Comparative Study of Gas and Liquid Chromatography Methods for the Determination of Underivatised Neutral and Acidic Cannabinoids and Cholesterol.

The aim of our study was to develop a gas chromatographic method coupled with mass spectrometry (GC-MS) for the determination of underivatised neutral (CBDs-N) and acidic (CBDs-A) cannabinoids (CBDs) and cholesterol (Chol). Emphasis was also placed on comparing our original GC-MS method with the currently developed C18-high-performance liquid chromatography with photodiode detection (C18-HPLC-DAD). A combination of a long GC column, shallow temperature column programme, and mass-spectrometry was employed to avoid issues arising from the overlap between CBDs and Chol and background fluctuations. The pre-column procedure for CBDs and Chol in egg yolks consisted of hexane extractions, whereas the pre-column procedure for CBDs in non-animal samples involved methanol and hexane extractions. CBDs-A underwent decarboxylation to CBDs during GC-MS analyses, and pre-column extraction of the processed sample with NaOH solution allowed for CBD-A removal. No losses of CBDs-N were observed in the samples extracted with NaOH solution. GC-MS analyses of the samples before and after extraction with NaOH solution enabled the quantification of CBDs-A and CBDs-N. CBDs-A did not undergo decarboxylation to CBDs-N during C18-HPLC-DAD runs. The use of the C18-HPLC-DAD method allowed simultaneous determination of CBDs-N and CBDs-A. In comparison to the C18-HPLC-DAD method, our GC-MS technique offered improved sensitivity, precision, specificity, and satisfactory separation of underivatised CBDs and Chol from biological materials of endogenous species, especially in hemp and hen egg yolk. The scientific novelty of the present study is the application of the GC-MS method for quantifying underivatised CBDs-A, CBDs-N, and Chol in the samples of interest.

Preparation and characterization of hydrophobic and hydrophilic amidated derivatives of carboxymethyl chitosan and carboxymethyl ß-glucan.

Chitosan and ß-glucan are substances that are very widely used in the pharmaceutical and food industries, medicine and other areas. These polysaccharides have immense significance in human metabolism. They are able to affect the levels of cholesterol and lipids. Chemical modification of these polysaccharides allows the support of these attributes. A main goal is to prepare a group of amide derivatives of carboxymethyl chitosan and carboxymethyl ß-glucan to obtain a new group of polysaccharide derivatives and increase the benefits and attributes of these polysaccharides. The aim of carboxymethylation and subsequent two-step amidation is to achieve a high degree of substitution in the prepared derivatives. The amidation consisted methyl esterification followed by amino-de-alkoxylation with amidation reagents (n-alkylamines, hydrazine and hydroxylamine). The purity and substitution degree of the prepared derivatives were monitored by vibration spectroscopic methods (FTIR and FT Raman) and organic elemental analysis. The main, which was due to the presence of a secondary component (chitin) of polysaccharides, was the calculation of the substitution degree, which was based on the organic elemental analysis in combination with FTIR spectroscopy. These analytic methods confirmed the preparation of the substituted N-alkylamides, hydrazide and hydroxamic acid of carboxymethyl chitosan and carboxymethyl ß-glucan.

Performance and Meat Quality of Dual-Purpose Cockerels of Dominant Genotype Reared on Pasture.

