Quantification of hydroxycinnamic acids and lignin in perennial forage and energy grasses by Fourier-transform infrared spectroscopy and partial least squares regression

Levels of lignin and hydroxycinnamic acid wall components in three genera of forage grasses (Lolium,Festuca and Dactylis) have been accurately predicted by Fourier-transform infrared spectroscopy using partial least squares models correlated to analytical measurements. Different models were derived that predicted the concentrations of acid detergent lignin, total hydroxycinnamic acids, total ferulate monomers plus dimers, p-coumarate and ferulate dimers in independent spectral test data from methanol extracted samples of perennial forage grass with accuracies of 92.8%, 86.5%, 86.1%, 59.7% and 84.7% respectively, and analysis of model projection scores showed that the models relied generally on spectral features that are known absorptions of these compounds. Acid detergent lignin was predicted in samples of two species of energy grass, (Phalaris arundinacea and Pancium virgatum) with an accuracy of 84.5%.

Measurement of key compositional parameters in two species of energy grass by Fourier transform infrared spectroscopy

Two energy grass species, switch grass, a North American tuft grass, and reed canary grass, a European native, are likely to be important sources of biomass in Western Europe for the production of biorenewable energy. Matching chemical composition to conversion efficiency is a primary goal for improvement programmes and for determining the quality of biomass feed-stocks prior to use and there is a need for methods which allow cost effective characterisation of chemical composition at high rates of sample through-put. In this paper we demonstrate that nitrogen content and alkali index, parameters greatly influencing thermal conversion efficiency, can be accurately predicted in dried samples of these species grown under a range of agronomic conditions by partial least square regression of Fourier transform infrared spectra (R2 values for plots of predicted vs. measured values of 0.938 and 0.937, respectively). We also discuss the prediction of carbon and ash content in these samples and the application of infrared based predictive methods for the breeding improvement of energy grasses.

Systems biology approach to study permeability of paracetamol and its solid dispersion

Physiological changes that take place at cellular level are usually reflective of their level of gene expression. Different formulation excipients have an impact on physiological behavior of the exposed cells and in turn affect transporter genes, enterocyte-mediated metabolism and toxicity biomarkers. The aim of this study was to prepare solid dispersion of paracetamol and evaluate genetic changes that occur in Caco-2 cell lines during the permeability of paracetamol alone and paracetamol solid dispersion formulations. Paracetamol-PEG 8000 solid dispersion was prepared by melt fusion method and the formulation was characterised using differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Formulation of solid dispersion resulted in the conversion of crystalline drug into an amorphous form. Permeability studies showed that paracetamol absorption was higher from the solid dispersion formulation. DNA microarrays analysis was carried out in order to investigate the involvement of any efflux/uptake transporters in paracetamol or its solid dispersion permeability. Neither transporter carriers nor efflux proteins were found to be involved in the absorption of paracetamol or its PEG solid dispersion. Gene expression analysis established that paracetamol toxicity was potentially reduced upon formulation into solid dispersion when ATP binding cassette (ABC) and solute carrier transporter (SLC) genes were analyzed.

Matrix-free mass spectrometric imaging using laser desorption ionisation Fourier transform ion cyclotron resonance mass spectrometry

Mass spectrometry imaging (MSI) is a powerful tool in metabolomics and proteomics for the spatial localization and identification of pharmaceuticals, metabolites, lipids, peptides and proteins in biological tissues. However, sample preparation remains a crucial variable in obtaining the most accurate distributions. Common washing steps used to remove salts, and solvent-based matrix application, allow analyte spreading to occur. Solvent-free matrix applications can reduce this risk, but increase the possibility of ionisation bias due to matrix adhesion to tissue sections. We report here the use of matrix-free MSI using laser desorption ionisation performed on a 12 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. We used unprocessed tissue with no post-processing following thaw-mounting on matrix-assisted laser desorption ionisation (MALDI) indium-tin oxide (ITO) target plates. The identification and distribution of a range of phospholipids in mouse brain and kidney sections are presented and compared with previously published MALDI time-of-flight (TOF) MSI distributions.

