Traditional medicines used by Q'eqchi' Maya to treat diabetic symptoms and their antiglycation potential.

ETHNOPHARMACOLOGICAL RELEVANCE: Because of the recent increase in type 2 diabetes and the need for complementary treatments in remote communities in many parts of the world, we undertook a study of treatments for diabetic symptoms used by traditional Q'eqchi' Maya healers of Belize. We used quantitative ethnobotany to rank culturally important taxa and subsequent pharmacological and phytochemical studies to assess bioactivity. MATERIALS AND METHODS: Antidiabetic plants identified in field interviews with traditional healers were ranked by syndromic importance value (SIV) based on 15 symptoms of diabetes. Species ranked with high SIV were tested in an assay relevant to many diabetes complications, the advanced glycation endproduct (AGE) inhibition assay. Active principles were identified by phytochemical analysis and bioassay. RESULTS: We collected over 70 plant species having a promising SIV score. The plants represented a broad range of neotropical taxa. Selected Q'eqchi' antidiabetic plants with high SIV were collected in bulk and tested in the advanced glycation endproduct (AGE) inhibition assay. All plant extracts showed AGE inhibition and the half maximal inhibitory concentration (IC50) ranged from 40.8 to 733 µg/mL, while the most active species was Tynanthus guatemalensis Donn (Bignoniaceae). A linear regression showed a significant relationship between 1/ IC50 and SIV. Phytochemical analysis revealed the presence of verbascoside, as a major component and active principle of the T guatemalensis which had an IC50 = 5.1 µg/mL, comparable to the positive control quercetin. CONCLUSION: The results reveal a rich botanical tradition of antidiabetic symptom treatments among the Q'eqchi'. Study of highly ranked plants revealed their activity in AGE inhibition correlated with SIV. T. guatemalensis was identified as a promising species for further evaluation and local use.

MUC1 inhibition leads to decrease in PD-L1 levels via upregulation of miRNAs.

The PD-L1/PD-1 pathway is a critical component of the immunosuppressive tumor microenvironment in acute myeloid leukemia (AML), but little is known about its regulation. We investigated the role of the MUC1 oncoprotein in modulating PD-L1 expression in AML. Silencing of MUC1 in AML cell lines suppressed PD-L1 expression without a decrease in PD-L1 mRNA levels, suggesting a post-transcriptional mechanism of regulation. We identified the microRNAs miR-200c and miR-34a as key regulators of PD-L1 expression in AML. Silencing of MUC1 in AML cells led to a marked increase in miR-200c and miR-34a levels, without changes in precursor microRNA, suggesting that MUC1 might regulate microRNA-processing. MUC1 signaling decreased the expression of the microRNA-processing protein DICER, via the suppression of c-Jun activity. NanoString (Seattle, WA, USA) array of MUC1-silenced AML cells demonstrated an increase in the majority of probed microRNAs. In an immunocompetent murine AML model, targeting of MUC1 led to a significant increase in leukemia-specific T cells. In concert, targeting MUC1 signaling in human AML cells resulted in enhanced sensitivity to T-cell-mediated lysis. These findings suggest MUC1 is a critical regulator of PD-L1 expression via its effects on microRNA levels and represents a potential therapeutic target to enhance anti-tumor immunity.

Anxiolytic activity and active principles of Piper amalago (Piperaceae), a medicinal plant used by the Q'eqchi' Maya to treat susto, a culture-bound illness.

