Study of the physical properties of kafirin during the fabrication of tablets for pharmaceutical applications

Kafirin and protein bodies were extracted from a condensed tannin-free white Sudanese cultivar of sorghum (Dabar). The extracted materials were characterized by SDS-PAGE. The potential of kafirin as a tablet matrix for pharmaceutical applications was studied. Tablets composed of kafirin, calcium hydrogen orthophosphate, caffeine and magnesium stearate were prepared by direct compression. The tablets showed appropriate levels of hardness and friability. Drug release studies showed that caffeine dissolution was greater in 0.1 M HCl than in either phosphate buffer (pH = 6.8) or distilled water. Deamidation of the protein in acid conditions might explain this observation.FTIR analysis showed that the secondary structure of kafirin was found to be mainly governed by α helices with some β sheets. Upon tabletting, there was a change in protein conformation, which was recovered upon dissolution irrespective of the dissolution media. This might be explained by the loss of protein coil to coil interaction during tabletting (possibly due to the diluting effect of calcium hydrogen orthophosphate). This was later recovered when tablets were dissolved due to the hydrophobic interactions between the kafirin proteins.In summary, this work has shown that kafirin has a potential for use as a tablet for drug delivery.     

Increased Osteogenic Potential of Pre-Osteoblasts on Three-Dimensional Printed Scaffolds Compared to Porous Scaffolds for Bone Regeneration

Background:One of the main challenges with conventional scaffold fabrication methods is the inability to control scaffold architecture. Recently, scaffolds with controlled shape and architecture have been fabricated using 3D-printing. Herein, we aimed to determine whether the much tighter control of microstructure of 3DP PLGA/β-TCP scaffolds is more effective in promoting osteogenesis than porous scaffolds produced by solvent casting/porogen leaching. Methods:Physical and mechanical properties of porous and 3DP scaffolds were studied. The response of pre-osteoblasts to the scaffolds was analyzed after 14 days. Results:The 3DP scaffolds had a smoother surface (R_a: 22 ± 3 µm) relative to the highly rough surface of porous scaffolds (R_a: 110 ± 15 µm). Water contact angle was 112 ± 4° on porous and 76 ± 6° on 3DP scaffolds. Porous and 3DP scaffolds had the pore size of 408 ± 90 and 315 ± 17 µm and porosity of 85 ± 5% and 39 ± 7%, respectively. Compressive strength of 3DP scaffolds (4.0 ± 0.3 MPa) was higher than porous scaffolds (1.7 ± 0.2 MPa). Collagenous matrix deposition was similar on both scaffolds. Cells proliferated from day 1 to day 14 by fourfold in porous and by 3.8-fold in 3DP scaffolds. ALP activity was 21-fold higher in 3DP scaffolds than porous scaffolds. Conclusion:The 3DP scaffolds show enhanced mechanical properties and ALP activity compared to porous scaffolds in vitro, suggesting that 3DP PLGA/β-TCP scaffolds are possibly more favorable for bone formation. Key Words: Alkaline phosphatase, β-tricalcium phosphate, Poly(lactic-co-glycolic) acid copolymer     

Release of doxycycline through cellulose acetate symmetric and asymmetric membranes produced from recycled agroindustrial residue: Sugarcane bagasse

Cellulose acetate is one of the components employed in drug controlled-release systems in the form of membranes. The aim of this study was to examine the controlled-release of doxycycline employing cellulose acetate symmetric and asymmetric membranes as matrices. The cellulose triacetate was produced from sugarcane bagasse through a homogeneous acetylation reaction, using acetic acid as the solvent, acetic anhydride as the acetylating agent and sulfuric acid as the catalyst. The viscosity average molecular weight of the cellulose acetate produced was 39,000 g mol⁻¹. The symmetric membranes were produced using a system solvent of dichloromethane/ethanol (9:1, v/v) and the asymmetric membranes were produced from the same solvent system and 10% of water. For the formulation of both, 5% of doxycycline was used. The membranes were characterized by thermal analysis (DSC and TGA) and scanning electron microscopy SEM. The release of doxycycline through cellulose triacetate matrices was examined using spectrophotometric analysis in the ultraviolet-visible region, at 275 nm. The results revealed that asymmetric membranes release 80% of the drug in 100 min, while symmetric membranes release 14% of the drug during the same time interval.     