Tailored laminin-332 alpha3 sequence is tethered through an enzymatic linker to a collagen scaffold to promote cellular adhesion

Surface modification techniques have been used to develop biomimetic scaffolds by incorporating cell adhesion peptides, which facilitates cell adhesion, migration and proliferation. In this study, we evaluated the cell adhesion properties of a tailored laminin-332 alpha3 chain tethered to a type I collagen scaffold using microbial transglutaminase (mTGase) by incorporating transglutaminase substrate peptide sequences containing either glutamine (peptide A: PPFLMLLKGSTREAQQIVM) or lysine (peptide B: PPFLMLLKGSTRKKKKG). The degree of cross-linking was studied by amino acid analysis following proteolytic digestion and the structural changes in the modified scaffold further investigated using Fourier transform infrared spectroscopy and atomic force microscopy. Fibroblasts were used to evaluate the cellular behaviour of the functionalized collagen scaffold. mTGase supports cell growth but tethering of peptide A and peptide B to the mTGase cross-linked collagen scaffold caused a significant increase in cell proliferation when compared with native and mTGase cross-linked collagen scaffolds. Both peptides enabled cell-spreading, attachment and normal actin cytoskeleton organization with slight increase in the cell proliferation was observed in peptide A when compared with the peptide B and mTGase cross-linked scaffold. An increase in the amount of epsilon(gamma-glutamyl) lysine isopeptide was observed in peptide A conjugated scaffolds when compared with peptide B conjugated scaffolds, mTGase cross-linked scaffold without peptide. Changes in D-spacing were observed in the cross-linked scaffolds with tethered peptides. These results demonstrate that mTGase can play a bifunctional role in both conjugation of the glutamine and lysine containing peptide sequences and also in the cross-linking of the collagen scaffold, thus providing a suitable substrate for cell growth.

Tethering a laminin peptide to a crosslinked collagen scaffold for biofunctionality

Cell adhesion peptide regulates various cellular functions like proliferation, attachment, and spreading. The cellular response to laminin peptide (PPFLMLLKGSTR), a motif of laminin-5 alpha3 chain, tethered to type I collagen, crosslinked using microbial transglutaminase (mTGase) was investigated. mTGase is an enzyme that initiates crosslinking by reacting with the glutamine and lysine residues on the collagen fibers stabilizing the molecular structure. In this study that tethering of the laminin peptide in a mTGase crosslinked collagen scaffold enhanced cell proliferation and attachment. Laminin peptide tethered crosslinked scaffold showed unaltered cell morphology of 3T3 fibroblasts when compared with collagen and crosslinked scaffold. The triple helical structure of collagen remained unaltered by the addition of laminin peptide. In addition a dose-dependent affinity of the laminin peptide towards collagen was seen. The degree of crosslinking was measured by amino acid analysis, differential scanning calorimeter and fourier transform infrared spectroscopy. Increased crosslinking was observed in mTGase crosslinked group. mTGase crosslinking showed higher shrinkage temperature. There was alteration in the fibrillar architecture due to the crosslinking activity of mTGase. Hence, the use of enzyme-mediated linking shows promise in tethering cell adhesive peptides through biodegradable scaffolds.

Assessment of cell viability in a three-dimensional enzymatically cross-linked collagen scaffold

Microbial transglutaminase (mTGase) is an enzyme that introduces a covalent bond between peptide bound glutamine and lysine residues. Proteins cross-linked in this manner are often more resistant to proteolytic degradation and show increased tensile strength. This study evaluates the effects of mTGase mediated cross-linking of collagen on the cellular morphology, behaviour and viability of murine 3T3 fibroblasts following their seeding into collagen scaffolds. Additionally, cell mediated scaffold contraction, porosity and level of cross-linking of the scaffold has been analysed using image analysis software, scanning electron microscopy (SEM), colorimetric assays, and Fourier transform infrared spectroscopy (FTIR). We demonstrate that the biocompatibility and cellular morphology, when comparing cultures of fibroblasts integrated in mTGase cross-linked collagen scaffolds with the native collagen counterparts, remained unaffected. It has been also elicited that the structural characteristics of collagen have been preserved while introducing enzymatically resistant covalent bonds.

Dissolution rate enhancement, in vitro evaluation and investigation of drug release kinetics of chloramphenicol and sulphamethoxazole solid dispersions

Formulation of solid dispersions is one of the effective methods to increase the rate of solubilization and dissolution of poorly soluble drugs. Solid dispersions of chloramphenicol (CP) and sulphamethoxazole (SX) as model drugs were prepared by melt fusion method using polyethylene glycol 8000 (PEG 8000) as an inert carrier. The dissolution rate of CP and SX were rapid from solid dispersions with low drug and high polymer content. Characterization was performed using fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). FTIR analysis for the solid dispersions of CP and SX showed that there was no interaction between PEG 8000 and the drugs. Hyper-DSC studies revealed that CP and SX were converted into an amorphous form when formulated as solid dispersion in PEG 8000. Mathematical analysis of the release kinetics demonstrated that drug release from the various formulations followed different mechanisms. Permeability studies demonstrated that both CP and SX when formulated as solid dispersions showed enhanced permeability across Caco-2 cells and CP can be classified as well-absorbed compound when formulated as solid dispersions. © 2013 Informa Healthcare USA, Inc.