ETHNOPHARMACOLOGICAL RELEVANCE: The medicinal plant, Piper amalago L. (Piperaceae), is used traditionally by Q'eqchi' Maya healers for the treatment of "susto" a culture-bound syndrome. Previous research suggests that susto symptoms may be a manifestation of anxiety. The objectives were to characterize the effect of ethanolic extract of P. amalago in behavioral assays of anxiety at doses representative of traditional use and to isolate active principles. MATERIALS AND METHODS: Rats treated orally with low dose ethanolic extracts of P. amalago leaves (8-75mg/kg) were tested in several behavioral paradigms including the elevated plus maze (EPM), social interaction (SI), and conditioned emotional response (CER) tests, and compared to diazepam, a positive control. The active anxiolytic principle was isolated by bioassay guided isolation using an in vitro GABAA competitive binding assay. RESULTS: Extracts had significant anxiolytic activity in all behavioral tests, with the strongest activity in the SI and the CER paradigms. In an in vitro GABAA competitive binding assay, a 66.5µg/mL concentration of P. amalago ethanol extract displaced 50% of the GABAA-BZD receptor ligand [(3)H]-Flunitrazepam. Bioassay-guided fractionation identified a furofuran lignan, a molecule with structural similarity to yangambin, with high affinity for the GABAA-BZD receptor as the principle bioactive. CONCLUSION: The results suggest that the ethnobotanical use of this plant may have a pharmacological basis in its anxiolytic activity, as demonstrated in animal behaviour tests.

Effects of short-day treatment on long-term growth performance and maturation of farmed Arctic charr Salvelinus alpinus reared in brackish water.

The effects of a 6 week short-day photoperiod followed by continuous light, applied during the juvenile phase of Arctic charr Salvelinus alpinus in fresh water on smoltification and on the long-term growth and maturity following transfer to brackish water (BW) (constant salinity of either 17 and 27 or increasing salinity in steps from 17 to 27) were investigated. Prior to salinity transfer, the juveniles were either reared at continuous light (C group) or reared for 6 weeks on a short day (8L:16D, S group) followed by continuous light (24L:0D). Increased salinity had negative effect on growth, with female fish reared at 17 salinity weighing 19 and 27% more than the salinity-step group (17-27) and the 27 salinity group, respectively. The stepwise acclimation to salinity had limited advantage in terms of growth rate. Short photoperiod for 6 weeks (November to January) followed by continuous light improved growth, but not seawater (SW) tolerance. Gill Na(+) , K(+) -ATPase activity and plasma Na(+) levels changed with time, indicating some variation in osmoregulatory capacity during the experimental period. Overall, there appear to be interactive effects on maturation from applying short-day photoperiod followed by rearing at higher salinities. Plasma leptin varied with time and may be linked to stress caused by the observed variations in osmoregulatory ability. It is concluded that changes in growth rates observed in this study are mainly related to rearing salinity with higher growth rates at lower salinities. Short-day photoperiod has some growth-inducing effects but did not improve SW tolerance. Farmers of S. alpinus using BW for land-based rearing should keep salinity at moderate and stable levels according to these results to obtain best growth.

Assessment of in vitro pharmacological effect of Neotropical Piperaceae in GABAergic bioassays in relation to plants traditionally used for folk illness by the Yanesha (Peru).

ETHNOPHARMACOLOGICAL RELEVANCE: A previous pilot ethnobotanical and ethnopharmacological study with the Q'echi׳ Maya identified the family Piperaceae, as an important taxonomic group traditionally used for the treatment of epileptic and culture-bound anxiety disorders and possessing activity in the GABA system. Following that lead, a botanical survey was conducted in Peru, where 47 species of Piperaceae were collected including 21 plants traditionally used for folk illnesses by the Yanesha of Peru, an indigenous Amazonian group. MATERIALS AND METHODS: Two high throughput bioassays were used to quantify the in vitro activity of botanical extracts on the GABA system. RESULTS: Plant extracts demonstrated moderate to high affinity to the γ-aminobutyric acid benzodiazepine (GABA-BZD) receptor. In addition, extracts demonstrated low to moderate activity in the inhibition of the GABA-transaminase, with select plants exhibiting significant activity. Plants indicated by the Yanesha showed comparable activity to the other Piperaceae plants collected. Piper cremii was the most active plant in the GABA-BZD receptor assay, and Drymaria cordata (Caryophyllaceae) in the GABA-T assay. CONCLUSION: The study provides evidence that there is a pharmacological basis behind the use of plants in the treatment of susto and mal aire in both Central and South America, and we propose that the possible mechanism of action includes an interaction with the GABA-T enzyme and/or the GABAA-BZD receptor.

Dillapiol: a pyrethrum synergist for control of the Colorado potato beetle.