Preferential binding of sorghum tannins with γ-kafirin and the influence of tannin binding on kafirin digestibility and biodegradation

Kafirins, sorghum prolamins bind with sorghum condensed tannins (CTs). The binding of different kafirin species with sorghum CTs was investigated. Analysis by chemical assay and by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), reversed-phase high-performance liquid chromatography (RP-HPLC), and free zone capillary electrophoresis (FZCE), showed that γ-kafirin bound more CTs than the other kafirin species. SDS–PAGE suggested that the γ-kafirin-bound tannins were in the form of aggregates of molecular size >200k. RP-HPLC and FZCE revealed that sample preparation and drying the kafirins prior to the binding assays had a significant impact on γ-kafirin solubility. The effect of tannin binding on kafirin and kafirin film digestibility and film biodegradation was determined. Kafirins bound to tannins had lower digestibilities than unbound kafirins. Films made from tannin-bound kafirin had much lower digestibility and were less biodegradable than films made from unbound kafirin. The increase in kafirin film life by tannin modification appears to be due to a decrease in protein digestibility caused by kafirin–tannin binding. These findings suggest that γ-kafirin content in sorghum may be manipulated to either reduce or increase tannin binding in order to change the functionality of the kafirin in food, feed or film applications.     

Transgenic sorghum with suppressed synthesis of kafirin subclasses: Effects on flour and dough rheological characteristics

Arising from work showing that conventionally bred high protein digestibility sorghum types have improved flour and dough functionality, the flour and dough properties of transgenic biofortified sorghum lines with increased protein digestibility and high lysine content (TG-HD) resulting from suppressed synthesis of several kafirin subclasses, especially the cysteine-rich γ-kafirin, were studied. TG-HD sorghums had higher flour water solubility at 30 °C (p < 0.05) and much higher paste viscosity (41% higher) than their null controls (NC). TG-HD doughs were twice as strong as their NC and dynamic rheological analysis indicated that the TG doughs were somewhat more elastic up to 90 °C. CLSM of doughs and pastes indicated that TG-HD had a less compact endosperm protein matrix surround the starch compared to their NC. The improved flour and dough functional properties of the TG-HD sorghums seem to be caused by reduced endosperm compactness resulting from suppression of synthesis of several kafirin subclasses which modifies protein body and protein matrix structure, and to improved protein-starch interaction through hydrogen bonding specifically caused by reduction in the level of the hydrophobic γ-kafirin. The improved flour functionality of these transgenic biofortified sorghums can increase their commercial utility by complementing their improved nutritional quality.     

Rheological properties of kafirin and zein prolamins

The possibility of forming dough from kafirin was investigated and laboratory prepared kafirin was formed into a viscoelastic dough system. Measurements with Contraction Flow showed that dough systems prepared from kafirin and from commercial zein had the required extensional rheological properties for baking of leavened bread. The extensional viscosity and strain hardening of the kafirin and zein dough systems were similar to those of gluten and wheat flour doughs. The kafirin dough system, however, unlike the zein dough system rapidly became very stiff. The stiffening behaviour of the kafirin dough system was presumed to be caused by cross-linking of kafirin monomers. SDS-PAGE showed that the kafirin essentially only contained α- and γ-kafirin, whereas the zein essentially only contained α-zein. Since γ-kafirin contains more cysteine residues than the α-prolamin it is more likely to form disulphide cross-links, which probably caused the differences in stiffening behaviour between kafirin and zein dough systems. Overall the kafirin dough system displayed rheological properties sufficient for baking of porous bread. Kafirin like zein appears to have promising properties for making non-gluten leavened doughs.     

