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.

Phytochemistry in the Ethnopharmacology of North and Central America.

Traditionally the role of phytochemistry in the ethnopharmacology of North and Central America has been to characterize plant materials so that they can be produced reproducibly for commercial use or to identify active principles in unstudied traditional medicines for drug discovery. With new decolonial objectives coming from Indigenous communities, emphasis has shifted to evaluating the safety and efficacy of traditional medicines and preparations for community use. With new techniques and technologies available, scientific focus has shifted from individual bioactives to more rapid and comprehensive chemical characterizations and polypharmacy of traditional medicines. Untargeted metabolomics and associated statistical treatments have greatly expanded identification of components, improved species and cultivar identification and provided means for identifying multiple activity biomarkers, via chemometric and biochemometric analysis. New integrated techniques are available for identifying multiple active principles and synergists. The recent explosion of information is not without problems that need to be addressed including many unconfirmed tentative identifications of phytochemicals, lack of quantitative testing, superficial chemical activity testing and continuing need for dereplication.

Interactions of Echinacea spp. Root Extracts and Alkylamides With the Endocannabinoid System and Peripheral Inflammatory Pain.

Historical ethnobotanies of indigenous peoples of the North American prairies reveal treatment of many painful conditions by Echinacea spp. Recent evidence suggests a pharmacological basis for such use as the bioactivity of E. angustifolia and E. purpurea is mediated, in part, through activation of the endocannabinoid system (ECS). Whereas the cannabimimetic effects of individual echinacea products and alkylamides have been described, the activity of crude extracts has not been compared between cannabinoid (CB) receptors or across species or genotypes. Moreover, few studies have explored echinacea's engagement of the ECS for historic treatments or new therapeutic applications in peripheral inflammatory pain. We hypothesized that 1) the in vitro effects of root extracts on CB receptor internalization would vary with species and phytochemistry, and that echinacea root extracts would reduce inflammatory pain in vivo through activation of the ECS. Root extracts of different E. angustifolia and E. purpurea accessions were prepared, analyzed by HPLC-DAD to quantify caffeic acid derivatives and alkylamides (AKA), and tested for agonist and antagonist activities using receptor redistribution assays. Linear regression of activity relative to phytochemistry identified predictive compounds that were assessed individually in redistribution assays. Extracts were evaluated in the Hargreaves model of chronic inflammatory pain in rats with co-administration of selective CB1/2 antagonists to gauge involvement of the ECS. CB receptor agonist activity varied among accessions of both species with linear regression revealing a significant relationship between CB1 activity and AKA2 for E angustifolia, and AKA 9 + 10 for E purpurea. CB2 activity was positively related with AKA 9 + 10 and total AKAs in E. angustifolia. Four isolated AKA demonstrated agonist activity in the CB2, but not CB1, assay. In the inflammatory pain model, oral administration of either E angustifolia or E. purpurea root extract produced dose-dependent analgesic effects that were partially reversed by co-administration of CB receptor antagonists. This study demonstrates that in vitro effects of crude echinacea root extracts on CB receptors is predicted by phytochemistry. In vivo, echinacea has potential applications for peripheral inflammatory pain such as arthritis and burns, reflecting the traditional uses of Indigenous North Americans.

Efficacy of Souroubea-Platanus Dietary Supplement Containing Triterpenes in Beagle Dogs Using a Thunderstorm Noise-Induced Model of Fear and Anxiety.

A novel botanical dietary supplement, formulated as a chewable tablet containing a defined mixture of Souroubea spp. vine and Platanus spp. Bark, was tested as a canine anxiolytic for thunderstorm noise-induced stress (noise aversion). The tablet contained five highly stable triterpenes and delivered 10 mg of the active ingredient betulinic acid (BA) for an intended 1 mg/kg dose in a 10 kg dog. BA in tablets was stable for 30 months in storage at 23 °C. Efficacy of the tablets in reducing anxiety in dogs was assessed in a blinded, placebo-controlled study by recording changes in blood cortisol levels and measures of behavioral activity in response to recorded intermittent thunder. Sixty beagles were assigned into groups receiving: placebo, 0.5×, 1×, 2×, and 4× dose, or the positive control (diazepam), for five days. Reduction in anxiety measures was partially dose-dependent and the 1× dose was effective in reducing inactivity time (p = 0.0111) or increased activity time (p = 0.0299) compared with placebo, indicating a decrease in anxiety response. Cortisol measures also showed a dose-dependent reduction in cortisol in dogs treated with the test tablet.

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.

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.

Potent CYP3A4 Inhibitors Derived from Dillapiol and Sesamol.