Dillapiol, the main constituent in dill Anethum sowa Roxb. ex Fleming (Apiaceae) oil and wild pepper, Piper aduncum L. (Piperaceae), is an effective cytochrome P450 inhibitor similar to piperonylbutoxide (PBO). Laboratory and field trials with pyrethrum Chrysanthemum cinerariifolium (Trevir.) vis. extracts combined with dillapiol (1:5 and 1:16 ratio) were effective against both insecticide-susceptible and -resistant Colorado potato beetle Leptinotarsa decemlineata (Say). In the laboratory, pyrethrum efficacy was increased 2.2-fold with the SS strain and 9.1-fold with the RS strains by using pyrethrum + dillapiol. Two field trials with the pyrethrum + dillapiol formulation demonstrated efficacy > or = 10 times than that of pyrethrum alone. The residual activity (half-life) of the combination exposed to direct sunlight was 3 h but it increased to 10.7 h by adding 2% of the sunscreen octylmethoxycinnamate.

Alkamides from Echinacea disrupt the fungal cell wall-membrane complex.

We tested the hypothesis that alkamides from Echinacea exert antifungal activity by disrupting the fungal cell wall/membrane complex. Saccharomyces cerevisiae cells were treated separately with each of seven synthetic alkamides found in Echinacea extracts. The resulting cell wall damage and cell viability were assessed by fluorescence microscopy after mild sonication. Membrane disrupting properties of test compounds were studied using liposomes encapsulating carboxyfluorescein. Negative controls included hygromycin and nourseothricin (aminoglycosides that inhibit protein synthesis), and the positive control used was caspofungin (an echinocandin that disrupts fungal cell walls). The results show that yeast cells exposed to sub-inhibitory concentrations of each of the seven alkamides and Echinacea extract exhibit increased frequencies of cell wall damage and death that were comparable to caspofungin and significantly greater than negative controls. Consistent with effects of cell wall damaging agents, the growth inhibition by three representative alkamides tested and caspofungin, but not hygromycin B, were partially reversed in sorbitol protection assays. Membrane disruption assays showed that the Echinacea extract and alkamides have pronounced membrane disruption activity, in contrast to caspofungin and other controls that all had little effect on membrane stability. A Quantitative Structure-Activity Relationship (QSAR) analysis was performed to study the effect of structural substituents on the antifungal activity of the alkamides. Among the set studied, diynoic alkamides showed the greatest antifungal and cell wall disruption activities while an opposite trend was observed in the membrane disruption assay where the dienoic group was more effective. We propose that alkamides found in Echinacea act synergistically to disrupt the fungal cell wall/membrane complex, an excellent target for specific inhibition of fungal pathogens. Structure-function relationships provide opportunities for synthesis of alkamide analogs with improved antifungal activities.

Expression of polyhydroxybutyric acid as a model for metabolic engineering of soybean seed coats.

The feasibility of genetically engineering soybean seed coats to divert metabolism towards the production of novel biochemicals was tested. The genes phbA, phbB, phbC from Ralstonia eutropha each under the control of the seed coat peroxidase promoter were introduced into soybean and the production of polyhydroxybutyrate (PHB) was assayed. The analysis of seed coats arising from 4 independent transformation events demonstrated that PHB was produced at a mean of 0.12% seed coat dried weight with individual values up to 0.36%. These values demonstrate that it is possible to metabolically engineer soybean seed coats.

Selection of natural health products for clinical trials: a preclinical template.

In preparation for a clinical trial on the efficacy of Echinacea products with a pediatric population, a rational method for selection of test products was developed, based on phytochemical and bioassay evaluation. Ten currently available commercial products of Echinacea angustifolia (EA) or Echinacea purpurea (EP) were selected, and 3 bottles of each of 2 different lots were purchased for each product. Investigators were blinded to product identity before phytochemical analysis. Lot-to-lot variation was small, but product variation due to species and formulation was large. Products derived from ethanol extracts had low polysaccharide content and high levels of alkamides (EA), echinacoside (EA), cynarin (EA), cichoric acid (EP), and caftaric acid (EP). These products possessed high antiviral activities that differed between EA and EP products, but limited immune activation properties. In contrast, products derived without ethanol extraction had higher polysaccharide levels, but low levels of other components. These aqueous compounds showed immunostimulant activity as measured in a mouse macrophage model and a somewhat different antiviral profile. The choice of Echinacea product for clinical trial must therefore consider the impact of immune enhancement, the specific viral infection targeted, and the potential to reduce symptoms via antiinflammatory activity. Product selection may also depend on whether the intent of the trial is prophylaxis or treatment.

