Non-enzymatic Degradation of (1→3)(1→4)-β-D-glucan in Aqueous Processing of Oats

Cereal water-soluble β-glucan [(1→3)(1→4)-β-D-glucan] has well-evidenced health benefits and it contributes to the texture properties of foods. These functions are characteristically dependent on the excellent viscosity forming ability of this cell wall polysaccharide. The viscosity is affected by the molar mass, solubility and conformation of β-glucan molecule, which are further known to be altered during food processing. This study focused on demonstrating the degradation of β-glucan in water solutions following the addition of ascorbic acid, during heat treatments or high pressure homogenisation. Furthermore, the motivation of this study was in the non-enzymatic degradation mechanisms, particularly in oxidative cleavage via hydroxyl radicals. The addition of ascorbic acid at food-related concentrations (2-50 mM), autoclaving (120°C) treatments, and high pressure homogenisation (300-1000 bar) considerably cleaved the β-glucan chains, determined as a steep decrease in the viscosity of β-glucan solutions and decrease in the molar mass of β-glucan. The cleavage was more intense in a solution of native β-glucan with co-extracted compounds than in a solution of highly purified β-glucan. Despite the clear and immediate process-related degradation, β-glucan was less sensitive to these treatments compared to other water-soluble polysaccharides previously reported in the literature. In particular, the highly purified β-glucan was relatively resistant to the autoclaving treatments without the addition of ferrous ions. The formation of highly oxidative free radicals was detected at the elevated temperatures, and the formation was considerably accelerated by added ferrous ions. Also ascorbic acid pronounced the formation of these oxidative radicals, and oxygen was simultaneously consumed by ascorbic acid addition and by heating the β-glucan solutions. These results demonstrated the occurrence of oxidative reactions, most likely the metal catalysed Fenton-like reactions, in the β-glucan solutions during these processes. Furthermore, oxidized functional groups (carbonyls) were formed along the β-glucan chain by the treatments, including high pressure homogenisation, evidencing the oxidation of β-glucan by these treatments. The degradative forces acting on the particles in the high pressure homogenisation are generally considered to be the mechanical shear, but as shown here, carbohydrates are also easily degraded during the process, and oxidation may have a role in the modification of polysaccharides by this technique. In the present study, oat β-glucan was demonstrated to be susceptible to degradation during aqueous processing by non-enzymatic degradation mechanisms. Oxidation was for the first time shown to be a highly relevant degradation mechanism of β-glucan in food processing.

Solubility and physical stability improvement of natiral xanthine derivatives

Use of natural xanthine derivates in medicine is complicated with their physical properties. Theobromine is poorly soluble while theophylline is highly sensitive to hydration. The aim of this study was to improve bioavailability of xanthines by co-crystallization, theophylline was also cocrystallized with carboxylic acids (capric, citric, glutaric, malenic, malonic, oxalic, stearic, succinic) and HPMC. Co-crystallization was performed by slow evaporation and ball milling. Physical stability was checked by wet granulation and water sorption methods, solubility was measured by intrinsic tablet dissolution. Theobromine formed co-crystal with other xanthines and theophylline interacted with all acids except stearic and HPMC, the latter showed alternative interactions based on hydrogen bonding. Hydration resistance was good in theophylline:succinic acid co-crystal and excellent in complexes containing capric, stearic acids and HPMC. Theophylline:HPMC showed improved solubility. The reported approach can promote use of xanthines and can be recommended for other compounds with similar problems.

Mesoporous silica- and silicon-based materials as carriers for poorly water soluble drugs

