Ammar Saleem

Stimulation of AMP-activated protein kinase and enhancement of basal glucose uptake in muscle cells by quercetin and quercetin glycosides, active principles of the antidiabetic medicinal plant Vaccinium vitis-idaea

Several medicinal plants that stimulate glucose uptake in skeletal muscle cells were identified from among species used by the Cree of Eeyou Istchee of northern Quebec to treat symptoms of diabetes. This study aimed to elucidate the mechanism of action of one of these products, the berries of Vaccinium vitis idaea, as well as to isolate and identify its active constituents using a classical bioassay-guided fractionation approach. Western immunoblot analysis in C2C12 muscle cells revealed that the ethanol extract of the berries stimulated the insulin-independent AMP-activated protein kinase (AMPK) pathway. The extract mildly inhibited ADP-stimulated oxygen consumption in isolated mitochondria, an effect consistent with metabolic stress and the ensuing stimulation of AMPK. This mechanism is highly analogous to that of Metformin. Fractionation guided by glucose uptake activity resulted in the isolation of ten compounds. The two most active, quercetin-3-O-glycosides, enhanced glucose uptake by 38-59% (50 muM; 18 h treatment) in the absence of insulin. Quercetin aglycone, a minor constituent, stimulated uptake by 37%. The quercetin glycosides and the aglycone stimulated the AMPK pathway at concentrations of 25-100 muM, but only the aglycone inhibited ATP synthase in isolated mitochondria (by 34 and 79% at 25 and 100 muM, respectively). This discrepancy suggests that the activity of the glycosides may require hydrolysis to the aglycone form. These findings indicate that quercetin and quercetin 3-O-glycosides are responsible for the antidiabetic activity of V. vitis crude berry extract mediated by AMPK. These common plant products may thus have potential applications for the prevention and treatment of insulin resistance and other metabolic diseases.

Bioaccumulation of the pharmaceutical 17α-ethinylestradiol in shorthead redhorse suckers (Moxostoma macrolepidotum) from the St. Clair River, Canada.

17alpha-ethynylestradiol (EE2), a synthetic estrogen prescribed as a contraceptive, was measured in Shorthead Redhorse Suckers (ShRHSs) (Moxostoma macrolepidotum) collected near a wastewater treatment plant (WWTP) in the St. Clair River (Ontario, Canada). We detected EE2 in 50% of the fish samples caught near the WWTP (Stag Island), which averaged 1.6+/-0.6ng/g (wet weight) in males and 1.43+/-0.96ng/g in females. No EE2 was detected in the samples from the reference site (Port Lambton) which was 26km further downstream of the Stag Island site. Only males from Stag Island had VTG induction, suggesting the Corunna WWTP effluent as a likely source of environmental estrogen. EE2 concentrations were correlated with total body lipid content (R(2)=0.512, p<0.01, n=10). Lipid normalized EE2 concentrations were correlated with delta(15)N (R(2)=0.436, p<0.05, n=10), suggesting higher EE2 exposures in carnivores. Our data support the hypothesis of EE2 bioaccumulation in wild fish.

Root colonization by an arbuscular mycorrhizal (AM) fungus increases growth and secondary metabolism of purple coneflower, Echinacea purpurea (L.) Moench.

Purple coneflower, Echinacea purpurea (L.) Moench, is an important phytomedicinal species that contains phenolics and alkamides with antipathogenic properties. This study aimed to examine the effect of arbuscular mycorrhizal (AM) colonization on the physiology and biochemistry of E. purpurea. It was hypothesized that AM colonization enhances the growth and secondary metabolism in E. purpurea. In this regard, a 13-week factorial greenhouse experiment was performed with E. purpurea, inoculated (or not) with the AM fungus Glomus intraradices Schenck & Smith. Overall, the results indicated that AM colonization significantly increased the mass of shoots and roots and the concentrations of proteins and most of the phenolics in the roots. Hence, the selected trait of mycorrhiza could play an important role in optimizing the growth of E. purpurea by inducing the production of secondary phytomedicinal metabolites.

Functional characterization of a UDP-glucose:flavonoid 3-O-glucosyltransferase from the seed coat of black soybean (Glycine max (L.) Merr.)