Modulation of immune response gene expression by echinacea extracts: results of a gene array analysis.

Echinacea extracts have traditionally been used in the treatment of many infectious and other diseases (such as rhinovirus colds), and research has revealed the presence of various bioactivities in these extracts, particularly those connected with immune responses. We examined the effects of Echinacea by using gene expression analysis in a line of human bronchial epithelial cells, with or without rhinovirus infection. More than 13 000 human genes were evaluated. From these analyses we focused primarily on immune response genes and found that both Echinacea extracts, one predominantly rich in polysaccharides and the other rich in alkylamides and caffeic acid derivatives, stimulated the expression of numerous genes. These included a number of cytokines and chemokines, although the pattern of stimulation was different. In addition, Echinacea extracts tended to neutralize the effects of the rhinovirus. When the immune response gene pathways were analyzed with the Ingenuity Pathway program, it became apparent that many of them were interconnected through a major node, the transcription factor C/EBPbeta (CAAT/enhancer-binding protein beta) and its related C/EBP proteins. This suggests that Echinacea can bring about important biological responses in cells by virtue of interactions between components of the extract and a small number of intracellular factors involved in multiple signaling pathways.

In vitro activity of uva-ursi against cytochrome P450 isoenzymes and P-glycoprotein.

Some natural health products (NHPs) affect drug metabolism enzymes and transport proteins, potentially affecting the safety and efficacy of the drug or other NHPs. This study was undertaken to characterize the effect of uva-ursi (Arctostaphylos uva-ursi) on cytochrome P450 isozyme (3A4, 3A5, 3A7, 2C19, and 19)-mediated metabolism and P-glycoprotein (P-gp) transport. Three bulk and 2 capsulated uva-ursi samples were obtained from commercial outlets. The capsules were batched, and herbal samples were ground to a common consistency. Aqueous and methanol extracts were freshly prepared. Cytochrome P450 isozyme-mediated metabolism was determined by using in vitro bioassays. P-gp transport function was determined by using a rhodamine 123 (Rh123) uptake test in human (THP-1) monocytes and human Caco-2 cells. All products were analyzed by HPLC for arbutin, gallic acid, myricitrin, and isoquercetin. A large variation was observed in the biomarkers found between the bulk and capsulated samples. Our data indicate that both the aqueous and methanol extracts of all 5 uva-ursi products showed high cytochrome P450 isozyme inhibition, with the exception of the methanol extracts against cytochromes P3A4 and P19, which had low to moderate activity. The aqueous extracts of uva-ursi showed an inhibitory effect on Rh123 efflux by P-gp at 1 h and an inductive effect at 18 h for both cell lines. Our results show that the uva-ursi herbal products tested here have pharmacological properties, including the potential capacity to affect drug safety and efficacy. Further studies are warranted against a wider range of cytochrome P450 isozymes and to determine whether these effects are clinically significant.

Effects of traditionally used anxiolytic botanicals on enzymes of the gamma-aminobutyric acid (GABA) system.

In Canada, the use of botanical natural health products (NHPs) for anxiety disorders is on the rise, and a critical evaluation of their safety and efficacy is required. The purpose of this study was to determine whether commercially available botanicals directly affect the primary brain enzymes responsible for gamma-aminobutyric acid (GABA) metabolism. Anxiolytic plants may interact with either glutamic acid decarboxylase (GAD) or GABA transaminase (GABA-T) and ultimately influence brain GABA levels and neurotransmission. Two in vitro rat brain homogenate assays were developed to determine the inhibitory concentrations (IC50) of aqueous and ethanolic plant extracts. Approximately 70% of all extracts that were tested showed little or no inhibitory effect (IC50 values greater than 1 mg/mL) and are therefore unlikely to affect GABA metabolism as tested. The aqueous extract of Melissa officinalis (lemon balm) exhibited the greatest inhibition of GABA-T activity (IC50 = 0.35 mg/mL). Extracts from Centella asiatica (gotu kola) and Valeriana officinalis (valerian) stimulated GAD activity by over 40% at a dose of 1 mg/mL. On the other hand, both Matricaria recutita (German chamomile) and Humulus lupulus (hops) showed significant inhibition of GAD activity (0.11-0.65 mg/mL). Several of these species may therefore warrant further pharmacological investigation. The relation between enzyme activity and possible in vivo mode of action is discussed.

