Antidiabetic Activity of Extracts from Needle, Bark, and Cone ofPicea glauca.: Organ-Specific Protection from Glucose Toxicity and Glucose Deprivation

Abstract

The incidence of type 2 diabetes mellitus has reached epidemic proportions worldwide. Canadian aboriginal communities, particularly the Cree Nation of Eeyou Istchee, have been identified as a high-risk population. Culturally acceptable treatment options are limited notably for diabetic complications resulting in peripheral neuropathy. Here, we describe results of an ongoing collaborative research project with Cree of Eeyou Istchee to identify botanicals capable of protecting peripheral neuronal precursors from glucose toxicity and glucose deprivation in vitro. Polar fractions of three plant organs (needles, cone, and bark) collected from Picea glauca (Moench) Voss (Pinaceae), were tested for toxicity under normoglucose, glucotoxicity, and glucose deprivation conditions. The profile of phenolic metabolites in each extract was first characterized by high-performance liquid chromatography-diode array detection-atmospheric pressure chemical ionisation mass spectrometry (HPLC-DAD-APCI/MS). We report here that these fractions are well-tolerated by PC12 neuronal precursors under normoglucose conditions. LD 50 concentrations of needle extracts exceeded 100 µg/mL, whereas the LD 50 of bark and cone extracts was 40 and 36.4 µg/mL, respectively. We further show that the cytoprotective properties of minhikw after glucose challenge are concentration-dependent and organ-specific. Needle * Dedicated to John Thor Arnason of the University of Ottawa, Department of Biology, on the occasion of his sixtieth birthday. extracts protected PC12 cells from both glucotoxicity and glucose deprivation. Bark extracts had negligible activity. Cone extracts further impaired PC12 cell glucose tolerance. This study provides the first validation of antidiabetic activity of minhikw organs at the cellular level relevant to the management of diabetic peripheral neuropathy.

Characterizing the cytoprotective activity of Sarracenia purpurea L., a medicinal plant that inhibits glucotoxicity in PC12 cells

“…Consistent with previous reports using the current protocol [29], the viability of vehicle-treated PC12-AC cells exposed to elevated glucose concentrations (150 mM) for 96 h was reduced by 40-50% relative to vehicle-treated cells under normal glucose conditions (Figure 1, left panel). This cell loss is glucose-specific and not due to osmotic stress as substitution of d -glucose for l -glucose abolishes toxicity [18,30]. Other studies have demonstrated a similar loss (30%) of PC12 cells exposed to 75 mM high glucose media once differentiated to a neuronal phenotype [28].…”

“…Leaves were cut open and rinsed clean of insects and dirt. Leaf and root tissues were then dehydrated using an electric food dehydrator (Nesco/American Hervest WI, USA) set to 40°C and processed using a Wiley Mill (2 mm filter) prior to extraction with 80% ethanol as previously described [18]. Dried extracts were prepared for experimental use as stock solutions in dimethyl sulfoxide (DMSO), filtered through a 0.2 μm nylon membrane filter (Chromatographic Specialties Inc., Brockville, ON, Canada), and serially diluted as required on the day of use to ensure all cultures were exposed to a final concentration of 0.1% DMSO (vehicle).…”

“…Prior to experimental use, cells were seeded in 96-well plates at a density of 6.25x10 3 cells/well and incubated overnight at 37°C in 5% CO 2 to allow adherence. As described previously [18], extract toxicity (IC 50 ) was determined by treating cultures for 96 h in serum-free RPMI 1640 containing 11 mM glucose, 0.025% bovine serum albumin (BSA) to facilitate intracellular passage of hydrophobic compounds, and either 0.1% DMSO (vehicle control) or increasing concentrations of plant extract (0-100 μg/mL). Similarly, for the glucose toxicity assay, cells were treated for 96 h in serum-free medium supplemented with glucose (to a final concentration of 150 mM), 0.025% BSA, and vehicle control or various concentrations of plant extract below its determined IC 50 value.…”

“…To assess viable cell number, the formazan dye WST (Roche Diagnostics, Laval, QC) was added to each well following 96 h of treatment in normal (11 mM) or high glucose (150 mM), as described by Harris et al [18]. After a 60 min incubation with WST, spectrophotometric analysis at 420 nm (formazan) and at 620 nm (reference) was performed using a Tecan Spectra Shell platereader model A-5082 (Durham, NC) and WinSelect software (Tecan US, Inc.).…”

Picea mariana bark: A new source of trans-resveratrol and other bioactive polyphenols

“…Indeed, compounds such as quercetin glycoside, kaempferol glycoside, dihydroquercetin-3 0 -O-b-D-glucopyranoside and isorhamnetin-3-O-(6 00 -O-acetyl)-b-D-glucopyranoside have been found in barks, needles and cones of the Picea spp. (Harris et al, 2008;Pan & Lundgren, 1995).…”