Effect of sample preparation on the thermal degradation of metal-added biomass

The present study investigates the effect of different sample preparation methods on the pyrolysis behaviour of metal-added biomass; Willow samples were compared in the presence of two salts of zinc and lead containing sulphate and nitrate anions which were added to the wood samples with three different techniques as dry-mixing, impregnation and ion-exchange. The effect of acid and water wash as common demineralisation pre-treatments were also analysed to evaluate their roles in the thermal degradation of the biomass. Results from thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FT-IR) and pyrolysis-mass spectrometry (Py-MS) measurements indicated that these pre-treatments change the matrix and the physical-chemical properties of wood. Results suggested that these structural changes increase the thermal stability of cellulose during pyrolysis. Sample preparation was also found to be a crucial factor during pyrolysis; different anions of metal salts changed the weight loss rate curves of wood material, which indicates changes in the primary degradation process of the biomass. Results also showed that dry-mixing, impregnation or ion-exchange influence the thermal behaviour of wood in different ways when a chosen metal salt was and added to the wood material. © 2011 Elsevier B.V. All rights reserved.

Polymeric films for buccal drug delivery

The aim of this research project is to evaluate whether or not pullulan films are suitable to buccal drug delivery of a phosphodiesterase5 (PDE5) inhibitor yonkenafil, which was discovered in our research group and currently is under phase II clinical trial for treatment of erectile dysfunction. Variable formulations of pullulan films were designed and the films were prepared. Mechanical properties of the films, in vitro drug release and polymer dissolution, in vitro drug penetration through porcine esophageal mucosa were investigated. The plasticization effects of solvents, polyols and acids to the films were studied by tensile test, and differential scanning calorimetry, thermogravimetric analysis, fourier transform-infrared, scanning electron microscopy, optical microscopy was applied to analyse the structure and chemical-bonding between pullulan and the additives within the films. Release mathematics models were used in the study of the mechanism of drug releases and polymer dissolutions. Ethanol, menthol, fatty acids, and sodium dodecyl sulphate were employed as penetration enhancers to pretreat the tissue. Various plasticizers and acids were applied into the films and the result showed polyethylene glycol 400 and 600 had the excellent plasticization effect on the drug-free pullulan films, while lactic acid was the best plasticizer for the drug-loaded films. Both PEG400 and lactic acid had a great effect on the drug release from the films in vitro, and all the results indicated that the hydroxyl and carboxyl groups of pullulan and the additives influenced the mechanical properties of the films significantly, and also altered drug release mechanisms. Ethanol shows the greatest enhancing ability on the drug permeation through the porcine esophageal mucosa. A possible mechanism for this is that ethanol interferes with the structure of the lipids in the mucosa, resulting in increased partitioning of the drug into the membrane.

Potassium catalysis in the pyrolysis behaviour of short rotation willow coppice

Short rotation willow coppice (SRC) and a synthetic biomass, a mixture of the basic biomass components (cellulose, hemicellulose and lignin), have been investigated for the influence of potassium on their pyrolysis behaviours. The willow sample was pre-treated to remove salts and metals by hydrochloric acid, and this demineralised sample was impregnated with potassium. The same type of pre-treatment was applied to components of the synthetic biomass. Characterisation was performed using thermogravimetric analysis with measurement of products by means of Fourier transform infrared spectroscopy (TGA-FTIR) and pyrolysis-gas chromatography-mass spectrometry (PY-GC-MS). A comparison of product distributions and kinetics are reported. While the general features of decomposition of SRC are described well by an additive behaviour of the individual components, there are some differences in the magnitude of the influence of potassium, and on the products produced. For both SRC and the synthetic biomass, TGA traces indicate catalytic promotion of both of the two-stages of biomass decomposition, and potassium can lower the average apparent first-order activation energy for pyrolysis by up to 50 kJ/mol. For both SRC and synthetic biomass the yields and distribution of pyrolysis products have been influenced by the presence of the catalyst. Potassium catalysed pyrolysis increases the char yields markedly and this is more pronounced for synthetic biomass than SRC. Gas evolution profiles during pyrolysis show the same general features for both SRC and synthetic biomass. Relative methane yields increase during the char formation stage of pyrolysis of the potassium doped samples. The evolution profiles of acetic acid and formaldehyde change, and these products are seen in lower relative amounts for both the demineralised samples. A greater variation in pyrolysis products is observed from the treated SRC samples compared to the different synthetic biomass samples. Furthermore, substituted phenols from lignin pyrolysis are more dominant in the pyrolysis profiles of the synthetic biomass than of the SRC, implying that the extracted lignins used in the synthetic biomass yield a greater fraction of monomeric type species than the lignocellulosic cell wall material of SRC. For both types of samples, PY-GS-MS analyses show that potassium has a significant influence on cellulose decomposition markers, not just on the formation of levoglucosan, but also other species from the non-catalysed mechanism, such as 3,4-dihydroxy-3-cyclobutene-1,2-dione. © 2007 Elsevier Ltd. All rights reserved.