Synthesis of 50 analogues of the natural insecticide synergists, dillapiol and sesamol, is reported. These were evaluated as potential insecticide synergists based on their inhibition of human CYP3A4. The most potent inhibitors have a relatively large hydrophobic substituent at either position 5 or 6 of these molecules. For example, 5-(benzyloxy)-6-(3-phenylsulfonyl)propyl)benzo[d][1,3]dioxole (18) and the diphenyl acetate of (6,7-dimethoxybenzo[d][1,3]dioxol-5-yl)propan-1-ol (5n) show inhibitory concentrations for 50% activity IC50 values of 0.086 and 0.2 µM, respectively. These compounds are 106 and 46 times more potent than dillapiol whose IC50 for the inhibition of CYP3A4 is 9.2 µM. The ortho-chloro analogue (8f), whose activity is 86 times the activity of dillapiol, is the most potent of the fourteen 5-(benzyloxy-6-(2 propenyl)benzo[d][1,3]dioxoles prepared for this study.

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.

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.

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.

Extract and Active Principal of the Neotropical Vine Souroubea sympetala Gilg. Block Fear Memory Reconsolidation.

Background: Souroubea sympetala Gilg. is a neotropical vine native to Central America, investigated as part of a targeted study of the plant family Marcgraviaceae. Our previous research showed that extract of S. sympetala leaf and small branch extract had anxiolytic effects in animals and acts as an agonist for the GABAA receptor at the benzodiazepine binding site. To date, the potential effects of S. sympetala and its constituents on reconsolidation have not been assessed. Reconsolidation, the process by which formed memories are rendered labile and susceptible to change, may offer a window of opportunity for pharmacological manipulation of learned fear. Here, we assessed the effects of S. sympetala crude extract and isolated phytochemicals (orally administered) on the reconsolidation of conditioned fear. In addition, we explored whether betulin (BE), a closely related molecule to betulinic acid (BA, an active principal component of S. sympetala), has effects on reconsolidation of learned fear and whether BE may synergize with BA to enhance attenuation of learned fear. Method: Male Sprague-Dawley rats received six 1.0-mA continuous foot shocks (contextual training). Twenty-four hours later, rats were re-exposed to the context (but in the absence of foot shocks). Immediately following memory retrieval (recall), rats received oral administration of S. sympetala extract at various doses (8-75 mg/kg) or diazepam (1 mg/kg). In separate experiments, we compared the effects of BA (2 mg/kg), BE (2 mg/kg), and BA + BE (2 mg/kg BA + 2 mg/kg BE). The freezing response was assessed either 24 h later (day 3) or 5 days later (day 7). Effects of phytochemicals on fear expression were also explored using the elevated plus maze paradigm. Results: S. sympetala leaf extract significantly attenuated the reconsolidation of contextual fear at the 25- and 75-mg/kg doses, but not at the 8-mg/kg dose. Furthermore, BA + BE, but not BA or BE alone, attenuated the reconsolidation of learned fear and exerted an anxiolytic-like effect on fear expression.

Effect of an anxiolytic botanical containing Souroubea sympetala and Platanus occidentalis on in-vitro diazepam human cytochrome P450-mediated metabolism.

OBJECTIVES: A novel anxiolytic natural health product (NHP) containing Souroubea sympetala and Platanus occidentalis is available for the companion animal market and is currently being developed for clinical evaluation. Addressing the risk of potential NHP-drug interactions, this study investigated S. sympetala and P. occidentalis plant extracts, and their identified bioactive compounds, for effects on the activity of cytochrome P450 (CYP) isozymes and the metabolism of the conventional anti-anxiety medication diazepam. METHODS: Souroubea sympetala and P. occidentalis extracts, a 1 : 1 blend of the two extracts, and five triterpenes were tested for inhibitory effects on human recombinant CYP3A4, CYP2D6, CYP2C9 and CYP2C19 activity using a fluorometric plate assay. Direct effects on the metabolism of diazepam were evaluated using human liver microsomes with drug and metabolite quantification by ultra-high-pressure liquid chromatography and mass spectroscopy. KEY FINDINGS: The active substances betulinic acid (BA) and ursolic acid (UA) strongly inhibited CYP3A4 activity while UA and lupeol moderately inhibited CYP2C19. All extracts exhibited strong activity against the tested isozymes at 50-100 µg/ml. BA and all plant extracts blocked the formation of major diazepam metabolites. CONCLUSIONS: Betulinic acid, UA and both the extracts and blended product are expected to affect the metabolism of diazepam when given in high dose.

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).

Sacred Maya incense, copal (Protium copal - Burseraceae), has antianxiety effects in animal models.