New chemical entities with unfavorable water solubility properties are continuously emerging in drug discovery. Without pharmaceutical manipulations inefficient concentrations of these drugs in the systemic circulation are probable. Typically, in order to be absorbed from the gastrointestinal tract, the drug has to be dissolved. Several methods have been developed to improve the dissolution of poorly soluble drugs. In this study, the applicability of different types of mesoporous (pore diameters between 2 and 50 nm) silicon- and silica-based materials as pharmaceutical carriers for poorly water soluble drugs was evaluated. Thermally oxidized and carbonized mesoporous silicon materials, ordered mesoporous silicas MCM-41 and SBA-15, and non-treated mesoporous silicon and silica gel were assessed in the experiments. The characteristic properties of these materials are the narrow pore diameters and the large surface areas up to over 900 m²/g. Loading of poorly water soluble drugs into these pores restricts their crystallization, and thus, improves drug dissolution from the materials as compared to the bulk drug molecules. In addition, the wide surface area provides possibilities for interactions between the loaded substance and the carrier particle, allowing the stabilization of the system. Ibuprofen, indomethacin and furosemide were selected as poorly soluble model drugs in this study. Their solubilities are strongly pH-dependent and the poorest (< 100 µg/ml) at low pH values. The pharmaceutical performance of the studied materials was evaluated by several methods. In this work, drug loading was performed successfully using rotavapor and fluid bed equipment in a larger scale and in a more efficient manner than with the commonly used immersion methods. It was shown that several carrier particle properties, in particular the pore diameter, affect the loading efficiency (typically ~25-40 w-%) and the release rate of the drug from the mesoporous carriers. A wide pore diameter provided easier loading and faster release of the drug. The ordering and length of the pores also affected the efficiency of the drug diffusion. However, these properties can also compensate the effects of each other. The surface treatment of porous silicon was important in stabilizing the system, as the non-treated mesoporous silicon was easily oxidized at room temperature. Different surface chemical treatments changed the hydrophilicity of the porous silicon materials and also the potential interactions between the loaded drug and the particle, which further affected the drug release properties. In all of the studies, it was demonstrated that loading into mesoporous silicon and silica materials improved the dissolution of the poorly soluble drugs as compared to the corresponding bulk compounds (e.g. after 30 min ~2-7 times more drug was dissolved depending on the materials). The release profile of the loaded substances remained similar also after 3 months of storage at 30°C/56% RH. The thermally carbonized mesoporous silicon did not compromise the Caco-2 monolayer integrity in the permeation studies and improved drug permeability was observed. The loaded mesoporous silica materials were also successfully compressed into tablets without compromising their characteristic structural and drug releasing properties. The results of this research indicated that mesoporous silicon/silica-based materials are promising materials to improve the dissolution of poorly water soluble drugs. Their feasibility in pharmaceutical laboratory scale processes was also confirmed in this thesis.

Lewis Acid-Lewis Base Mediated Metal-Free Hydrogen Activation and Catalytic Hydrogenation

Organocatalysis, the use of organic molecules as catalysts, is attracting increasing attention as one of the most modern and rapidly growing areas of organic chemistry, with countless research groups in both academia and the pharmaceutical industry around the world working on this subject. The literature review of this thesis mainly focuses on metal-free systems for hydrogen activation and organocatalytic reduction. Since these research topics are relatively new, the literature review also highlights the basic principles of the use of Lewis acid-Lewis base pairs, which do not react irreversibly with each other, as a trap for small molecules. The experimental section progresses from the first observation of the facile heterolytical cleavage of hydrogen gas by amines and B(C6F5)3 to highly active non-metal catalysts for both enantioselective and racemic hydrogenation of unsaturated nitrogen-containing compounds. Moreover, detailed studies of structure-reactivity relationships of these systems by X-ray, neutron diffraction, NMR methods and quantum chemical calculations were performed to gain further insight into the mechanism of hydrogen activation and hydrogenation by boron-nitrogen compounds.

