Role of a Mixture of Polyphenol Compounds Released after Blueberry Fermentation in Chemoprevention of Mammary Carcinoma: In Vivo Involvement of miR-145.

Epigenetic mechanisms such as microRNA (miRNA) deregulation seem to exert a central role in breast cancer initiation and progression. Therefore, targeting epigenetics deregulation may be an effective strategy for preventing and halting carcinogenesis. Studies have revealed the significant role of naturally occurring polyphenolic compounds derived from fermented blueberry fruits in cancer chemoprevention by modulation of cancer stem cell development through the epigenetic mechanism and regulation of cellular signaling pathways. In this study, we first investigated the phytochemical changes during the blueberry fermentation process. Fermentation favored the release of oligomers and bioactive compounds such as protocatechuic acid (PCA), gallic acid, and catechol. Next, we investigated the chemopreventive potentials of a polyphenolic mixture containing PCA, gallic acid, and catechin found in fermented blueberry juice in a breast cancer model by measuring miRNA expression and the signaling pathways involved in breast cancer stemness and invasion. To this end, 4T1 and MDA-MB-231 cell lines were treated with different doses of the polyphenolic mixture for 24 h. Additionally, female Balb/c mice were fed with this mixture for five weeks; two weeks before and three weeks after receiving 4T1 cells. Mammosphere formation was assayed in both cell lines and the single-cell suspension obtained from the tumor. Lung metastases were counted by isolating 6-thioguanine-resistant cells present in the lungs. In addition, we conducted RT-qPCR and Western blot analysis to validate the expression of targeted miRNAs and proteins, respectively. We found a significant reduction in mammosphere formation in both cell lines treated with the mixture and in tumoral primary cells isolated from mice treated with the polyphenolic compound. The number of colony-forming units of 4T1 cells in the lungs was significantly lower in the treatment group compared to the control group. miR-145 expression significantly increased in the tumor samples of mice treated with the polyphenolic mixture compared to the control group. Furthermore, a significant increase in FOXO1 levels was noted in both cell lines treated with the mixture. Overall, our results show that phenolic compounds found in fermented blueberry delay the formation of tumor-initiating cells in vitro and in vivo and reduce the spread of metastatic cells. The protective mechanisms seem to be related, at least partly, to the epigenetic modulation of mir-145 and its signaling pathways.

Proline as a Sparker Metabolite of Oxidative Metabolism during the Flight of the Bumblebee, Bombus impatiens.

Several insect species use the amino acid proline as a major energy substrate. Although initially thought to be limited to blood-feeding dipterans, studies have revealed this capability is more widespread. Recent work with isolated flight muscle showed that the bumblebee Bombus impatiens can oxidize proline at a high rate. However, its role as a metabolic fuel to power flight is unclear. To elucidate the extent to which proline is oxidized to power flight and how its contribution changes during flight, we profiled 14 metabolites central to energy and proline metabolism at key time points in flight muscle and abdominal tissues. Ultra-high performance liquid chromatography-electrospray ionization-quadrupole time of flight mass spectrometry (UPLC-ESI-QTOF MS) analysis revealed that proline is likely used as a sparker metabolite of the tricarboxylic acid cycle at the onset of flight, whereby it supplements the intermediates of the cycle. Carbohydrates are the major energy substrates, which is evidenced by marked decreases in abdominal glycogen stores and a lack of alanine accumulation to replenish flight muscle proline. The time course of fuel stores and metabolites changes during flight highlights homeostatic regulation of energy substrates and patterns of changes in metabolic intermediates within pathways. This study clarifies the role of proline and carbohydrate metabolism during flight in hymenopterans, such as B. impatiens.

A Multivariate Approach to Ethnopharmacology: Antidiabetic Plants of Eeyou Istchee.