Modulation of human cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp) in Caco-2 cell monolayers by selected commercial-source milk thistle and goldenseal products.

In this study, we used an in vitro Caco-2 cell monolayer model to evaluate aqueous extracts of commercial-source goldenseal (Hydrastis canadensis) and milk thistle (Silybum marianum) capsule formulations, their marker phytochemicals (berberine and silibinin, respectively), as well as dillapiol, vinblastine, and the HIV protease inhibitor saquinavir for their ability to modulate CYP3A4 and ABCB1 expression after short-term exposure (48 h). Both upregulation and downregulation of CYP3A4 expression was observed with extracts of varying concentrations of the two natural health products (NHPs). CYP3A4 was highly responsive in our system, showing a strong dose-dependent modulation by the CYP3A4 inhibitor dillapiol (upregulation) and the milk thistle flavonolignan silibinin (downregulation). ABCB1 was largely unresponsive in this cellular model and appears to be of little value as a biomarker under our experimental conditions. Therefore, the modulation of CYP3A4 gene expression can serve as an important marker for the in vitro assessment of NHP-drug interactions.

In vitro evaluation of human cytochrome P450 and P-glycoprotein-mediated metabolism of some phytochemicals in extracts and formulations of African potato.

African potato (Hypoxis hemerocallidea, AP) is a traditional herbal medicine widely used as an immune booster and also for the treatment of various ailments such as urinary diseases, prostrate hypertrophy and cancer. Amongst the chemical components contained in AP, the norlignan glycoside, hypoxoside (HYP) is purported to be the most important phytochemical in terms of AP's medicinal value. Additional constituents in AP include the sterols, beta-sitosterol (BSS), stigmasterol (STG), and the stanol, stigmastanol (STN). The potential of extracts of AP, AP formulations as well as HYP, its aglycone rooperol (ROP) and the sterols to inhibit in vitro metabolism of drug marker substrates by human cytochrome P450 (CYP) enzymes such as CYP3A4, 3A5 and CYP19 were investigated. Samples were also assessed for their effect on drug transport proteins such as P-glycoprotein (P-gp). The effects on CYP-mediated metabolism were studied by fluorometric microtitre plate assay. The potential interaction with P-gp was investigated by measuring the efflux of the fluorescent dye rhodamine 123 (Rh 123) in the CaCo-2 (colon carcinoma) cell line. Various extracts of AP, AP formulations, only STG and the norlignans, in particular the aglycone ROP, exhibited inhibitory effects on CYP3A4-, 3A5- and 19-mediated metabolism. The extracts and the formulations that contained a significant amount of HYP showed high induction of P-gp compared to the positive control, ritonavir. Whilst extrapolation of the current in vitro findings to clinical effects may well be considered speculative, these in vitro data should be heeded as a signal of possible in vivo interactions. Appropriate measures are therefore necessary to explore the possibility of such in vitro-in vivo correlations.

Investigation of the antioxidant activity of African potato (Hypoxis hemerocallidea).

African potato (AP) is widely used as an immune booster for the treatment of various ailments. The norlignan glycoside hypoxoside, a major phytoconstituent of AP, its aglycon rooperol, and an aqueous preparation of lyophilized AP corms were screened for in vitro antioxidant activity using the DPPH (1,1-diphenyl-2-picryl hydrazine) and FRAP (ferric reducing ability of plasma) tests. Inhibition of quinolinic acid (QA) induced lipid peroxidation in rat liver tissue was studied in vitro using the thiobarbituric assay (TBA). Superoxide free radical scavenging activity was determined by the nitroblue tetrazolium assay. An isocratic HPLC method was developed to quantitatively determine both hypoxoside and rooperol concurrently. While rooperol and AP extracts reduced QA-induced lipid peroxidation in rat liver homogenates and significantly scavenged the superoxide anion at pharmacological doses, in comparison, hypoxoside was virtually devoid of activity. Since hypoxoside is converted to rooperol in vivo following administration of AP, the results indicate that the hypoxoside component in AP could have value as an antioxidant prodrug.