The seed coats of black soybean (Glycine max (L.) Merr.) accumulate red (cyanidin-), blue (delphinidin-), purple (petunidin-), and orange (pelargonidin-based) anthocyanins almost exclusively as 3-O-glucosides; however, the responsible enzyme has not been identified. In this study, the full-length cDNA which encodes the enzyme that catalyzes the final step in anthocyanin biosynthesis, namely UDP-glucose:flavonoid 3-O-glucosyltransferase (UGT78K1), was isolated from the seed coat tissue of black soybean using rapid amplification of cDNA ends (RACE). Of the 28 flavonoid substrates tested, the purified recombinant protein glucosylated only anthocyanidins and flavonols, and demonstrated strict 3-OH regiospecificity. Galactose could also be transferred with relatively low activity to the 3-position of cyanidin or delphinidin in vitro. These findings are consistent with previous reports of mainly 3-O-glucosylated and minor amounts of 3-O-galactosylated anthocyanins in the seed coat of black soybean. The recombinant enzyme exhibited pronounced substrate inhibition by cyanidin at 100 microM acceptor concentration. Transfer of UGT78K1 into the Arabidopsis T-DNA mutant (ugt78d2) deficient in anthocyanidin and flavonol 3-O-glucosyltransferase activity, restored the accumulation of anthocyanins and flavonols, suggesting the in vivo function of the enzyme as a flavonoid 3-O-glucosyltransferase. Genomic and phylogenetic analyses suggest the existence of three additional soybean sequences with high similarity to UGT78K1. RT-PCR confirmed the co-expression of one of these genes (Glyma08g07130) with UGT78K1 in the seed coat of black soybean, suggesting possible functional redundancies in anthocyanin biosynthesis in this tissue.

Seasonal phytochemical variation of anti-glycation principles in lowbush blueberry (Vaccinium angustifolium).

Diabetic hyperglycaemia promotes the production of advanced glycation end-products (AGEs), which play a significant role in the development of complications associated with type 2 diabetes mellitus. Vaccinium angustifolium, a medicinal plant used for the treatment of diabetes, produces a variety of phenolic metabolites with putative anti-diabetic activities. To assess optimal cultivation time, seasonal changes in the concentration of six phenolic compounds in leaves and twelve compounds in stems were examined using HPLC-DAD and examined in relation to seasonal changes in AGE inhibition activity, assessed with a fluorescence-based assay. A seasonal decline occurred in the concentration of chlorogenic acid, rutin, and quercetin 3-arabinoside in leaves and chlorogenic acid in stems. The concentration of (+)-catechin, and (-)-epicatechin in stems declined within two weeks before rising and fluctuating insignificantly. AGE inhibition activity of leaves was significantly greater at the final compared to the initial collection date whereas the activity of stems did not change significantly. Relative to the leaf extract, the stem was a more potent inhibitor of AGE formation, which could be a result of the unique phytochemistry of stems. Together, these results revealed significant seasonal variation in the phenolic profile and anti-glycation effects of V. angustifolium extracts and indicated late summer as the collection time yielding optimal activity.

Inhibitory effect of the Cree traditional medicine wiishichimanaanh (Vaccinium vitis-idaea) on advanced glycation endproduct formation: identification of active principles.

Like many aboriginal populations, First Nations communities such as the Cree of Eeyou Istchee are facing continuously increasing rates of diabetes and related complications. Advanced glycation endproducts (AGEs), which readily form and accumulate with sustained hyperglycemia, contribute to the development of diabetic complications and, as such, are considered a potential therapeutic target. In the present study, the inhibition of AGE formation by ethanolic extracts of the Cree medicinal plant Vaccinium vitis‐idaea L. was assessed by fluorometric detection of fluorescent AGEs and immunodetection of Nϵ‐(carboxymethyl)lysine adducts of albumin. Extracts from V. vitis‐idaea berries demonstrated a concentration‐dependent inhibition of AGE formation in both measures. High performance liquid chromatography mass spectrometry (HPLC/MS) identified nine main phenolic constituents. Four were selected for further testing, of which catechin, quercetin‐3‐O‐galactoside and cyanidin‐3‐O‐glucoside but not para‐coumaric acid displayed antiglycation activities. These results demonstrate that the flavonoid components of the berry extract are potent antiglycation agents and provide pharmacological validation for the traditional use of V. vitis‐idaea as an antidiabetic remedy. Copyright