Spray Drying of Chitosan Acid Salts: Process Development,Scaling Up and Physicochemical Material Characterization

We investigated a spray drying process for preparing water-soluble salts of high molecular weight chitosan (CH) intended for pharmaceutical excipient applications. CH was derived from chitin of marine lobster origin (Panulirus argus). The effects of organic acid (acetic or lactic acid) and the ratio (difference) of inlet/outlet air temperature (140/90 °C or 160/100 °C) on spray drying were studied. The yield of spray-dried CH salt powders ranged from 50% to 99% in laboratory and industrial-scale processes. The spray-dried dry powder of CH salts consisted of spherical agglomerated particles with an average diameter of 36.2 ± 7.0 µm (CH acetate) and 108.6 ± 11.5 µm (CH lactate). After dispersing the spray-dried CH salt powder samples in purified water, the mean particle sizes obtained for the CH acetate salts were 31.4 nm (batch A001), 33.0 nm (A002) and 44.2 nm (A003), and for the CH lactate salts 100.8 nm (batch L001), 103.2 nm (L002) and 121.8 nm (L003). The optimum process conditions for spray drying were found: an inlet air temperature of 160 ± 5 °C, an outlet temperature of 100 ± 5 °C and an atomizer disk rotational speed of 18,200 min⁻¹. The X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) results confirmed the amorphous state of the CH salts. The ¹H nuclear magnetic resonance (NMR) and Fourier transform infrared (FT-IR) spectra of CH acetate and lactate salts verified that the spray drying process does not affect the polymer backbone. In conclusion, both laboratory and industrial-scale spray drying methods for preparing water-soluble acid salts of CH are reproducible, and the physicochemical properties of the corresponding CH acid salts are uniform.     

Mucoadhesive Microparticles for Gastroretentive Delivery: Preparation,Biodistribution and Targeting Evaluation

The aim of this research was to prepare and characterize alginate-chitosan mucoadhesive microparticles containing puerarin. The microparticles were prepared by an emulsification-internal gelatin method using a combination of chitosan and Ca²⁺ as cationic components and alginate as anions. Surface morphology, particle size, drug loading, encapsulation efficiency and swelling ratio, in vitro drug released, in vitro evaluation of mucoadhesiveness and Fluorescence imaging of the gastrointestinal tract were determined. After optimization of the formulation, the encapsulation efficiency was dramatically increased from 70.3% to 99.2%, and a highly swelling ratio was achieved with a change in particle size from 50.3 ± 11.2 μm to 124.7 ± 25.6 μm. In ethanol induced gastric ulcers, administration of puerarin mucoadhesive microparticles at doses of 150 mg/kg, 300 mg/kg, 450 mg/kg and 600 mg/kg body weight prior to ethanol ingestion significantly protected the stomach ulceration. Consequently, significant changes were observed in inflammatory cytokines, such as prostaglandin E₂ (PGE₂), tumor necrosis factor (TNF-α), interleukin 6 (IL-6), and interleukin1β (IL-1β), in stomach tissues compared with the ethanol control group. In conclusion, core-shell type pH-sensitive mucoadhesive microparticles loaded with puerarin could enhance puerarin bioavailability and have the potential to alleviate ethanol-mediated gastric ulcers.     

Porous Laponite/Poly(L-lactic acid) Membrane with Controlled Release of TCH and Efficient Antibacterial Performance