The culling of layer cockerels due to economic inefficiency is an ethical problem. Organic or free-range fattening of these cockerels or dual-purpose genotypes breeding is a possible solution to this problem. The aim of the study was to assess the differences in performance and meat quality characteristics in dual-purpose cockerels Dominant of three genotypes (Dominant Sussex D 104, Dominant Brown D 102 and Dominant Tinted D 723, 100 cockerels per genotype) with access to pasture. The cockerels were housed in mobile boxes on the pasture herbage from the 50th to 77th day of age (stocking density: 0.108 m2/bird). The highest body weight on the 77th day of age (p < 0.001) and the nonsignificantly lowest feed conversion was achieved by Dominant Brown D 102 cockerels (1842 g and 2.79, respectively). Non-significantly higher pasture herbage intake on the 70th day of age was recorded in genotype Dominant Brown D 102 (7.41 g dry matter (DM)/bird/day) and Dominant Tinted D 723 (7.52 g DM/bird/day). The pasture herbage contained 56.9 mg/kg DM α-tocopherol, 170.3 mg/kg DM zeaxanthin and 175.0 mg/kg DM lutein and had a favourable n6/n3 ratio (0.26). The boiled meat of cockerels Dominant Tinted D723 showed the highest tenderness based on both the sensory evaluation (p = 0.022) and the value of shear force (p = 0.049). This corresponds with a higher (p < 0.001) cross-sectional area and muscle fibre diameter in these chickens. The highest content of n3 fatty acids (eicosapentaenoic, clupanodonic and docosahexaenoic acids) in breast meat were found in Dominant Sussex D104 chickens (p < 0.001). In contrast, a significantly higher α-tocopherol content (p < 0.001) and higher oxidative stability (p = 0.012) were found in Dominant Brown D102 (4.52 mg/kg and 0.282 mg/kg) and Dominant Tinted D 723 chickens (4.64 mg/kg and 0.273 mg/kg) in comparison with the Dominant Sussex D104 genotype (3.44 mg/kg and 0.313 mg/kg). The values of the atherogenic and thrombogenic indexes were the lowest (p < 0.001) in meat from Dominant Brown D102 chickens. Moreover, a lower cholesterol content (p < 0.001) was recorded from the genotypes Dominant Brown D102 (396 mg/kg) and Dominant Tinted D723 (306 mg/kg) chickens, contrary to the Dominant Sussex D104 cockerels (441 mg/kg). It can be concluded that cockerels Dominant Brown D102 are a suitable genotype for free-range rearing due to higher performance and higher pasture herbage intake, which positively influences meat quality, whereas the meat of Dominant Sussex D104 cockerels shows higher amounts of n3 fatty acids and lower n6/n3 ratios.

Effects of Dietary Hemp Seed and Flaxseed on Growth Performance, Meat Fatty Acid Compositions, Liver Tocopherol Concentration and Bone Strength of Cockerels.

The aim of the study is to determine the effect of hemp seed (HS) of the nonpsychotropic variety Futura and extruded flaxseed (EF) in the diet of cockerels on cockerel growth performance, breast muscle and liver α- and γ-tocopherol concentrations, breast muscle fatty acid concentrations and tibia strength. Five hundred and forty one-day-old male Ross 308 cockerels are equally allocated into six groups. Each group has three replicates of 30 cockerels in pens with litter. The formulated diets are isoenergetic (the metabolisable energy ranged from 12.4 to 12.8 MJ/kg) and isonitrogenic (the protein concentration ranged from 209.7 to 210.9 g/kg) and provided ad libitum. During the experiment, which lasts 35 days, the control group is fed a diet without EF or HS. Rapeseed oil was the lipid source in the control diet. The diet for the second group contains EF at 60 g/kg, the diet for the third group contains HS at 40 g/kg, and the diets for the fourth to sixth groups contain HS and EF at 30 and 60 g/kg, 40 and 60 g/kg and 50 and 60 g/kg, respectively. At the end of the experiment, 15 cockerels of average weight are slaughtered per group, and the breast muscle, liver and tibia bone are dissected for chemical analyses. The all dietary combination of HS and EF increases (p < 0.001) cockerel body weight (2375-2493 g) more than HS alone (2174 g) or EF alone (2254 g). A similar finding is observed for the diet composition and tocopherol content in the liver, but the doses of HS required to achieve this effect are higher (40 and 50 g/kg). The tocopherol content in the breast muscle is not influenced by the diet. The dietary combination of 60 g/kg EF and 40 g/kg HS results in the most promising findings of the experiment, since it leads to the lowest n-6/n-3 polyunsaturated fatty acids ratio (p < 0.001; 1.75). Incorporation of HS into the diet increases cockerel tibia strength (p < 0.001), which is of great practical importance due to the frequent occurrence of limb fractures. To conclude, the dietary supplementation with 40 g/kg HS and 60 g/kg EF improves cockerel performance, meat and bone quality and deposition of α-tocopherol in the liver.

Effect of amidated alginate on faecal lipids, serum and hepatic cholesterol in rats fed diets supplemented with fat and cholesterol.