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

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

A RP‐HPLC‐DAD‐APCI/MSD method for the characterisation of medicinal Ericaceae used by the Eeyou Istchee Cree First Nations

INTRODUCTION Ericaceae medicinal plants are traditionally used by the Eeyou Istchee Cree and other northern peoples of North America to treat type 2 diabetic symptoms. Because of the importance of phenolics as potential cures for degenerative diseases including type 2 diabetes, an analytical method was developed to detect them in the leaf extracts of 14 Ericaceae plants. OBJECTIVE To develop an optimised method which is applicable to a relatively large number of Ericaceae plants using their leaf extracts. For this purpose phenolics with a wide range of polarity, including a glucosylated benzoquinone, two phenolic acids, three flavanols, a flavanone, a flavone and five flavonols, were included in this study. METHODOLOGY Characterisation of phytochemicals in extracts was undertaken by automated matching to the UV spectra to those of an in house library of plant secondary metabolites and the authentication of their identity was achieved by reversed phase-high-performance chromatography-diode array detection-atmospheric pressure chemical ionisation/mass selective detection. RESULTS Twenty-six phenolics were characterised within 26 min of chromatographic separation in 80% ethanol extracts of 14 Ericaceae plants. The calibration curves were linear within 0.5-880 microg/g dry mass of the plant with regression values better than 0.995. The limits of detection ranged from 0.3 for microg/mL for (+)-catechin to 2.6 microg/mL for chlorogenic acid. This is a first study dealing with relatively large number of Ericaceae extracts and is applicable to other plants of same family.

Anti-adipogenic activities of Alnus incana and Populus balsamifera bark extracts, part II: bioassay-guided identification of actives salicortin and oregonin.

Among modern day metabolic diseases, obesity has reached epidemic proportions worldwide and novel therapeutic support strategies are urgently needed. Adipocytes are interesting targets in this context. Using ethnobotanical and bioassay screening techniques, we have identified two Boreal Forest plants used by the James Bay Cree that potently inhibit adipogenesis, namely ALNUS INCANA ssp. RUGOSA (Speckled Alder) and POPULUS BALSAMIFERA (Balsam Poplar). The mode of action of this inhibitory activity was reported in a companion paper. The current study report the results of a classical bioassay-guided fractionation approach aimed at identifying the active principles responsible for the inhibition of adipogenesis, as measured using triglyceride accumulation in the 3T3-L1 adipocyte model cell line. The glycosides oregonin and salicortin were isolated and identified as the respective active principles for ALNUS INCANA and POPULUS BALSAMIFERA. These compounds thus offer promise as novel agents to mitigate the incidence or the progression of obesity.

Identification of two anthocyanidin reductase genes and three red-brown soybean accessions with reduced anthocyanidin reductase 1 mRNA, activity, and seed coat proanthocyanidin amounts.

Anthocyanidin reductase (ANR; EC 1.3.1.77) catalyzes a key step in the biosynthesis of proanthocyanidins (PAs; also known as condensed tannins), flavonoid metabolites responsible for the brown pigmentation of seeds. Here, two ANR genes (ANR1 and ANR2) from the seed coat of brown soybean (Glycine max (L.) Merr.) have been isolated and their enzymatic function confirmed for the reduction of cyanidin to (-)-epicatechin in vitro. Biochemical and genetic comparisons of soybean lines differing in seed coat color revealed three red-brown lines to exhibit major reductions in the amounts of soluble PAs in the seed coat compared to brown soybean lines. Two spontaneous mutants with red-brown grain color had reduced ANR1 gene expression in the seed coat, and an EMS-mutagenized red-brown mutant had nonsynonymous substitutions that resulted in slightly reduced ANR1 activity in vitro. These results suggest that defects in the ANR1 gene can be associated with red-brown soybean grain color. These results suggest that suppressing ANR1 gene expression or activity may be a rational approach toward engineering seed coat color to enable the visual identification of genetically modified soybean grains.