An ethnopharmacological metanalysis was conducted with a large database available on antidiabetic activities of plant foods and medicines from the northern boreal forest, which are traditionally used by the indigenous Cree of James Bay, Quebec, Canada. The objective was to determine which bioassays are closely associated with the traditional knowledge of the Cree and which pharmacological metrics and phytochemical signals best define these plants and their groups. Data from 17 plant species, ethnobotanically ranked by syndromic importance value for treatment of 15 diabetic symptoms, was used along with 49 bioassay endpoints reported across numerous pharmacological studies and a metabolomics dataset. Standardized activities were separated into primary, secondary and safety categories and summed to produce a Pharmacological Importance Value (PIV) in each of the three categories for each species. To address the question of which pharmacological metrics and phytochemical signals best define the CEI anti-diabetes plants, multivariate analyses were undertaken to determine groupings of plant families and plant parts. The analysis identified Larix larcina as the highest PIV species in primary assays, Salix planifolia in secondary assays, and Kalmia angustifolia in safety assays, as well as a ranking of other less active species by PIV. Multivariate analysis showed that activity in safety PIV monitored mainly with cytochrome P450 inhibition patterns best reflected patterns of traditional medicine importance in Cree traditional knowledge, whereas potent primary bioactivities were seen in individual plants determined to be most important to the Cree for anti-diabetes purposes. In the secondary anti-diabetes assays, pharmacological variability was better described by plant biology, mostly in terms of the plant part used. Key signal in the metabolomics loadings plots for activity were phenolics especially quercetin derivatives. Traditional Indigenous knowledge in this analysis was shown to be able to guide the identification of plant pharmacological qualities in scientific terms.

Translational Molecular Ecology in practice: Linking DNA-based methods to actionable marine environmental management.

Molecular-based approaches can provide timely biodiversity assessments, showing an immense potential to facilitate decision-making in marine environmental management. However, the uptake of molecular data into environmental policy remains minimal. Here, we showcase a selection of local to global scale studies applying molecular-based methodologies for environmental management at various stages of implementation. Drawing upon lessons learned from these case-studies, we provide a roadmap to facilitate applications of DNA-based methods to marine policies and to overcome the existing challenges. The main impediment identified is the need for standardized protocols to guarantee data comparison across spatial and temporal scales. Adoption of Translational Molecular Ecology - the sustained collaboration between molecular ecologists and stakeholders, will enhance consensus with regards to the objectives, methods, and outcomes of environmental management projects. Establishing a sustained dialogue among stakeholders is key to accelerating the adoption of molecular-based approaches for marine monitoring and assessment.

Metabolome Variation between Strains of Microcystis aeruginosa by Untargeted Mass Spectrometry.

Cyanobacteria are notorious for their potential to produce hepatotoxic microcystins (MCs), but other bioactive compounds synthesized in the cells could be as toxic, and thus present interest for characterization. Ultra performance liquid chromatography and high-resolution accurate mass spectrometry (UPLC-QTOF-MS/MS) combined with untargeted analysis was used to compare the metabolomes of five different strains of the common bloom-forming cyanobacterium, Microcystis aeruginosa. Even in microcystin-producing strains, other classes of oligopeptides including cyanopeptolins, aeruginosins, and aerucyclamides, were often the more dominant compounds. The distinct and large variation between strains of the same widespread species highlights the need to characterize the metabolome of a larger number of cyanobacteria, especially as several metabolites other than microcystins can affect ecological and human health.

Identifying novel treeline biomarkers in lake sediments using an untargeted screening approach.