Reactive processing of polymers:functionalisation of ethylene-propylene diene terpolymer (EPDM) in the presence and absence of a co-agent and effect of functionalised EPDM on compatibilisation of poly(ethylene terephthalate)/EPDM blends

Ethylene-propylene diene terpolymer (EPDM) was functionalized with glycidyl methacrylate (GMA) during melt processing by free radical grafting with peroxide initiation in the presence and absence of a reactive comonomer trimethylolpropane triacrylate (Tris). Increasing the peroxide concentration resulted in an increase in the GMA grafting yield, albeit the overall grafting level was low and was accompanied by higher degree of crosslinking of EPDM which was found to be the major competing reaction. The presence of Tris in the grafting system gave rise to higher grafting yield produced at a much lower peroxide concentration though the crosslinking reactions remained high but without the formation of GMA-homopolymer in either of the two systems. The use of these functionalized EPDM (f-EPDM) samples with PET as compatibilisers in binary and ternary blends of PET/EPDM/f-EPDM was evaluated. The influence of the different functionalisation routes of the rubber phase (in presence and absence of Tris) and the effect of the level of functionality and microstructure of the resultant f-EPDM on the extent of the interfacial reaction, morphology and mechanical properties was also investigated. It is suggested that the mechanical properties of the blends are strongly influenced by the performance of the graft copolymer, which is in turn, determined by the level of functionality, molecular structure of the functionalized rubber and the interfacial concentration of the graft copolymer across the interface. The cumulative evidence obtained from torque rheometry, scanning electron microscopy, SEM, dynamic mechanical analysis (DMA), tensile mechanical tests and Fourier transform infrared (FTIR) supports this. It was shown that binary and ternary blends prepared with f-EPDM in the absence of Tris and containing lower levels of g-GMA effected a significant improvement in mechanical properties. This increase, particularly in elongation to break, could be accounted for by the occurrence of a reaction between the epoxy groups of GMA and the hydroxyl/carboxyl end groups of PET that would result in a graft copolymer which could, most probably, preferentially locate at the interface, thereby acting as an 'emulsifier' responsible for decreasing the interfacial tension between the otherwise two immiscible phases. This is supported by results from FTIR analysis of the fractionated PET phase of these blends which confirm the formation of an interfacial reaction, DMA results which show a clear shift in the T s of the blend components and SEM results which reveal very fine morphology, suggesting effective compatibilisation that is concomitant with the improvement observed in their tensile properties. Although Tris has given rise to highest amount of g-GMA, it resulted in lower mechanical properties than the optimized blends produced in the absence of Tris. This was attributed to the difference in the microstructure of the graft and the level of functionality in these samples resulting in less favourable structure responsible for the coarser dispersion of the rubber phase observed by SEM, the lower extent of T shift of the PET phase (DMA), the lower height of the torque curve during reactive blending and FTIR analysis of the separated PET phase that has indicated a lower extent of the interfacial chemical reaction between the phases in this Tris-containing blend sample. © 2005 Elsevier Ltd. All rights reserved.

Reactive processing of polymers:effect of in situ compatibilisation on characteristics of blends of polyethylene terephthalate and ethylene-propylene rubber