The effect of octadecylamide of alginic acid on serum and hepatic cholesterol, and the faecal output of fat and sterols was examined in female rats fed diets containing cholesterol and palm fat at 10 and 50 g kg-1, respectively. Cholesterol supplementation significantly increased serum and hepatic cholesterol concentrations, and faecal output of cholesterol and coprostanol. Cholesterol and amidated alginate supplementations changed the profile of fatty acids in the faeces. Cholesterol increased molar percentages of saturated fatty acids and amidated alginate reversed this effect. Amidated alginate, supplied at 10, 20 and 40 g kg-1, significantly decreased serum cholesterol from 2.82 to 2.00, 1.95, and 1.63 µmol mL-1, respectively, and significantly decreased hepatic cholesterol from 13.8 to 9.33, 7.81 and 6.3 µmol g-1, respectively. Amidated alginate increased the faecal output of fat and neutral sterols in a dose-dependent manner. In contrast, the output of bile acids was significantly decreased. The faecal outputs of fat and serum cholesterol were negatively correlated. At the highest concentration tested, amidated alginate significantly reduced the serum concentration of triacylglycerols. It can be concluded that amidated alginate is an effective cholesterol-lowering agent and sorbent of dietary fat.

Preparation and characterization of amidated derivatives of alginic acid.

Alginic acid is a suitable material for modification to prepare new derivatives because of presence of its carboxyl groups. The high content of carboxyl groups over the entire length of its chain renders it an easily modifiable material with a possibility of achieving a high degree of substitution in the prepared derivatives. The salt of alginic acid (sodium alginate) is readily commercially available and is widely used in many branches of chemistry. Alginic acid was thus selected as the substrate for amidation. The amidation used two-steps: methyl esterification followed by amino-de-alkoxylation. The aim of this study was to prepare highly substituted derivatives with different polysaccharide chain characteristics. As such, the alginic acid was modified by the two-step amidation based on the esterification of the alginic acid carboxyl groups by reaction with methanol and further amino-de-alkoxylation (aminolysis) of the obtained methyl ester with amidation reagents (n-alkylamines, hydrazine and hydroxylamine). The purity and substitution degree of the prepared derivatives were monitored by vibration spectroscopic methods (FTIR and FT Raman) and organic elemental analysis. These analytical methods confirmed the preparation of highly or moderately substituted N-alkylamides, hydrazide and hydroxamic acid of alginic acid.

Comparative study of the hypocholesterolemic and hypolipidemic activity of alginate and amidated alginate in rats.

Alginate is a copolymer of ß-d-mannuronate and α-l-guluronate, which are present in the cell wall of brown algae. The hypocholesterolemic and hypolipidemic activities of alginate and its derivative, which is prepared by a reaction with octadecylamine, were compared in rats fed diets containing cholesterol and palm fat at 10 and 50g/kg, respectively. Amidated alginate at 20g/kg significantly decreased serum cholesterol from 2.93 to 2.00µmol/mL, serum triacylglycerols from 1.66 to 0.92µmol/mL, hepatic cholesterol from 17.5 to 5.9µmol/g, and total hepatic lipids from 67.4 to 51.7mg/g. Alginate at 20g/kg significantly reduced hepatic cholesterol to 13.1µmol/g, but did not influence serum cholesterol, triacylglycerols, and total hepatic lipids. Amidated alginate significantly increased the faecal concentrations of neutral sterols from 98.7 to 122.4µmol/g DM, but decreased faecal concentration of bile acids from 19.4 to 14.0µmol/g DM. In samples of intestinal contents, taurine-conjugated bile acids dominated glycine conjugates. The supplementation of diets with cholesterol significantly increased the expression of hepatic cholesterol 7α-hydroxylase, especially in rats that received cholesterol without alginate or amidated alginate. In conclusion, amidated alginate is an effective hypocholesterolemic agent that is more efficient than its parent polysaccharide.

Preparation of amidated derivatives of carboxymethylcellulose.

Carboxymethylcellulose (CMC) was selected as substrate for amidation based on previous results described for monocarboxy cellulose (MCC) with the aim to prepare highly substituted products. In comparison with MCC containing uronic carboxyl groups at C-6 position, O-carboxymethyl groups in CMC should be more accessible for reagents because they are more distant from the polysaccharide chain. Two-step way of amidation was based on the esterification of CMC carboxyls by reaction with methanol and further amino-de-alkoxylation (aminolysis) of the obtained methyl ester with amidation reagents (n-alkylamines, hydrazine and hydroxylamine). Purity and substitution degree of the products were monitored by the vibration spectroscopic methods (FTIR and Raman) and organic elemental analysis. Analytical methods confirmed the preparation of highly or moderately substituted N-alkylamides, hydrazide and hydroxamic acid of CMC.