Paleolimnology uses sedimentary biomarkers as proxies to reconstruct long-term changes in environmental conditions from lake sediment cores. This work describes an untargeted metabolomics-based approach and uniquely applies it to the field of paleolimnology to identify novel sediment biomarkers to track long-term patterns in treeline dynamics. We identified new potential biomarkers across the Canadian northern Arctic, non-alpine, treeline using high-resolution accurate mass spectrometry, and pattern recognition analysis. This method was applied to 120 sediment core extracts from 14 boreal, 25 forest-tundra, and 21 tundra lakes to assess long-term fluctuations in treeline position. High resolution accurate mass spectrometry resolved many compounds from complex mixtures with low mass accuracy errors. This generated a large dataset that required metabolomics styled statistical analyses to identify potential biomarkers. In total, 29 potential biomarkers discriminated between boreal and tundra lakes. Tetrapyrrole-type phorbides and squalene derivatives dominated in boreal regions, while biohopane-type lipids were in the tundra regions. Tetrapyrroles were in both surface and subsurface sediments of boreal lakes indicating these compounds can survive long-term burial in sediments. At the ecozone level, tetrapyrroles were more abundant in boreal Taiga Shield, and Taiga Plains. Boreal plant extracts belonging to Pinaceae and Ericaceae also contained tetrapyrroles. Squalene derivatives demonstrated long-term preservation, but wider distribution than tetrapyrroles. Hopanoids were present in tundra and forest-tundra lake regions, specifically the Low Arctic and Taiga Shield, and were absent in all boreal lake sediments. Herein, we describe a method that can systematically identify new paleolimnological biomarkers. Novel biomarkers would facilitate multi-proxy paleolimnological studies and potentially lead to more accurate paleoenvironmental reconstructions.

Safety evaluation of a new anxiolytic product containing botanicals Souroubea spp. and Platanus spp. in dogs.

Separation anxiety and noise aversion are common behavioral problems in dogs. They elicit fear responses such as cowering, seeking out the owner, and attempting to escape. This can result in property damage, injury to the dog, and disruption of the owner-pet bond, possibly leading to pet abandonment or euthanasia. A novel botanical anxiolytic product was evaluated for safety in dogs as the target animal species. Its intended use is for the treatment and prevention of anxiety and noise aversion in dogs. It contains a defined mixture of Souroubea spp. vine and Platanus spp. bark, delivering the active principle, betulinic acid, at a recommended dose of 1 mg/kg body weight (BW). In the current target animal safety study, 16 healthy male beagle dogs were administered either a placebo or the newly formulated botanical tablets at 0.5×, 2.5×, or 5× the recommended dose (1 mg/kg BW) over 28 d. The dogs were monitored for occurrence of any systemic or local adverse events. In the investigation presented here, there were no clinically significant adverse effects following treatment, as determined by clinical observations, physical examinations, BW, hematology, clinical biochemistry, and urinalysis. Pharmacokinetic analysis demonstrated that the concentration of betulinic acid in serum was below 0.020 µg/mL in treated animals. Under the conditions of these studies, the formulated blend of S. sympetala and P. occidentalis, when administered up to 5× the intended dose for 28 consecutive d, showed no adverse effects on the health of dogs.

L'anxiété de séparation et une aversion au bruit sont des problèmes de comportement fréquents chez les chiens. Elles élicitent des réponses de peur telles que des tremblements, la recherche du propriétaire, et une tentative de fuite. Elles peuvent résulter en des dommages à la propriété, des blessures au chien, et un bris du lien propriétaire-animal, pouvant potentiellement mener à l'abandon de l'animal ou l'euthanasie. Un nouveau produit anxiolytique botanique a été évalué pour sa sécurité chez les chiens, l'espèce animale cible. Son utilisation visée est pour le traitement et la prévention de l'anxiété et de l'aversion au bruit chez les chiens. Le produit contient un mélange défini de vigne de Souroubea spp. et d'écorce de Platanus spp., fournissant le principe actif, l'acide bétulinique, à un dosage recommandé de 1 mg/kg de poids corporel (PC). Dans l'étude de sécurité chez l'espèce animale cible, 16 chiens mâles de race beagle en santé ont reçu soit un placebo ou les nouvelles tablettes botaniques à 0,5×, 2,5×, ou 5× la dose recommandée (1 mg/kg PC) pendant 28 jours. Les chiens ont été observés pour l'apparition de manifestions adverses systémiques ou locales. Dans l'étude présentée ici, il n'y eut aucun effet clinique adverse significatif suivant le traitement, tel que déterminé par les observations cliniques, les examens physiques, le PC, et les résultats des analyses hématologiques, de biochimie clinique et urinaires. L'analyse pharmacocinétique a démontré que la concentration d'acide bétulinique dans le sérum était moins de 0,020 µg/mL chez les animaux traités. Dans les conditions des présentes études, le mélange de S. sympetala et de P. occidentalis, lorsqu'administré jusqu'à 5× le dosage prévu pendant 28 jours consécutifs, n'a démontré aucun effet adverse sur la santé des chiens.(Traduit par Docteur Serge Messier).