Immunochemical analysis of prolamins in gluten-free foods

People with coeliac disease have to maintain a gluten-free diet, which means excluding wheat, barley and rye prolamin proteins from their diet. Immunochemical methods are used to analyse the harmful proteins and to control the purity of gluten-free foods. In this thesis, the behaviour of prolamins in immunological gluten assays and with different prolamin-specific antibodies was examined. The immunoassays were also used to detect residual rye prolamins in sourdough systems after enzymatic hydrolysis and wheat prolamins after deamidation. The aim was to characterize the ability of the gluten analysis assays to quantify different prolamins in varying matrices in order to improve the accuracy of the assays. Prolamin groups of cereals consist of a complex mixture of proteins that vary in their size and amino acid sequences. Two common characteristics distinguish prolamins from other cereal proteins. Firstly, they are soluble in aqueous alcohols, and secondly, most of the prolamins are mainly formed from repetitive amino acid sequences containing high amounts of proline and glutamine. The diversity among prolamin proteins sets high requirements for their quantification. In the present study, prolamin contents were evaluated using enzyme-linked immunosorbent assays based on ω- and R5 antibodies. In addition, assays based on A1 and G12 antibodies were used to examine the effect of deamidation on prolamin proteins. The prolamin compositions and the cross-reactivity of antibodies with prolamin groups were evaluated with electrophoretic separation and Western blotting. The results of this thesis research demonstrate that the currently used gluten analysis methods are not able to accurately quantify barley prolamins, especially when hydrolysed or mixed in oats. However, more precise results can be obtained when the standard more closely matches the sample proteins, as demonstrated with barley prolamin standards. The study also revealed that all of the harmful prolamins, i.e. wheat, barley and rye prolamins, are most efficiently extracted with 40% 1-propanol containing 1% dithiothreitol at 50 °C. The extractability of barley and rye prolamins was considerably higher with 40% 1-propanol than with 60% ethanol, which is typically used for prolamin extraction. The prolamin levels of rye were lowered by 99.5% from the original levels when an enzyme-active rye-malt sourdough system was used for prolamin degradation. Such extensive degradation of rye prolamins suggest the use of sourdough as a part of gluten-free baking. Deamidation increases the diversity of prolamins and improves their solubility and ability to form structures such as emulsions and foams. Deamidation changes the protein structure, which has consequences for antibody recognition in gluten analysis. According to the resuts of the present work, the analysis methods were not able to quantify wheat gluten after deamidation except at very high concentrations. Consequently, deamidated gluten peptides can exist in food products and remain undetected, and thus cause a risk for people with gluten intolerance. The results of this thesis demonstrate that current gluten analysis methods cannot accurately quantify prolamins in all food matrices. New information on the prolamins of rye and barley in addition to wheat prolamins is also provided in this thesis, which is essential for improving gluten analysis methods so that they can more accurately quantify prolamins from harmful cereals.

Effects of gender, and SLCO1B1 and CYP7A1 polymorphisms on plasma bile acids in humans and the pharmacokinetics of therapeutic ursodeoxycholic acid

Bile acids are important steroid-derived molecules essential for fat absorption in the small intestine. They are produced in the liver and secreted into the bile. Bile acids are transported by bile flow to the small intestine, where they aid the digestion of lipids. Most bile acids are reabsorbed in the small intestine and return to the liver through the portal vein. The whole recycling process is referred to as the enterohepatic circulation, during which only a small amount of bile acids are removed from the body via faeces. The enterohepatic circulation of bile acids involves the delicate coordination of a number of bile acid transporters expressed in the liver and the small intestine. Organic anion transporting polypeptide 1B1 (OATP1B1), encoded by the solute carrier organic anion transporter family, member 1B1 (SLCO1B1) gene, mediates the sodium independent hepatocellular uptake of bile acids. Two common SNPs in the SLCO1B1 gene are well known to affect the transport activity of OATP1B1. Moreover, bile acid synthesis is an important elimination route for cholesterol. Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme of bile acid production. The aim of this thesis was to investigate the effects of SLCO1B1 polymorphism on the fasting plasma levels of individual endogenous bile acids and a bile acid synthesis marker, and the pharmacokinetics of exogenously administered ursodeoxycholic acid (UDCA). Furthermore, the effects of CYP7A1 genetic polymorphism and gender on the fasting plasma concentrations of individual endogenous bile acids and the bile acid synthesis marker were evaluated. Firstly, a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of bile acids was developed (Study I). A retrospective study examined the effects of SLCO1B1 genetic polymorphism on the fasting plasma concentrations of individual bile acids and a bile acid synthesis marker in 65 healthy subjects (Study II). In another retrospective study with 143 healthy individuals, the effects of CYP7A1 genetic polymorphism and gender as well as SLCO1B1 polymorphism on the fasting plasma levels of individual bile acids and the bile acid synthesis marker were investigated (Study III). The effects of SLCO1B1 polymorphism on the pharmacokinetics of exogenously administered UDCA were evaluated in a prospective genotype panel study including 27 healthy volunteers (Study IV). A robust, sensitive and simple HPLC-MS/MS method was developed for the simultaneous determination of 16 individual bile acids in human plasma. The method validation parameters for all the analytes met the requirements of the FDA (Food and Drug Administration) bioanalytical guidelines. This HPLC-MS/MS method was applied in Studies II-IV. In Study II, the fasting plasma concentrations of several bile acids and the bile acid synthesis marker seemed to be affected by SLCO1B1 genetic polymorphism, but these findings were not replicated in Study III with a larger sample size. Moreover, SLCO1B1 polymorphism had no effect on the pharmacokinetic parameters of exogenously administered UDCA. Furthermore, no consistent association was observed between CYP7A1 genetic polymorphism and the fasting plasma concentrations of individual bile acids or the bile acid synthesis marker. In contrast, gender had a major effect on the fasting plasma concentrations of several bile acids and also total bile acids. In conclusion, gender, but not SLCO1B1 or CYP7A1 polymorphisms, has a major effect on the fasting plasma concentrations of individual bile acids. Moreover, the common genetic polymorphism of CYP7A1 is unlikely to influence the activity of CYP7A1 under normal physiological conditions. OATP1B1 does not play an important role in the in vivo disposition of exogenously administered UDCA.