Fabrication of porous polymer membrane with controlled drug release and efficient antibacterial performances is of great interest in biomedical fields. In this study, Laponite (LAP) nanodisks were first used to encapsulate a model antibiotic drug, tetracycline hydrochloride (TCH). Then, drug-loaded LAP nanodisks with an optimized loading efficiency (85.3 %) were mixed with poly(L-lactic acid) (PLLA) polymer to form drug-loaded composite porous membrane via solvent coasting. The structure, morphology and swelling property of the porous membranes formed with varied solvent ratio of methylene dichloride (DCM) and dimethyl formamide (DMF) in the mixture solvent were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy and swelling test. In vitro drug release behavior, the cytotoxicity and the antibacterial activity of drug-loaded composite membranes were evaluated. Results showed that the TCH release was dependent on the physical structure of PLLA membrane and the presence of LAP nanodisks effectively weakened the initial burst release of TCH, and improved the sustained release property of porous PLLA membrane. The released TCH of TCH/LAP/PLLA_3:1 and TCH/LAP/PLLA_4:1 was 10.0 % and 5.3 % within initial 1 h, respectively. More importantly, the porous TCH/LAP/PLLA membrane was cytocompatible and displayed considerable antibacterial activity, solely associated with the loaded TCH drug, confirming its potential utility in wound dressings and tissue engineering.     

Solvent crystallization of palm based dihydroxystearic acid with isopropyl alcohol: Effects of solvent quantity and concentration on particle size distribution, crystal habit and morphology, and resultant crystal purity

Crude dihydroxystearic acid was prepared from palm based oleic acid and was then solvent purified with isopropyl alcohol in a custom fabricated simultaneous batch crystallizer unit. The crystallized dihydroxystearic acid was a functional ingredient that acted as multipurpose intermediate for synthesis of various fine chemicals, cosmetics and personal care products. The effects of solvent quantity and concentration on particle size distribution, crystal habit and morphology, and resultant crystal purity were studied. The crystals were purer but smaller and the span of the distribution curve was wider at higher solvent quantity and concentration. Through scanning electron microscopy and X-ray diffraction, it was observed that the crystals agglomerated into plate-like (flaky) habit with triclinic crystal structure. Solvent crystallization with 80% IPA at 20 °C and solute:solvent ratio of 1.0:1.0 was the most optimized and efficient, producing β-DHSA crystals that has high resistance against fat exudation during vacuum filtration process.     

Type II Collagen and Gelatin from Silvertip Shark (Carcharhinus albimarginatus) Cartilage: Isolation,Purification, Physicochemical and Antioxidant Properties

Type II acid soluble collagen (CIIA), pepsin soluble collagen (CIIP) and type II gelatin (GII) were isolated from silvertip shark (Carcharhinus albimarginatus) cartilage and examined for their physicochemical and antioxidant properties. GII had a higher hydroxyproline content (173 mg/g) than the collagens and cartilage. CIIA, CIIP and GII were composed of two identical α₁ and β chains and were characterized as type II. Amino acid analysis of CIIA, CIIP and GII indicated imino acid contents of 150, 156 and 153 amino acid residues per 1000 residues, respectively. Differing Fourier transform infrared (FTIR) spectra of CIIA, CIIP and GII were observed, which suggested that the isolation process affected the secondary structure and molecular order of collagen, particularly the triple-helical structure. The denaturation temperature of GII (32.5 °C) was higher than that of CIIA and CIIP. The antioxidant activity against 1,1-diphenyl-2-picrylhydrazyl radicals and the reducing power of CIIP was greater than that of CIIA and GII. SEM microstructure of the collagens depicted a porous, fibrillary and multi-layered structure. Accordingly, the physicochemical and antioxidant properties of type II collagens (CIIA, CIIP) and GII isolated from shark cartilage were found to be suitable for biomedical applications.     

Structural changes in kafirin extracted from a white type II tannin sorghum during germination

Kafirin proteins were extracted from a Sudanese cultivar of sorghum (Feterita). The extracted materials were characterized by SDS PAGE. Under non-reducing conditions as germination was progressing from 0 to 72 h, there was visually a decrease in β-kafirins whereas a small effect was observed on α-kafirins. The occurrence of interactions between tannins and kafirin might explain this behavior when compared to other cultivars. Thermogravimetric analysis (TGA) revealed that the first change in weight between room temperature and 100 °C was due to the loss of water. The protein extract started to degrade thereafter with the decomposition rate increasing with germination time. Fourier Transform Infrared (FT-IR) analysis showed that the secondary structure of kafirin was found to be mainly governed by α-helices with some β-sheets. Upon germination, there was a change in protein conformation, mainly an increase in α-helices. Scanning Electron Microscopy (SEM) showed that after extraction, kafirin proteins formed aggregated particles. This study is the first to show in the case of Feterita cultivar that upon germination, the physical properties of the kafirin proteins were significantly affected and relied on composition. This could shed some light on the effect of processing such as germination on sorghum kafirin proteins originated from Feterita.     