Effects of long-term cadmium exposure on urinary metabolite profiles in mice.

Cadmium (Cd) is a common environmental pollutant with known toxic effects on the kidney. Urinary metabolomics is a promising approach to study mechanism by which Cd-induced nephrotoxicity. The aim of this study was to elucidate the mechanism of Cd toxicity and to develop specific biomarkers by identifying urinary metabolic changes after a long-term of Cd exposure and with the critical concentration of Cd in the kidney. Urine samples were collected from wild-type 129/Sv mice after 67 weeks of 300 ppm Cd exposure and analyzed by ultra performance liquid chromatography connected with quadrupole time of flight mass spectrometer (UPLC-QTOF-MS) based metabolomics approach. A total of 40 most differentiated metabolites (9 down-regulated and 31 up-regulated) between the control and Cd-exposed group were identified. The majority of the regulated metabolites are amino acids (glutamine, L-aspartic acid, phenylalanine, tryptophan, and D-proline) indicating that amino acid metabolism pathways are affected by long-term exposure of Cd. However, there are also some nucleotides (guanosine, guanosine monophosphate, cyclic AMP, uridine), amino acid derivatives (homoserine, N-acetyl-L-aspartate, N-acetylglutamine, acetyl-phenylalanine, carboxymethyllysine), and peptides. Results of pathway analysis showed that the arginine and proline metabolism, purine metabolism, alanine, aspartate and glutamate metabolism, and aminoacyl-tRNA biosynthesis were affected compared to the control. This study demonstrates that metabolomics is useful to elucidate the metabolic responses and biological effects induced by Cd-exposure.

Anti-apoptotic potential of several antidiabetic medicinal plants of the eastern James Bay Cree pharmacopeia in cultured kidney cells.

BACKGROUND: Our team has identified 17 Boreal forest species from the traditional pharmacopeia of the Eastern James Bay Cree that presented promising in vitro and in vivo biological activities in the context of type 2 diabetes (T2D). We now screened the 17 plants extracts for potential anti-apoptotic activity in cultured kidney cells and investigated the underlying mechanisms. METHODS: MDCK (Madin-Darnby Canine Kidney) cell damage was induced by hypertonic medium (700 mOsm/L) in the presence or absence of maximal nontoxic concentrations of each of the 17 plant extracts. After 18 h' treatment, cells were stained with Annexin V (AnnV) and Propidium iodide (PI) and subjected to flow cytometry to assess the cytoprotective (AnnV-/PI-) and anti-apoptotic (AnnV+/PI-) potential of the 17 plant extracts. We then selected a representative subset of species (most cytoprotective, moderately so or neutral) to measure the activity of caspases 3, 8 and 9. RESULTS: Gaultheria hispidula and Abies balsamea are amongst the most powerful cytoprotective and anti-apoptotic plants and appear to exert their modulatory effect primarily by inhibiting caspase 9 in the mitochondrial apoptotic signaling pathway. CONCLUSION: We conclude that several Cree antidiabetic plants exert anti-apoptotic activity that may be relevant in the context of diabetic nephropathy (DN) that affects a significant proportion of Cree diabetics.

