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.

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).

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.

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.

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).

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.

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.

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.

Cytochrome P450 3A4 and 2D6-mediated metabolism of leisure and medicinal teas.

PURPOSE: Thirty-five commercially available Camellia sinensis (black and green) and herbal leisure teas and an assortment of Traditional Chinese medicinal teas were randomly selected and examined for their potential to inhibit the drug metabolizing enzyme cytochrome P450 3A4 (CYP3A4). The study was then extended to examine CYP2D6*1 and CYP2D6*10. METHODS: Microtiter fluorometric assays were utilized to examine the potential for the teas to inhibit CYP-mediated metabolism. Aqueous or alcoholic extracts of the dried tea plant material were examined. METHODS: Most of the black and green leisure teas generally inhibited CYP3A4 more than the Chinese medicinal teas. The medicinal Chinese teas were generally more inhibitory towards CYP3A4 compared to the CYP2D6 isozymes, and the aqueous extracts displayed more potency than the alcoholic extracts. CONCLUSIONS: Tea whether used for leisure or medicinal purposes has the potential to inhibit CYP3A4-mediated drug metabolism particularly black tea.

The effect of Cree traditional medicinal teas on the activity of human cytochrome P450-mediated metabolism.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhododendron groenlandicum (Bog Labrador tea), Rhododendron tomentosum (Marsh Labrador tea) and Juniperus communis (Juniper) are used in medicinal teas by Canadian aboriginal cultures alone and in combination with conventional drug products. The safety of this combination had not been previously examined and this study was initiated to examine the potential of medicinal teas to inhibit the major human drug metabolizing enzyme, cytochrome P450 3A4 (CYP3A4). MATERIALS AND METHODS: The decoctions of Rhododendron groenlandicum and Rhododendron tomentosum leaves and Juniperus communis berries were examined in a microtiter fluorometric assay to examine their potential to inhibit CYP-mediated metabolism. RESULTS: The decoctions showed progressive inhibition towards CYP3A4 the longer the leaves or berries were brewed. R. Rhododendron groenlandicum and Juniperus communis may have the potential to inhibit CYP3A4-mediated metabolism. CONCLUSIONS: The findings of this study with these traditional medicines are significant in that they provide mechanistic support that these products have the potential to affect the safety and efficacy of other health and medicinal products. As this study only examined CYP3A4, it is possible that these medicinals contain substances that could also affect other metabolic enzymes.

Canadian boreal pulp and paper feedstocks contain neuroactive substances that interact in vitro with GABA and dopaminergic systems in the brain.

Pulp and paper wood feedstocks have been previously implicated as a source of chemicals with the ability to interact with or disrupt key neuroendocrine endpoints important in the control of reproduction. We tested nine Canadian conifers commonly used in pulp and paper production as well as 16 phytochemicals that have been observed in various pulp and paper mill effluent streams for their ability to interact in vitro with the enzymes monoamine oxidase (MAO), glutamic acid decarboxylase (GAD), and GABA-transaminase (GABA-T), and bind to the benzodiazepine-binding site of the GABA(A) receptor (GABA(A)-BZD). These neuroendocrine endpoints are also important targets for treatment of neurological disorders such as anxiety, epilepsy, or depression. MAO and GAD were inhibited by various conifer extracts of different polarities, including major feedstocks such as balsam fir, black spruce, and white spruce. MAO was selectively stimulated or inhibited by many of the tested phytochemicals, with inhibition observed by a group of phenylpropenes (e.g. isoeugenol and vanillin). Selective GAD inhibition was also observed, with all of the resin acids tested being inhibitory. GABA(A)-BZD ligand displacement was also observed. We compiled a table identifying which of these phytochemicals have been described in each of the species tested here. Given the diversity of conifer species and plant chemicals with these specific neuroactivities, it is reasonable to propose that MAO and GAD inhibition reported in effluents is phytochemical in origin. We propose disruption of these neuroendocrine endpoints as a possible mechanism of reproductive inhibition, and also identify an avenue for potential research and sourcing of conifer-derived neuroactive natural products.

