Maturity and Temperature Influence on Lycopene Distribution during Filtration Processing of Red-fleshed Watermelons

Our objectives were to evaluate filtration processing and extraction steps necessary for purification of lycopene from red-fleshed watermelons. Maturity and temperature influence on lycopene segregation during processing were investigated. Purification of lycopene was evaluated with a filtration procedure on ground flesh and subsequent centrifugation of the produced filtrate. Temperature treatments included 25OC, 60OC and 85OC, applied on ground flesh before filtration. Ground flesh filtration segregated lycopene in two fractions; a filter cake and a filtrate. At 25OC segregation was maturity dependent, with overripe melons segregating more in the filtrate. About 75-85% of the filtrate lycopene could be recovered as pellet for undermature and mature melons, while only 55% for overripe. This loss could be partially avoided with heat application on ground flesh, which resulted in lycopene retention to the filter cake fraction. Further rinse of the filter cakes and subsequent filtrations was not recommended, due to excessive lycopene loss.Horticulture and Landscape Architecture Departmen

Influence of solvent extraction and winemaking steps on antioxidant activity of red grapes

Scope and Method of Study: Compare different solvent combinations on antioxidant extraction from Cynthiana pomace for potential industrial scale use. Measure the antioxidant activity of Cabernet franc grapes at various steps during wine production. Identify major compounds present in pomace and wine; correlate antioxidant activity with total phenolics present.Findings and Conclusions: Out of the 32 solvent treatments, 50% Acetone/Water mix proved to be a good extraction medium when used for 2h at 2:1 solvent:sample ratio. Yield of 31micromoles TE/g tissue suggest that Cynthiana pomace is a good source of natural antioxidants, with anthocyanins cyanidin 3-O-glucoside and malvidin 3-O-glucoside being two of the major compounds in the extracts.Both wine and pomace of Cabernet franc showed high antioxidant activity of approximately 27micromoles TE/mL wine and 56-83micromoles TE/g pomace respectively. Antioxidant activity was also positively correlated with total phenolic content, both for wine and pomace, with average of 762mg Gallic acid equivalents/L of wine and 2mg Gallic acid equivalents/g pomace respectively. The major compounds identified in wine with HPLC were gallic acid and epicatechin, with malvidin 3-O-glucoside in smaller amounts. Pomace extracts contained mainly catechin and epicatechin, with malvidin 3-O-glucoside as the major anthocyanin. Several major peaks were not identified, which suggests that additional compounds might also be responsible for the antioxidant properties of Cabernet franc.Both experiments suggest that grape pomace from red grape varieties has good antioxidant properties and thus may be a good source of valuable natural compounds. Our method presents a potentially industrial-scale, single-solvent extraction process for screening antioxidants from grape pomace - and possibly other waste streams. Quantification and identification of all major compounds present in the grape pomace is needed to give a better understanding of the true value of the waste streams and also to help determine if further extract processing would be beneficial and/or economically feasible

Structure based inhibitor design targeting glycogen phosphorylase b. Virtual screening, synthesis, biochemical and biological assessment of novel N-acyl-β-d-glucopyranosylamines

Glycogen phosphorylase (GP) is a validated target for the development of new type 2 diabetes treatments. Exploiting the Zinc docking database, we report the in silico screening of 1888 β- D-glucopyranose-NH-CO-R putative GP inhibitors differing only in their R groups. CombiGlide and GOLD docking programs with different scoring functions were employed with the best performing methods combined in a “consensus scoring” approach to ranking of ligand binding affinities for the active site. Six selected candidates from the screening were then synthesized and their inhibitory potency was assessed both in vitro and ex vivo. Their inhibition constants’ values, in vitro, ranged from 5 to 377 µM while two of them were effective at causing inactivation of GP in rat hepatocytes at low µM concentrations. The crystal structures of GP in complex with the inhibitors were defined and provided the structural basis for their inhibitory potency and data for further structure based design of more potent inhibitors

Computationally motivated synthesis and enzyme kinetic evaluation of N-(β-d-glucopyranosyl)-1,2,4-triazolecarboxamides as glycogen phosphorylase inhibitors