Cymbopogon citratus and Camellia sinensis extracts selectively induce apoptosis in cancer cells and reduce growth of lymphoma xenografts in vivo.

Cancer cells are reported to have elevated levels of reactive oxygen species (ROS) and are highly dependent on cellular defense mechanisms against oxidative stress. Numerous nutraceuticals and natural polyphenolic compounds have a wide range of abilities to alter cellular redox states with potential implications in various diseases. Furthermore, therapeutic options for cancers are mostly nonselective treatments including genotoxic or tubulin-targeting compounds. Some of the natural extracts, containing multiple bioactive compounds, could target multiple pathways in cancer cells to selectively induce cell death. Cymbopogon citratus (lemongrass) and Camellia sinensis (white tea) extracts have been shown to have medicinal properties, however, their activity against lymphoma and leukemia, as well as mechanistic details, have not been fully characterized. Herein, we report potent anti-cancer properties in dose and time-dependent manners of ethanolic lemongrass and hot water white tea extracts in lymphoma and leukemia models. Both extracts were able to effectively induce apoptosis selectively in these human cancer cell types. Interestingly, ethanolic lemongrass extract induces apoptosis primarily by the extrinsic pathway and was found to be dependent on the generation of ROS. Conversely, apoptotic induction by hot water white tea extract was independent of ROS. Furthermore, both of these extracts caused mitochondrial depolarization and decreased rates of oxygen consumption in lymphoma and leukemia cells, leading to cell death. Most importantly, both these extracts were effective in reducing tumor growth in human lymphoma xenograft models when administered orally. Thus, these natural extracts could have potential for being nontoxic alternatives for the treatment of cancer.

Phytogeographic and genetic variation in Sorbus, a traditional antidiabetic medicine-adaptation in action in both a plant and a discipline.

Mountain ash (Sorbus decora and S. americana) is used by the Cree Nation of the James Bay region of Quebec (Eeyou Istchee) as traditional medicine. Its potential as an antidiabetic medicine is thought to vary across its geographical range, yet little is known about the factors that affect its antioxidant capacity. Here, we examined metabolite gene expression in relation to antioxidant activity, linking phytochemistry and medicinal potential. Samples of leaf and bark from S. decora and S. americana were collected from 20 populations at four different latitudes. Two genes known to produce antidiabetic substances, flavonol synthase and squalene synthase, were analyzed using quantitative real time PCR. Gene expression was significantly higher for flavonol synthase compared to squalene synthase and increased in the most Northern latitude. Corresponding differences observed in the antioxidant capacity of ethanolic extracts from the bark of Sorbus spp. confirm that plants at higher latitudes increase production of stress-induced secondary metabolites and support Aboriginal perceptions of their higher medicinal potential. Modern genetic techniques such as quantitative real time PCR offer unprecedented resolution to substantiate and scrutinise Aboriginal medicinal plant perception. Furthermore, it offers valuable insights into how environmental stress can trigger an adaptive response resulting in the accumulation of secondary metabolites with human medicinal properties.

Larix laricina bark, a traditional medicine used by the Cree of Eeyou Istchee: Antioxidant constituents and in vitro permeability across Caco-2 cell monolayers.