Cholesterol and fat lowering with hydrophobic polysaccharide derivatives.

Hydrophobic derivatives of highly methylated citrus pectin, chitosan and cellulose were prepared and tested as potential cholesterol lowering agents. Elemental analysis and spectroscopic methods confirmed high substitution degree for all of them. Substitution with long alkyl/acyl groups led to significant changes in physical and thermal properties of modified polysaccharides. Sorption of cholate and cholesterol by these polysaccharide-based sorbents was estimated in comparison with the synthetic drug cholestyramine. It was found that modified polysaccharides have high affinity to cholesterol. By contrast, cholestyramine was effective only in cholate sorption.

Structural Features and Anti-coagulant Activity of the Sulphated Polysaccharide SPS-CF from a Green Alga Capsosiphon fulvescens.

Previously, we reported that the sulphated polysaccharides (SPS)-CF, a water-soluble polysaccharide isolated and purified from Korean green alga Maesaengi (Capsosiphon fulvescens, Chlorophyta), is a glucuronogalactomannan based mainly on the monosaccharide composition determined by high-performance liquid chromatography (HPLC) analysis after 1-phenyl-3-methyl-5-pyrazolone (PMP) labelling of sugars in the acid (trifluoroacetic acid (TFA)) hydrolyzates of SPS-CF, which showed mannose (55.4 mol %), galactose (25.3 mol %) and glucuronic acid (16.3 mol %) as major sugars (Na et al., Int Immunopharmacol 10:364-370, 2010). However, the results of the present study re-performed for monosaccharide composition of this polysaccharide using, in addition to HPLC of PMP-labelled sugars, other separation methods, i.e. high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), gas chromatography with flame ionising detection (GC-FID) and thin-layer chromatography (TLC), clearly demonstrated that the most prominent neutral monosaccharides of SPS-CF are xylose (38.6-49.4 mol %) and rhamnose (39.6-45 mol %), while mannose and galactose are present at a much lesser extent or in negligible amount. These extensive monosaccharide analyses, correlation nuclear magnetic resonance (NMR), electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) measurements confirmed the sulphated glucuronorhamnoxylan (ulvan) type of SPS-CF polysaccharide, whose backbone is composed of alternating sequence of 4-linked L-rhamnose-3-sulphate and D-xylose residues (ulvobiose U3s) carrying monomeric D-glucuronic acid or D-glucuronic acid-3-sulphate on O-2 of some L-rhamnose-3-sulphate units as the side chains. The SPS-CF exhibited significant in vitro anti-coagulant activity by which the activated partial thromboplastin time (aPTT) and thrombin time (TT) were significantly prolonged. The results of this study demonstrated that the ulvan SPS-CF isolated from Korean Maesaengi C. fulvescens can be considered a potential anti-coagulant agent.

Dose-response efficacy and long-term effect of the hypocholesterolemic effect of octadecylpectinamide in rats.

The dose-response efficiency and long-term effect of the hypocholesterolemic effect of octadecylpectinamide was examined in female rats fed diets containing cholesterol at 10 g/kg. In our first experiment, amidated pectin supplied at 20 g/kg, 40 g/kg and 60 g/kg significantly decreased serum cholesterol from 3.32 µmol/ml (control) to 1.23 µmol/ml in a dose-dependent manner. In a second experiment, the hypocholesterolemic effect of amidated pectin supplied at 20 g/kg persisted after 3 months of feeding. In both experiments, the amidated pectin significantly decreased the concentrations of cholesterol in hepatic tissue and triacylglycerols in serum. The serum concentration of aspartate aminotransferase significantly increased in rats fed amidated pectin at 60 g/kg for 4 weeks, and at 20 g/kg for 3 months. In conclusion, amidated pectin at a low dose and used for a period shorter than 3 months might be considered as an effective hypocholesterolemic and lipid-lowering agent that may substitute typical antilipidemic drugs.