Ethylene-propylene rubber (EPR) functionalised with glycidyl methacrylate (GMA) (f-EPR) during melt processing in the presence of a co-monomer, such as trimethylolpropane triacrylate (Tris), was used to promote compatibilisation in blends of polyethylene terephthalate (PET) and f-EPR, and their characteristics were compared with those of PET/f-EPR reactive blends in which the f-EPR was functionalised with GMA via a conventional free radical melt reaction (in the absence of a co-monomer). Binary blends of PETand f-EPR (with two types of f-EPR prepared either in presence or absence of the co-monomer) with various compositions (80/20, 60/40 and 50/50 w/w%) were prepared in an internal mixer. The blends were evaluated by their rheology (from changes in torque during melt processing and blending reflecting melt viscosity, and their melt flow rate), morphology scanning electron microscopy (SEM), dynamic mechanical properties (DMA), Fourier transform infrared (FTIR) analysis, and solubility (Molau) test. The reactive blends (PET/f-EPR) showed a marked increase in their melt viscosities in comparison with the corresponding physical (PET/EPR) blends (higher torque during melt blending), the extent of which depended on the amount of homopolymerised GMA (poly-GMA) present and the level of GMA grafting in the f-EPR. This increase was accounted for by, most probably, the occurrence of a reaction between the epoxy groups of GMA and the hydroxyl/carboxyl end groups of PET. Morphological examination by SEM showed a large improvement of phase dispersion, indicating reduced interfacial tension and compatibilisation, in both reactive blends, but with the Tris-GMA-based blends showing an even finer morphology (these blends are characterised by absence of poly-GMA and presence of higher level of grafted GMA in its f-EPR component by comparison to the conventional GMA-based blends). Examination of the DMA for the reactive blends at different compositions showed that in both cases there was a smaller separation between the glass transition temperatures compared to their position in the corresponding physical blends, which pointed to some interaction or chemical reaction between f-EPR and PET. The DMA results also showed that the shifts in the Tgs of the Tris-GMA-based blends were slightly higher than for the conventional GMA-blends. However, the overall tendency of the Tgs to approach each other in each case was found not to be significantly different (e.g. in a 60/40 ratio the former blend shifted by up to 4.5 °C in each direction whereas in the latter blend the shifts were about 3 °C). These results would suggest that in these blends the SEM and DMA analyses are probing uncorrelatable morphological details. The evidence for the formation of in situ graft copolymer between the f-EPR and PET during reactive blending was clearly illustrated from analysis by FTIR of the separated phases from the Tris-GMA-based reactive blends, and the positive Molau test pointed out to graft copolymerisation in the interface. A mechanism for the formation of the interfacial reaction during the reactive blending process is proposed.

Novel sol–gel preparation of (P2O5)0.4–(CaO)0.25–(Na2O)X–(TiO2)(0.35−X) bioresorbable glasses (X = 0.05, 0.1, and 0.15)

Quaternary phosphate-based glasses in the P2O5–CaO–Na2O–TiO2 system with a fixed P2O5 and CaO content of 40 and 25 mol% respectively have been successfully synthesised via sol–gel method and bulk, transparent samples were obtained. The structure, elemental proportion, and thermal properties of stabilised sol–gel glasses have been characterised using X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDX), 31P nuclear magnetic resonance (31P NMR), titanium K-edge X-ray absorption near-edge structure (XANES), fourier transform infrared (FTIR) spectroscopy, and differential thermal analysis (DTA). The XRD results confirmed the amorphous nature for all stabilized sol–gel derived glasses. The EDX result shows the relatively low loss of phosphorus during the sol–gel process and Ti K-edge XANES confirmed titanium in the glass structure is in mainly six-fold coordination environment. The 31P NMR and FTIR results revealed that the glass structure consist of mainly Q1 and Q2 phosphate units and the Ti4+ cation was acting as a cross-linking between phosphate units. In addition DTA results confirmed a decrease in the glass transition and crystallisation temperature with increasing Na2O content. Ion release studies also demonstrated a decrease in degradation rates with increasing TiO2 content therefore supporting the use of these glasses for biomedical applications that require a degree of control over glass degradation. These sol–gel glasses also offer the potential to incorporate proactive molecules for drug delivery application due to the low synthesis temperature employed.

Preformulation studies on grewia gum as a formulation excipient

Grewia gum is a naturally occurring polysaccharide which has potential as a pharmaceutical excipient. Differential scanning calorimetry and Fourier transform infrared (FT-IR) spectroscopy techniques were used to examine the thermal and molecular behaviours, respectively, of mixtures of grewia gum with cimetidine, ibuprofen or standard excipients, to assess potential interactions. No disappearance or broadening of the melting endotherm was seen with cimetidine or ibuprofen. Similarly, there was no interaction between grewia gum and the standard excipients tested. The results obtained using thermal analyses were supported by FT-IR analysis of the material mixtures. Grewia gum is an inert natural polymer which can be used alone or in combination with other excipients in the formulation of pharmaceutical dosage forms. © 2011 Akadémiai Kiadó, Budapest, Hungary.