ETHNOPHARMACOLOGICAL RELEVANCE: The Maya have traditionally used copal, Protium copal, as incense during ceremonies since pre-Columbian times. Anecdotally, copal (when burned as incense), is thought to elicit mentally uplifting and calming effects. The main objective of this study was to determine whether the incense elicits anxiolytic-like behavior in animal models using rats. A second objective was to characterize active constituents and discern potential mechanism(s) of action, specifically the involvement of the GABAergic and endocannabinoid (eCB) systems. Despite the extensive Central American use of this resin, there are currently no known scientific behavioral or pharmacological studies done with the incense. MATERIALS AND METHODS: Quantification of the triterpenes in the copal resin and cold trapped incense was achieved by HPLC MS. Behavioral effects in rats were assessed using the elevated plus maze (EPM), social interaction (SI) test, conditioned emotion response (CER) and Novel object recognition (NOR) paradigms. Rats were exposed to burning copal (200 mg) over 5 min in a smoking chamber apparatus and then immediately tested in each behavioral paradigm. Follow-up SI tests were done using two antagonists flumazenil (1 mg/kg) and AM251 (1 mg/kg) administered systemically. Inhibition of MAGL (monoacylglycerol lipase) was measured by microplate assay with recombinant human enzyme and probe substrate. RESULTS: Phytochemical analysis revealed that copal resin and incense had high α- and ß-amyrins and low lupeol triterpene content. Exposure to Protium copal incense significantly reduced anxiety-like behavior in the SI and CER tests. In contrast, no anxiolytic effects were observed in the EPM. The CER effect was time dependent. Both flumazenil and AM251 blocked the anxiolytic activity of copal revealing the involvement of GABAergic and endocannabinoid systems. Copal, as well as the identified triterpenes, potently inhibited monoacylglycerol lipase (MAGL) activity in vitro (IC50 ≤ 811 ng/mL). CONCLUSIONS: This is the first study to show that copal incense from Protium copal elicits anxiolytic-like effects in fear and social interaction models as evidenced by a reduced learned fear behavior and an increase in active social interaction. It's high α and ß-amyrin content suggests behavioral effects may be mediated, in part, by the known action of these terpenes at the benzodiazepine receptor. Furthermore, P. copal's observed activity through the eCB system via MAGL offers a new potential mechanism underlying the anxiolytic activity.

Vaccinium angustifolium (lowbush blueberry) leaf extract increases extravillous trophoblast cell migration and invasion in vitro.

Perturbations to extravillous trophoblast (EVT) cell migration and invasion are associated with the development of placenta-mediated diseases. Phytochemicals found in the lowbush blueberry plant (Vaccinium angustifolium) have been shown to influence cell migration and invasion in models of tumorigenesis and noncancerous, healthy cells, however never in EVT cells. We hypothesized that the phenolic compounds present in V. angustifolium leaf extract promote trophoblast migration and invasion. Using the HTR-8/SVneo human EVT cell line and Boyden chamber assays, the influence of V. angustifolium leaf extract (0 to 2 × 104  ng/ml) on trophoblast cell migration (n = 4) and invasion (n = 4) was determined. Cellular proliferation and viability were assessed using immunoreactivity to Ki67 (n = 3) and trypan blue exclusion assays (n = 3), respectively. At 20 ng/ml, V. angustifolium leaf extract increased HTR-8/SVneo cell migration and invasion (p < .01) and did not affect cell proliferation or viability. Chlorogenic acid was identified as a major phenolic compound of the leaf extract and the most active compound. Evidence from Western blot analysis (n = 3) suggests that the effects of the leaf extract and chlorogenic acid on trophoblast migration and invasion are mediated through an adenosine monophosphate-activated protein (AMP) kinase-dependent mechanism. Further investigations examining the potential therapeutic applications of this natural health product extract and its major chemical compounds in the context of placenta-mediated diseases are warranted.

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.

New Botanical Anxiolytics for Use in Companion Animals and Humans.

As part of our ongoing research into botanical therapies for anxiety disorders, the neotropical vine Souroubea sympetala was chosen for study as a phytochemical discovery strategy focusing on rare Central American plant families. When orally administered to male Sprague-Dawley rats, the crude plant extract, its ethyl acetate fraction, supercritical carbon dioxide fraction, or its isolated triterpenes reduced anxiety and/or fear-related behavior in standardized behavioral models. Pharmacological studies showed that the extracts acted at the benzodiazepine GABAA receptor and reduced corticosterone levels. A preparation containing Souroubea fortified with a second triterpene containing plant, Platanus occidentalis, was shown to be safe in a 28-day feeding trial with beagles at 5 times the intended dose. Subsequent trials with beagles in a thunderstorm model of noise aversion showed that the material reduced anxiety behaviors and cortisol levels in dogs. The formulation has been released for the companion animal market in Canada and the USA under the Trademark "Zentrol." Ongoing research is exploring the use of the material in treatment of anxiety and post-traumatic stress in humans.

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.