In vitro inhibition of metabolism but not transport of gliclazide and repaglinide by Cree medicinal plant extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Interactions between conventional drug and traditional medicine therapies may potentially affect drug efficacy and increase the potential for adverse reactions. Cree traditional healing is holistic and patients may use medicinal plants simultaneously with the conventional drugs. However, there is limited information that these medicinal plants may interact with drugs and additional mechanistic information is required. In this study, extracts from traditionally used Cree botanicals were assessed for their potential interaction that could alter the disposition of two blood glucose lowering drugs, gliclazide (Diamicron) and repaglinide (Gluconorm) though inhibition of either metabolism or transport across cell membranes. MATERIALS AND METHODS: The effect of 17 extracts on metabolism was examined in a human liver microsome assay by HPLC and individual cytochrome P450s 2C9, 2C19, 2C8 and 3A4 in a microplate fluorometric assay. Gliclazide, rhaponticin and its aglycone derivative, rhapontigenin were also examined in the fluorometric assay. The effect on transport was examined with 11 extracts using the intestinal epithelial Caco-2 differentiated cell monolayer model at times up to 180 min. RESULTS: Both blood glucose lowering medications, gliclazide and repaglinide traversed the Caco-2 monolayer in a time-dependent manner that was not affected by the Cree plant extracts. Incubation of the Cree plant extracts inhibited CYP2C9, 2C19, 2C8 and 3A4-mediated metabolism, and the formation of four repaglinide metabolites: M4, m/z 451-A, m/z 451-B and the glucuronide of repaglinide in the human liver microsome assay. Gliclazide caused no significant inhibition. Likewise, rhaponticin had little effect on the enzymes causing changes of less than 10% with an exception of 17% inhibition of CYP2C19. By contrast, the aglycone rhapontigenin showed the greatest effects on all CYP-mediated metabolism. Its inhibition ranged from a mean of 58% CYP3A4 inhibition to 89% inhibition of CYP2C9. While rhaponticin and the aglycone did not show significant effects on repaglinide metabolism, they demonstrated inhibition of gliclazide metabolism. The aglycone significantly affected levels of gliclazide and its metabolites. CONCLUSION: These studies demonstrate that the Cree plant extracts examined have the potential in vitro to cause drug interactions through effects on key metabolic enzymes.

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.

Populus balsamifera Extract and Its Active Component Salicortin Reduce Obesity and Attenuate Insulin Resistance in a Diet-Induced Obese Mouse Model.

Populus balsamifera L. (BP) is a medicinal plant stemming from the traditional pharmacopoeia of the Cree of Eeyou Istchee (CEI-Northern Quebec). In vitro screening studies revealed that it strongly inhibited adipogenesis in 3T3-L1 adipocytes, suggesting potential antiobesity activity. Salicortin was identified, through bioassay-guided fractionation, as the active component responsible for BP's activity. The present study aimed to assess the potential of BP and salicortin at reducing obesity and features of the metabolic syndrome, in diet-induced obese C57Bl/6 mice. Mice were subjected to high fat diet (HFD) for sixteen weeks, with BP (125 or 250 mg/kg) or salicortin (12.5 mg/kg) introduced in the HFD for the last eight of the sixteen weeks. BP and salicortin effectively reduced whole body and retroperitoneal fat pad weights, as well as hepatic triglyceride accumulation. Glycemia, insulinemia, leptin, and adiponectin levels were also improved. This was accompanied by a small yet significant reduction in food intake in animals treated with BP. BP and salicortin (slightly) also modulated key components in signaling pathways involved with glucose regulation and lipid oxidation in the liver, muscle, and adipose tissue. These results confirm the validity of the CEI pharmacopoeia as alternative and complementary antiobesity and antidiabetic therapies.