Effect of suppressing the synthesis of different kafirin sub-classes on grain endosperm texture, protein body structure and protein nutritional quality in improved sorghum lines

To improve sorghum grain protein nutritional quality, improved sorghum lines were transformed to suppress the synthesis of different kafirin sub-classes, or backcrossed into transgenic lines with improved protein quality. Co-suppression of the alpha-, gamma- and delta-kafirin sub-classes and removal of the tannin trait resulted in transgenic sorghum lines with high cooked protein digestibility (±80%), improved Amino Acid Score (0.8) and Protein Digestibility Corrected Amino Acid Score (0.7) compared to the non-transgenic null controls (±50%, 0.4 and 0.2, respectively). These high protein quality lines had a floury endosperm. They also had modified protein body structure, where the protein bodies were irregular shaped with few to numerous invaginations and were less densely packed, with a dense protein matrix visible around the protein bodies. When fewer sub-classes were suppressed, i.e. gamma 1 and delta 2, the endosperm was corneous with normal protein body structure but the improvement in cooked protein digestibility appeared to be less. Apparently, co-suppression of several kafirin sub-classes is required to obtain high protein nutritional quality sorghum lines, but this seems to result in floury-type grain endosperm texture.     

Protein bodies ontogeny and localization of prolamin components in the developing endosperm of wheat caryopses

During caryopsis development, prolamins are initially stored in individual protein bodies, then generate a protein matrix in the ripe caryopsis. The ontogeny of the protein bodies was analyzed by fluorescence and electron microscopy from 7 to 43 days after anthesis (dAA), a period of time from the cellularization of endosperm to its desiccation. A series of antibodies specific to each prolamin type (α/β-, γ-, ω-gliadins, low-molecular weight and high-molecular weight glutenin subunits) made it possible to localize and co-localize the different prolamins in organelles of endosperm cells at different developmental stages. Protein bodies containing prolamins were observed as early as 7 dAA. At the early developmental stages, protein bodies were spherical with diameters around 1–2 μm. Later, around 15 dAA, the PBs enlarged, and aggregation and/or coalescence were prominent at 21 dAA. From 33 dAA, individual PBs were no longer visible, but a protein matrix was confined in the space between starch granules. All prolamins were found in the same protein bodies, without any segregation according to their types. Immunochemical labelling of prolamins failed to reveal in TEM analyses any particular internal organization in protein bodies. Glutenin subunits and gliadins were observed in the Golgi apparatus at the early stages of endosperm development.     

Compositional variation amongst sorghum hybrids: Effect of kafirin concentration on metabolizable energy

Kafirins are stored proteins that negatively affect the nutritional quality of sorghum grain. Kafirin concentration and other chemical characteristics were determined in 12 sorghum hybrids and varied significantly, from 58% (HB1) to 42% (HB12) as percent total protein. Kafirin concentration correlated negatively with crude protein (CP) (−0.49), with acid detergent fiber (−0.40), apparent metabolizable energy (−0.61), and true metabolizable energy corrected for N (−0.63). HB12 was the hybrid with the lowest content of kafirins, amylose and tannins, and the highest content of apparent metabolizable energy. No differences were observed in the concentration of starch, but differences were found in apparent metabolizable energy (3325–2944 kcal kg⁻¹) probably due to a greater availability of starch, related to differences in kafirin concentration.     