ETHNOPHARMACOLOGICAL RELEVANCE: Larix laricina, a native tree of North America, is a highly respected medicinal plant used for generations by Indigenous Peoples across its range, including the Cree of northern Québec who use the bark to treat symptoms of diabetes. This study investigates the antioxidant capacity and bioavailability of active constituents identified in L. laricina bark extracts. MATERIALS AND METHODS: (1) Oxygen radical absorbance capacity (ORAC) assay was employed to test antioxidant capacity of organic extracts (80% ethanol) from bark of L. laricina as well as fractions, isolated compounds, and media samples collected during permeability assays. (2) Caco-2 cell monolayer cultures were used to determine the permeability of identified antioxidants, which were quantified in basolateral media samples using liquid chromatography - tandem mass spectrometry (HPLC-ESI-MS/MS). RESULTS: Crude ethanolic extract possessed strong antioxidant potential in vitro (7.1±0.3 Trolox equivalents (TE) µM/mg). Among the 16 L. laricina fractions obtained by chromatographic separation, fraction 10 (F10) showed the highest antioxidant capacity (21.8±1.7µm TE/mg). Among other identified antioxidants, the stilbene rhaponticin (isolated from F10) was the most potent (24.6±1.1µm TE/mg). Caco-2 transport studies revealed that none of the identified compounds were detectable in basolateral samples after 2-h treatment with crude extract. In monolayers treated with F10 (60% rhaponticin), small quantities of rhaponticin were increasingly detected over time in basolateral samples with an apparent permeability coefficient (Papp) of 1.86×10-8cm/s (0-60min). To model potential effects on blood redox status, we evaluated the antioxidant capacity of collected basolateral samples and observed enhanced activity over time after exposure to both extract and F10 (75µg/mL) relative to control. CONCLUSIONS: By profiling the antioxidant constituents of L. laricina bark, we identified rhaponticin as the most potent oxygen radical scavenger and observed low permeability in Caco-2 cell monolayers but an increase in basolateral antioxidant capacity.

An HPLC-ELSD Method for the Determination of Triterpenes in Sorbus decora and Sorbus americana Bark Used by the Eeyou Istchee Cree First Nation.

Sorbus decora and Sorbus americana are used traditionally as medicine by the Eeyou Istchee Cree First Nation of the James Bay region of Quebec, Canada. Because the ethanol extracts of the bark and the isolated terpenes of these plants have shown promising in vivo antidiabetic effects, an analytical method was developed and validated by RP-HPLC-ELSD for the identification and quantification of eight lupane- and ursane-type terpenes. The extraction method reproducibly recovered the compounds above 70 % and the chromatographic separation of betulin, 23-hydroxy-betulin, 23,28-dihydroxylupan-20(29)-ene-3ß-caffeate, betulinic acid, α-amyrin, uvaol, 3ß,23,28-trihydroxy-12-ursene, and 23,28-dihydroxyursan-12-ene-3ß-caffeate was achieved within 27 min by linear gradient. The method produced highly reproducible quantitative data at interday and intraday levels. The limits of detection were in the ng level on-column with remarkable range and linearity. The target compounds were present at mg levels in the populations, collected from inland (Mistissini and Nemaska) and costal (Waskagnish and Chisasibi) Cree communities of northern Quebec. A triterpene, 23-hydroxybetulin, was the most abundant, while betulinic acid and uvaol were minor constituents. Overall, HPLC-ELSD analyses produced very similar profiles and contents of the eight compounds in the plants collected from four geographic locations. The developed HPLC-ELSD method can be used as a targeted analysis of triterpenes in these medicinal plants.

A combination of (+)-catechin and (-)-epicatechin underlies the in vitro adipogenic action of Labrador tea (Rhododendron groenlandicum), an antidiabetic medicinal plant of the Eastern James Bay Cree pharmacopeia.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhododendron groenlandicum (Oeder) Kron & Judd (Labrador tea) was identified as an antidiabetic plant through an ethnobotanical study carried out with the close collaboration of Cree nations of northern Quebec in Canada. OBJECTIVES: In a previous study the plant showed glitazone-like activity in a 3T3-L1 adipogenesis bioassay. The current study sought to identify the active compounds responsible for this potential antidiabetic activity using bioassay guided fractionation based upon an in vitro assay that measures the increase of triglycerides content in 3T3-L1 adipocyte. MATERIALS AND METHODS: Isolation and identification of the crude extract's active constituents was carried out. The 80% ethanol extract was fractionated using silica gel column chromatography. Preparative HPLC was then used to isolate the constituents. The identity of the isolated compounds was confirmed by UV and mass spectrometry. RESULTS: Nine chemically distinct fractions were obtained and the adipogenic activity was found in fraction 5 (RGE-5). Quercetins, (+)-catechin and (-)-epicatechin were detected and isolated from this fraction. While (+)-catechin and (-)-epicatechin stimulated adipogenesis (238±26% and 187±21% relative to vehicle control respectively) at concentrations equivalent to their concentrations in the active fraction RGE-5, none afforded biological activity similar to RGE-5 or the plant's crude extract when used alone. When cells were incubated with a mixture of the two compounds, the adipogenic activity was close to that of the crude extract (280.7±27.8 vs 311± 30%). CONCLUSION: Results demonstrate that the mixture of (+)-catechin and (-)-epicatechin is responsible for the adipogenic activity of Labrador tea. This brings further evidence for the antidiabetic potential of R. groenlandicum and provides new opportunities to profile active principles in biological fluids or in traditional preparations.