Following our recent study of N-(β-D-glucopyranosyl)-oxadiazole-carboxamides (Polyák et al., Biorg. Med. Chem. 2013, 21, 5738) revealed as moderate inhibitors of glycogen phosphorylase (GP), in silico docking calculations using Glide have been performed on N-(β-D-glucopyranosyl)-1,2,4-triazolecarboxamides with different aryl substituents predicting more favorable binding at GP. The ligands were subsequently synthesized in moderate yields using N-(2,3,4,6-terta-O-acetyl-β-D-glucopyranosyl)-tetrazole-5-carboxamide as starting material. Kinetics experiments against rabbit muscle glycogen phosphorylase b (RMGPb) revealed the ligands to be low µM GP inhibitors; the phenyl analogue (Ki = 1 µM) is one of the most potent N-(β-D-glucopyranosyl)-heteroaryl-carboxamide-type inhibitors of the GP catalytic site discovered to date. Based on QM and QM/MM calculations, the potency of the ligands is predicted to arise from favorable intra- and intermolecular hydrogen bonds formed by the most stable solution phase tautomeric (t2) state of the 1,2,4-triazole in a conformationally dynamic system. ADMET property predictions revealed the compounds to have promising pharmacokinetic properties without any toxicity. This study highlights the benefits of a computationally lead approach to GP inhibitor design

Metabolic sensitivity of pancreatic tumour cell apoptosis to glycogen phosphorylase inhibitor treatment

Inhibitors of glycogen breakdown regulate glucose homeostasis by limiting glucose production in diabetes. Here we demonstrate that restrained glycogen breakdown also inhibits cancer cell proliferation and induces apoptosis through limiting glucose oxidation, as well as nucleic acid and de novo fatty acid synthesis. Increasing doses (50-100 microM) of the glycogen phosphorylase inhibitor CP-320626 inhibited [1,2-(13)C(2)]glucose stable isotope substrate re-distribution among glycolysis, pentose and de novo fatty acid synthesis in MIA pancreatic adenocarcinoma cells. Limited oxidative pentose-phosphate synthesis, glucose contribution to acetyl CoA and de novo fatty acid synthesis closely correlated with decreased cell proliferation. The stable isotope-based dynamic metabolic profile of MIA cells indicated a significant dose-dependent decrease in macromolecule synthesis, which was detected at lower drug doses and before the appearance of apoptosis markers. Normal fibroblasts (CRL-1501) did not show morphological or metabolic signs of apoptosis likely due to their slow rate of growth and metabolic activity. This indicates that limiting carbon re-cycling and rapid substrate mobilisation from glycogen may be an effective and selective target site for new drug development in rapidly dividing cancer cells. In conclusion, pancreatic cancer cell growth arrest and death are closely associated with a characteristic decrease in glycogen breakdown and glucose carbon re-distribution towards RNA/DNA and fatty acids during CP-320626 treatment

Glucose utilization via glycogen phosphorylase sustains proliferation and prevents premature senescence in cancer cells.

Metabolic reprogramming of cancer cells provides energy and multiple intermediates critical for cell growth. Hypoxia in tumors represents a hostile environment that can encourage these transformations. We report that glycogen metabolism is upregulated in tumors in vivo and in cancer cells in vitro in response to hypoxia. In vitro, hypoxia induced an early accumulation of glycogen, followed by a gradual decline. Concordantly, glycogen synthase (GYS1) showed a rapid induction, followed by a later increase of glycogen phosphorylase (PYGL). PYGL depletion and the consequent glycogen accumulation led to increased reactive oxygen species (ROS) levels that contributed to a p53-dependent induction of senescence and markedly impaired tumorigenesis in vivo. Metabolic analyses indicated that glycogen degradation by PYGL is important for the optimal function of the pentose phosphate pathway. Thus, glycogen metabolism is a key pathway induced by hypoxia, necessary for optimal glucose utilization, which represents a targetable mechanism of metabolic adaptation