Asim Muhammad

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.

Bioassay‐guided fractionation of lemon balm (Melissa officinalis L.) using an in vitro measure of GABA transaminase activity

A novel pharmacological mechanism of action for the anxiolytic botanical Melissa officinalis L. (lemon balm) is reported. The methanol extract was identified as a potent in vitro inhibitor of rat brain GABA transaminase (GABA‐T), an enzyme target in the therapy of anxiety, epilepsy and related neurological disorders. Bioassay‐guided fractionation led to the identification and isolation of rosmarinic acid (RA) and the triterpenoids, ursolic acid (UA) and oleanolic acid (OA) as active principles. Phytochemical characterization of the crude extract determined RA as the major compound responsible for activity (40% inhibition at 100 µg/mL) since it represented approximately 1.5% of the dry mass of the leaves. Synergistic effects may also play a role. Copyright

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.

Structural constraints and the importance of lipophilicity for the mitochondrial uncoupling activity of naturally occurring caffeic acid esters with potential for the treatment of insulin resistance

Caffeic acid phenethyl ester (CAPE) has recently been shown to potently stimulate glucose uptake in cultured skeletal muscle cells through the AMPK pathway and therefore to have anti-diabetic potential. We report here that CAPE increases glucose uptake in C2C12 muscle cells by 225+/-21% at 50 microM, and that activation of AMPK is a consequence of the metabolic stress resulting from an uncoupling-type disruption of mitochondrial function (complete uncoupling at 50 microM). We also observe that the therapeutic potential of CAPE is offset by its high potential for toxicity. The purpose of this study was therefore to identify other active caffeic acid derivatives, evaluate their ratio of activity to toxicity, and elucidate their structure-activity relationship. Twenty naturally occurring derivatives were tested for glucose-uptake stimulating activity in C2C12 cells following 18 h of treatment and for uncoupling activity in isolated rat liver mitochondria. Cytotoxicity was assessed in C2C12 cells by the release of lactate dehydrogenase over 18 h. In addition to CAPE, four compounds were identified to be active, both stimulating glucose uptake and uncoupling isolated mitochondria. Activity required that the caffeic acid moiety be intact and that the compound not contain a strongly ionized group. Both activity and toxicity were found to be well-correlated to predicted lipophilicity. However, two compounds exhibited little to no toxicity while still stimulating glucose uptake by 65-72%. These results support a therapeutic potential for this family of compounds and provide the framework for the design of alternatives to Metformin with an optimized balance of safety and activity.

Bioassay-Guided Isolation of the Antidiabetic Principle from Sorbus decora (Rosaceae) Used Traditionally by the Eeyou Istchee Cree First Nations

Bioassay-guided fractionation of a crude extract (80% EtOH in H(2)O) of stem bark of Sorbus decora led the isolation of three new pentacycle triterpenes (compounds 1-3). The structures of 1-3 were established on the basis of spectroscopic methods (IR, HREIMS, 1D and 2D NMR) as 23,28-dihydroxyursan-12-ene-3β-caffeate, 23,28-dihydroxylupan-20(29)-ene-3β-caffeate, and 3β,23,28-trihydroxy-12-ursene, respectively. Compound 2 significantly enhanced glucose uptake in C2C12 cells, but compounds 1 and 3 did not. In addition, triterpenoids 4-8, catechin, and epicatechin were also isolated. This is the first comprehensive report of the phytochemical constituents of S. decora since the initial study by Narashmachari and von Rudloff (1962) and includes evaluation of their antidiabetic activity.

Adipogenic constituents from the bark of Larix laricina du Roi (K. Koch; Pinaceae), an important medicinal plant used traditionally by the Cree of Eeyou Istchee (Quebec, Canada) for the treatment of type 2 diabetes symptoms.

ETHNOPHARMACOLOGICAL RELEVANCE Diabetes is a growing epidemic worldwide, especially among indigenous populations. Larix laricina was identified through an ethnobotanical survey as a traditional medicine used by Healers and Elders of the Cree of Eeyou Istchee of northern Quebec to treat symptoms of diabetes and subsequent in vitro screening confirmed its potential. MATERIALS AND METHODS We used a bioassay-guided fractionation approach to isolate the active principles responsible for the adipogenic activity of the organic extract (80% EtOH) of the bark of Larix laricina. Post-confluent 3T3-L1 cells were differentiated in the presence or absence of the crude extract, fractions or isolates of Larix laricina for 7 days, then triglycerides content was measured using AdipoRed reagent. RESULTS We identified a new cycloartane triterpene (compound 1), which strongly enhanced adipogenesis in 3T3-L1 cells with an EC(50) of 7.7 μM. It is responsible for two thirds of the activity of the active fraction of Larix laricina. The structure of compound 1 was established on the basis of spectroscopic methods (IR, HREIMS, 1D and 2D NMR) as 23-oxo-3α-hydroxycycloart-24-en-26-oic acid. We also identified several known compounds, including three labdane-type diterpenes (compounds 2-4), two tetrahydrofuran-type lignans (compounds 5-6), three stilbenes (compounds 7-9), and taxifolin (compound 10). Compound 2 (13-epitorulosol) also potentiated adipogenesis (EC(50) 8.2 μM) and this is the first report of a biological activity for this compound. CONCLUSIONS This is the first report of putative antidiabetic principles isolated from Larix laricina, therefore increasing the interest in medicinal plants from the Cree pharmacopeia.

Phytochemical constituents of Sarracenia purpurea L. (pitcher plant)

From the leaves of Sarracenia purpurea, collected in Mistissini, Quebec, Canada, four goodyerosides and three phenolics and nine known compounds, were isolated. The structures of the compounds were determined by mass spectrometry, including HRMS, and by 1D and 2D NMR spectroscopy.

Bioactive Phytochemicals from Canadian Boreal Forest Species Used Traditionally by Eastern James Bay Cree Aboriginals to Treat Diabetes Mellitus

Type-2 diabetes mellitus is a chronic metabolic disorder characterized by impaired insulin secretion and sensitivity, and is more pronounced among some indigenous populations due to their transition from traditional to modern diets, as well as their cultural disconnection from modern pharmacological treatment regimes. This is the case for the Cree Nations of Eeyou Istchee (CEI) of northern Quebec, where the age-adjusted prevalence of disease reached 29 %. The Canadian Institutes of Health Research Team in Aboriginal Antidiabetic Medicines is a multidisciplinary team aimed at alleviating the impact of this disease in the CEI by using a culturally adapted approach. A quantitative ethnobotanical study of traditional medicines conducted by our team in collaboration with Healers and Elders resulted in the identification of several potential antidiabetic plants. The crude extracts of these plants were tested in a comprehensive platform of in vitro bioassays designed to detect potential antidiabetic biological activities including: stimulation of glucose uptake in C2C12 muscle cells and potentiation of differentiation of 3T3-L1 pre-adipocytes indicating enhanced insulin sensitivity. These procedures allowed us to identify the most significant species from the biological activity viewpoint, and they were considered for further phytochemical characterization. The present report provides a comprehensive summary of the major biological activities and phytochemistry of these key Canadian boreal forest plants that demonstrated significant ethnobotanical evidence of antidiabetic activity and associated symptoms.