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.

Selective Induction of Apoptosis by Azadarichta indica Leaf Extract by Targeting Oxidative Vulnerabilities in Human Cancer Cells.

PURPOSE: Natural products have been a great source of medications used in conventional medicines for the treatment of various diseases; more importantly, they have played a significant role in the development of anti-cancer drugs for a number of decades. The benefits to employing whole extracts of natural health products, rather than a single ingredient, for cancer treatment remains unexplored. Our research group has previously demonstrated the potential anti-cancer benefits of several natural health products (NHPs), prompting further studies into other NHPs, such as Neem (Azadarichta indica), a tree native to India and has been used in Ayurvedic medicine for over 4000 years. The objective of this study is to determine the possible anti-cancer potential of aqueous and ethanolic Neem leaf extracts (NLEs) and to identify the specific mode(s) of action. METHODS: Cells were treated with NLE and cell viability was then assessed using a water-soluble tetrazolium salt. Cell death was confirmed using the fluorescent dye propidium iodide and apoptosis was identified using the Annexin-V binding assay. Mitochondrial membrane permeabilization was visualized using JC-1 staining and the production of whole cell and mitochondrial ROS was measured with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and Amplex Red, respectively. In vivo efficacy of aqueous NLE was assessed in human tumour xenografts in CD-1 nu/nu immunocompromised mice. RESULTS: Results indicate that both ethanolic and aqueous extracts of Neem leaf were effective in inducing apoptosis in leukemia and colon cancer cells, following destabilization of the mitochondrial membrane. Furthermore, an increase in the production of reactive oxygen species (ROS) was observed in cancer cells treated with NLEs, indicating that oxidative stress may play a role in the mechanism of cell death. Additionally, in vivo results showed that aqueous NLE (delivered orally) was well tolerated and inhibited tumour growth of human xenografts in mice. CONCLUSIONS: These findings suggest the potential of NLEs as safer and effective alternatives to conventional chemotherapy. 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.

Novel Approach to Identify Potential Bioactive Plant Metabolites: Pharmacological and Metabolomics Analyses of Ethanol and Hot Water Extracts of Several Canadian Medicinal Plants of the Cree of Eeyou Istchee.

We evaluated and compared the antidiabetic potential and molecular mechanisms of 17 Cree plants' ethanol extracts (EE) and hot water extracts (HWE) on glucose homeostasis in vitro and used metabolomics to seek links with the content of specific phytochemicals. Several EE of medical plants stimulated muscle glucose uptake and inhibited hepatic G6Pase activity. Some HWE partially or completely lost these antidiabetic activities in comparison to EE. Only R. groenlandicum retained similar potential between EE and HWE in both assays. In C2C12 muscle cells, EE of R. groenlandicum, A. incana and S. purpurea stimulated glucose uptake by activating AMP-activated protein kinase (AMPK) pathway and increasing glucose transporter type 4 (GLUT4) expression. In comparison to EE, HWE of R. groenlandicum exhibited similar activities; HWE of A. incana completely lost its effect on all parameters; interestingly, HWE of S. purpurea activated insulin pathway instead of AMPK pathway to increase glucose uptake. In the liver, for a subset of 5 plants, HWE and EE activated AMPK pathway whereas the EE and HWE of S. purpurea and K. angustifolia also activated insulin pathways. Quercetin-3-O-galactoside and quercetin 3-O-α-L-arabinopyranoside, were successfully identified by discriminant analysis as biomarkers of HWE plant extracts that stimulate glucose uptake in vitro. More importantly, the latter compound was not identified by previous bioassay-guided fractionation.