Novel chemistry of invasive exotic plants.

Of the many exotic plants that have become naturalized in North America, only a small proportion are pests capable of invading and dominating intact natural communities. In the present study, we tested the hypothesis that the most invasive plants are phytochemically unique in their new habitats. A comparison of exotic plant species that are highly invasive in North America with exotics that are widespread, but non-invasive revealed that the invasive plants were more likely to have potent secondary compounds that have not been reported from North American native plants. On average, the compounds found in the invasive plants were reported from fewer species, fewer genera and fewer families than those from non-invasive plants. Many of the unique phytochemicals from invasive plants have been reported to have multiple activities, including antiherbivore, antifungal, antimicrobial and allelopathic (phytotoxic) effects, which may provide the plants with several advantages in their new environments.

Echinacea extracts modulate the production of multiple transcription factors in uninfected cells and rhinovirus-infected cells.

Extracts of Echinacea purpurea are widely used for the prevention and treatment of common colds, coughs, bronchitis and other upper respiratory infections, many of which are caused by rhinoviruses (RVs). Recent reports have indicated that rhinoviruses can stimulate the release of various pro-inflammatory cytokines and chemokines from cultured nasal and bronchial human epithelial cells, and several transcription factors (TFs) have been implicated in this process. The effects of Echinacea treatment and rhinovirus infection on the activation of a range of transcription factors were evaluated by means of a protein/DNA array analysis. The BEAS-2B cell line was used as the model, and nuclear extracts of uninfected cells and rhinovirus-14 infected cells were examined with and without treatment with one of two chemically different Echinacea extracts. It was found that both Echinacea extracts increased the nuclear content of more than 30 transcription factors, including the 12 pro-inflammatory factors examined, such as NFkB, AP-1, AP-2 and STATs 1-6. Virus infection resulted in a more dramatic increase in these same TFs. However, when RV-infected cells were treated with either of the two Echinacea extracts, TF levels were reduced to low levels, although the pattern of the reductions was different for the two extracts. These results indicate that rhinovirus infection of epithelial cells, and treatment with Echinacea extracts, led to profound effects on numerous transcription factors, which could explain the previously observed modulation of secreted cytokines and chemokines, as well as other signaling pathways. In addition, the results could help to explain the beneficial effects of Echinacea consumption.

Location, identification, and size distribution of depleted uranium grains in reservoir sediments.

The location, nature, and size distribution of uranium-rich grains in sediment layers can be identified by sunbursts of etched particle tracks if each sample is pressed against a track detector, next irradiated with thermal neutrons, and the detectors then chemically etched to reveal fission tracks. The total track abundance from the sample is a measure of the 235U content; hence, if the bulk uranium (mostly 238U) has been measured, the two sets of results give the depletion or enrichment of the uranium. Sunbursts of tracks mark the locations of low-abundance, high-uranium grains allowing them to be singled out for further study.

The effect of a synergistic concentration of a Piper nigrum extract used in conjunction with pyrethrum upon gene expression in Drosophila melanogaster.

An ethyl acetate extract of Piper nigrum L. (Piperaceae) peppercorns was tested as a synergist for the botanical insecticide pyrethrum. A high synergist ratio of 11.6 against Drosophila melanogaster was obtained for the combination of pyrethrum supplemented with P. nigrum. The effect of this combination was investigated using cDNA microarray analysis of gene expression profiles in D. melanogaster. Treatment of D. melanogaster with pyrethrum alone resulted in a large number of differentially expressed genes, principally associated with stress responses. Seven genes were identified as being commonly expressed in D. melanogaster treated with at least two of the following treatments: P. nigrum, pyrethrum or P. nigrum plus pyrethrum. These are likely implicated in Drosophila defence responses to toxins.