Evaluation of Ultrasound,Microwave, Ultrasound–Microwave,Hydrothermal and High Pressure Assisted Extraction Technologies for the Recovery of Phytochemicals and Antioxidants from Brown Macroalgae

This study aims to explore novel extraction technologies (ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), ultrasound–microwave-assisted extraction (UMAE), hydrothermal-assisted extraction (HAE) and high-pressure-assisted extraction (HPAE)) and extraction time post-treatment (0 and 24 h) for the recovery of phytochemicals and associated antioxidant properties from Fucus vesiculosus and Pelvetia canaliculata. When using fixed extraction conditions (solvent: 50% ethanol; extraction time: 10 min; algae/solvent ratio: 1/10) for all the novel technologies, UAE generated extracts with the highest phytochemical contents from both macroalgae. The highest yields of compounds extracted from F. vesiculosus using UAE were: total phenolic content (445.0 ± 4.6 mg gallic acid equivalents/g), total phlorotannin content (362.9 ± 3.7 mg phloroglucinol equivalents/g), total flavonoid content (286.3 ± 7.8 mg quercetin equivalents/g) and total tannin content (189.1 ± 4.4 mg catechin equivalents/g). In the case of the antioxidant activities, the highest DPPH activities were achieved by UAE and UMAE from both macroalgae, while no clear pattern was recorded in the case of FRAP activities. The highest DPPH scavenging activities (112.5 ± 0.7 mg trolox equivalents/g) and FRAP activities (284.8 ± 2.2 mg trolox equivalents/g) were achieved from F. vesiculosus. Following the extraction treatment, an additional storage post-extraction (24 h) did not improve the yields of phytochemicals or antioxidant properties of the extracts.     

Identification of Nanopillars on the Cuticle of the Aquatic Larvae of the Drone Fly (Diptera: Syrphidae)

Here, we describe a nano-scale surface structure on the rat-tailed maggot, the aquatic larva of the Drone fly Eristalis tenax (L.). Larvae of this syrphid hover fly live in stagnant, anaerobic water-courses that are rich in organic matter. The larvae burrow into fetid slurry and feed on microorganisms which they filter out from the organic material. This environment is rich in bacteria, fungi and algae with the capacity to form biofilms that might develop on the larval surface and harm them. Using transmission and scanning electron microscopy we have identified an array of slender (typically < 100 nm in diameter) nanopillars that cover the surface of the larvae. The high density and dimensions of these spine-like projections appear to make it difficult for bacteria to colonize the surface of the animal. This may interfere with the formation of biofilms and potentially act as a defence against bacterial infection.     

Highly hydrophobic nanofibrous surfaces genearated by poly(vinylidene fluoride)

Lotus-leaf-like nanofibrous surfaces were prepared by electrospinning hydrophobic poly(vinylidene fluoride) (PVDF) from a mixed solvent of N,N-dimethylformamide (DMF) and acetone. PVDF fibrous mats with a bead-on-string morphology were generated because the nonpolar acetone decresed the viscosity of the PVDF solution and promoted the evaporation of the solution during electrospinning process. The morphology of the nanofibirous surface was examined by scanning electron microscopy. Micron-sized beads were introduced to the electrospun PVDF mats, resulting in enhanced hydrophobicity of the electrospun mats. The addition of a small amount (0.05 vol%) of acetic acid to the polymer solution effectively improved the bead-on-string morphology of the electrospun mats, and led to a higher water contact angle (WCA). The electrospun PVDF fibrous mat showed a maximum WCA of 148.5° due to the appropriate surface roughness.     

Preparation and Characterization of Chitosan Poly(acrylic acid) Magnetic Microspheres

Spherical microparticles, capable of responding to magnetic fields, were prepared by encapsulating dextran-coated Fe₃O₄ nanoparticles into chitosan poly(acrylic acid) (PAA) microspheres template. The obtained magnetic microspheres were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), X-ray powder diffraction (XRD), and thermogravimetry (TG). The results showed that the microspheres were formed and demonstrated magnetic behavior in an applied magnetic field. In addition, magnetite particles were well encapsulated and the composite particles have high magnetite content, which was more than 40%.