Lipeäkäsitellyn vehnän vaikutus lypsylehmien syöntiin ja tuotokseen vapaalla seosrehuruokinnalla

Lipeä on vahva emäs, jonka on havaittu lisäävän hemiselluloosan ja ligniinin hydrolyysiä pötsissä. Näin ollen lipeäkäsittelyllä on mahdollista korvata viljan mekaaninen litistys ja jauhatus. Seosrehuruokinnalla, jonka osana on lipeäkäsitelty vilja, on mahdollista vähentää liiallisesta tärkkelyksestä aiheutuvia metabolisia ongelmia pötsissä. Tämän tutkielman tarkoituksena oli selvittää lipeäkäsitellyn vehnän vaikutusta lypsylehmien syöntiin ja tuotokseen ad libitum seosrehuruokinnoilla. Ruokinnoissa korvattiin kuivaa murskattua vehnää asteittain kokonaisella lipeäkäsitellyllä vehnällä. Kontrollina oli perinteisesti käytetty kuiva, murskattu ohra-kaura seos. Koe tehtiin Ruotsin maatalousyliopiston (SLU) maataloustieteiden laitoksella Uumajassa. Koe alkoi syyskuussa ja päättyi joulukuussa 2010. Kokeessa oli 17 useamman kerran poikinutta lehmää ja 6 ensikkoa (Ruotsin punainen -rotu). Lehmät olivat lämpimässä pihattonavetassa, jossa seosrehun syöntiä mitattiin vaakakuppien avulla. Koekäsittelyt olivat murskattu ohra-kaura seos (1:1), murskattu kuiva vehnä (1:0), murskatun kuivan vehnän ja kokonaisen lipeävehnän seos (1:1) ja kokonainen lipeävehnä (1:0). Ruokintojen kuiva-ainepitoisuudeksi asetettiin 370 g/kg ja raakavalkuaispitoisuudeksi 180 g/kg kuiva-ainetta. Näennäinen ravintoaineen sulavuus määritettiin happoon liukenemattoman tuhkan avulla. Typen hyväksikäyttöä arvioitiin laskennallisen typpitaseen avulla. Koe toteutettiin 4x4 latinalaisen neliön koemallin mukaisesti ja käsittelyjen väliset tilastolliset erot testattiin kontrastien avulla. Kuiva-aineen (PQ=0,02) ja orgaanisen aineen (PQ=0,02) syönnit lisääntyivät, samalla kun niiden sulavuudet paranivat korvattaessa puolet kuivasta vehnästä lipeävehnällä. Ruokintojen välillä ei ollut tilastollisesti merkitsevää eroa maitotuotoksessa eikä energiakorjatussa maitotuotoksessa. Maidon rasvatuotos lisääntyi vähän (PQ=0,04) ja rasvapitoisuus selvästi (PQ=0,004), kun kuivasta vehnästä korvattiin puolet lipeävehnällä. Kun kaikki kuiva vehnä korvattiin lipeävehnällä, maidon valkuaispitoisuus väheni (PL<0,001). Samoin kävi maidon ureapitoisuudelle (PL=0,002). Lipeäkäsittely ei tuottanut tässä kokeessa taloudellisesti kannattavaa tulosta, sillä maidon valkuaispitoisuus väheni ja syönti lisääntyi maitotuotoksen pysyessä samana. Vehnäruokinnoista paras tuotosvaste saatiin kuivan vehnän ja lipeävehnän seoksella.