest of the landraces by the increased level of ginsenoside R(b1). The Ontario P. quinquefolius was also compared with Asian P. ginseng by PCA, and clear separation between the two groups was detected in the PCA scores plot. The PCA loadings plot and a t-test NMR difference plot were able to identify an increased level of maltose and a decreased level of sucrose in the Asian ginseng compared with the Ontario ginseng. An overall decrease of ginsenoside content, especially ginsenoside R(b1), was also detected in the Asian ginseng's metabolic profile. This study demonstrates the potential of NMR-based metabolomics as a powerful high-throughput technique in distinguishing various closely related ginseng landraces and its ability to identify metabolic differences from Ontario and Asian ginseng. The results from this study will allow better understanding for quality assessment, species authentication, and the potential for developing a fully automated method for quality control.

Characterization of the anxiolytic activity of Nunavik Rhodiola rosea.

Rhodiola rosea is a medicinal plant used by the indigenous Inuit people of Nunavik and Nunatsiavut, Eastern Canada, as a mental and physical rejuvenating agent. This traditional use led to the present investigation of R. rosea in the context of anxiety disorders. An alcohol extract of R. rosea roots was characterized phytochemically and orally administered for three consecutive days to Sprague-Dawley rats at 8 mg/kg, 25 mg/kg, and 75 mg/kg body weight. The rats were subjected to three behavioral paradigms of anxiety, including the elevated plus maze, social interaction, and contextual conditioned emotional response tests. Rhodiola rosea showed dose-dependent anxiolytic activity in the elevated plus maze and conditioned emotional response tests, with moderate effects in the higher-anxiety SI test. The active dose varied according to the anxiety test. In order to elucidate a mechanism, the extract was further tested in an in vitro GABAA-benzodiazepine receptor-binding assay, where it demonstrated low activity. This study provides the first comparative assessment of the anxiolytic activity of Nunavik R. rosea in several behaviour models and suggests that anxiolytic effects may be primarily mediated via pathways other than the GABAA-benzodiazepine site of the GABAA receptor.

Coloring genetically modified soybean grains with anthocyanins by suppression of the proanthocyanidin genes ANR1 and ANR2.

Detection and quantification of the levels of adventitious presence of genetically modified (GM) soybeans in non-GM grain shipments currently requires sophisticated tests that can have issues with their reproducibility. We show here that pigment biosynthesis in the soybean seed coat can be manipulated to provide a distinct color that would enable the simple visible detection of the GM soybean grain. We observed that a distinct red-brown grain color could be engineered by the simultaneous suppression of two proanthocyanidin (PA) genes, ANTHOCYANIDIN REDUCTASE1 (ANR1) and ANR2. Multiple reaction monitoring by liquid chromatography tandem mass spectrometry was used to quantify differentially accumulated seed coat metabolites, and revealed the redirection of metabolic flux into the anthocyanin pigment pathway and unexpectedly the flavonol-3-O-glucoside pathway. The upregulations of anthocyanin isogenes (DFR1 and GST26) and the anthocyanin/flavonol-3-O-glycosyltransferase (UGT78K2) were identified by quantitative RT-PCR to be endogenous feedback and feedforward responses to overaccumulation of upstream flavonoid intermediates resulting from ANR1 and ANR2 suppressions. These results suggested the transcription of flavonoid genes to be a key component of the mechanism responsible for the redirection of metabolite flux. This report identifies the suppression of PA genes to be a novel approach for engineering pigmentation in soybean grains.

Evaluating the potential of effluents and wood feedstocks from pulp and paper mills in Brazil, Canada, and New Zealand to affect fish reproduction: chemical profiling and in vitro assessments.

This study investigates factors affecting reproduction in fish exposed to pulp and paper mill effluents by comparing effluents from countries with varying levels of documented effects. To explore the hypothesis of wood as a common source of endocrine disrupting compounds, feedstocks from each country were analyzed. Analyses included in vitro assays for androgenic activity (binding to goldfish testis androgen receptors), estrogenic activity (yeast estrogen screen), and neurotransmitter enzyme inhibition (monoamine oxidase and glutamic acid decarboxylase). Chemical analyses included conventional extractives, known androgens, and gas chromatograph index (GCI) profiles. All effluents and wood contained androgenic activity, particularly in nonpolar fractions, although known androgens were undetected. Effluents with low suspended solids, having undergone conventional biotreatment had lower androgenic activities. Estrogenic activity was only associated with Brazilian effluents and undetected in wood. All effluents and wood inhibited neurotransmitter enzymes, predominantly in polar fractions. Kraft elemental chlorine free mills were associated with the greatest neurotransmitter inhibition. Effluent and wood GCI profiles were correlated with androgenic activity and neurotransmitter enzyme inhibition. Differences in feedstock bioactivities were not reflected in effluents, implying mill factors mitigate bioactive wood components. No differences in bioactivities could be discerned on the basis of country of origin, thus we predict effluents in regions lacking monitoring would affect fish reproduction and therefore recommend implementing such programs.