Regulation of liver cell glucose homeostasis by dehydroabietic acid, abietic acid and squalene isolated from balsam fir (Abies balsamea (L.) Mill.) a plant of the Eastern James Bay Cree traditional pharmacopeia.

In our previous study, Abies balsamea (L.) Mill., a plant used in Cree traditional medicine, had a strong effect on the regulation of glucose homeostasis in liver cells. This study aimed to isolate and identify its active constituents using a bioassay-guided fractionation approach as well as to elucidate their mechanism(s) of action. The effect of the crude extract and its constituents was evaluated on the activity of Glucose-6-Phosphatase (G6Pase) and Glycogen Synthase (GS) and phosphorylation of three kinases, AMP-activated protein kinase (AMPK), Akt and Glycogen Synthase Kinase-3 (GSK-3). Three compounds, abietic acid, dehydroabietic acid and squalene, were isolated from the most active fraction in the bioassays (hexane). The compounds were able to decrease the activity of G6Pase and to stimulate GS. Their effect on G6Pase activity involved both Akt and AMPK phosphorylation with significant correlations between insulin-dependent and -independent pathways and the bioassay. In addition, the compounds were able to stimulate GS through GSK-3 phosphorylation with a significant correlation between the signaling pathway and the bioassay. Dehydroabietic acid stood out for its strongest effect in all the experiments close to that of the crude extract. These compounds may have potential applications in the treatment of type 2 diabetes and insulin resistance.

Analysis of intracellular and extracellular microcystin variants in sediments and pore waters by accelerated solvent extraction and high performance liquid chromatography-tandem mass spectrometry.

The fate and persistence of microcystin cyanotoxins in aquatic ecosystems remains poorly understood in part due to the lack of analytical methods for microcystins in sediments. Existing methods have been limited to the extraction of a few extracellular microcystins of similar chemistry. We developed a single analytical method, consisting of accelerated solvent extraction, hydrophilic-lipophilic balance solid phase extraction, and reversed phase high performance liquid chromatography-tandem mass spectrometry, suitable for the extraction and quantitation of both intracellular and extracellular cyanotoxins in sediments as well as pore waters. Recoveries of nine microcystins, representing the chemical diversity of microcystins, and nodularin (a marine analogue) ranged between 75 and 98% with one, microcystin-RR (MC-RR), at 50%. Chromatographic separation of these analytes was achieved within 7.5 min and the method detection limits were between 1.1 and 2.5 ng g(-1) dry weight (dw). The robustness of the method was demonstrated on sediment cores collected from seven Canadian lakes of diverse geography and trophic states. Individual microcystin variants reached a maximum concentration of 829 ng g(-1) dw on sediment particles and 132 ng mL(-1) in pore waters and could be detected in sediments as deep as 41 cm (>100 years in age). MC-LR, -RR, and -LA were more often detected while MC-YR, -LY, -LF, and -LW were less common. The analytical method enabled us to estimate sediment-pore water distribution coefficients (K(d)), MC-RR had the highest affinity for sediment particles (log K(d)=1.3) while MC-LA had the lowest affinity (log K(d)=-0.4), partitioning mainly into pore waters. Our findings confirm that sediments serve as a reservoir for microcystins but suggest that some variants may diffuse into overlying water thereby constituting a new route of exposure following the dissipation of toxic blooms. The method is well suited to determine the fate and persistence of different microcystins in aquatic systems.