Amphiphilic and thermosensitive copolymers based on pullulan and Jeffamine: Synthesis, characterization and physicochemical properties

The synthesis of thermosensitive copolymers based on pullulan and polyether amine was performed in water using a water-soluble carbodiimide and N-hydroxysuccinimide as activators. Jeffamine^® M2005 was chosen as a polyether to impart thermosensitive character to the copolymer. Pullulan was modified into carboxymethylpullulan, to bring carboxylate groups to the polysaccharide so as to further the grafting reaction. The copolymers were characterized by FT-IR, ¹H NMR spectroscopy and molecular weights measurements (by SEC coupled with MALS/DRI/Viscometer lines). The thermosensitive behaviour of CMP-g-M2005 copolymers was studied by fluorescence spectroscopy of pyrene, by rheometry and microDSC measurements. The sol-gel transition temperature was found dependent on the solvent, the grafting degree of M2005 and the concentration of the copolymer. For example it was 35 °C in water, 28 °C in acid buffer (0.1 M, pH 5.4) and 26 °C in saline phosphate buffer (0.15 M, pH 7.4) for a grafting degree of 0.20 at a concentration of 5 wt%.     

Free radical grafting kinetics of acrylamide onto a blend of starch/chitosan/alginate

Grafting of monomer onto polymer backbone is one of the effective and accessible methods for the chemical modification of polysaccharides. Grafting of acrylamide (AAm) onto polysaccharides blend (PsB) composed of starch, chitosan and alginate has been carried out using potassium persulfate (KPS) as an initiator. The kinetics of the grafting polymerization also has been studied. The grafting parameters have been evaluated by changing the initial concentrations of AAm from 8 to 16 g, PsB from 6 to 14 g and KPS from 0.2 to 1 g. Evidence of grafting has been obtained from FTIR, XRD and TGA. The kinetics of the grafting polymerization also has been studied. The grafting rate equation of the produced hydrogel (PsB-g-AAm) hydrogel has been expressed by: Rg = k[AAm] [PsB]^0.5 [KPS]^0.5. The grafting rate is a first order dependence to [AAm] initial concentration and square root to [PsB] and [KPS] initial concentrations in the used concentrations range.     

Preparation and characterization of chitosan-polyvinyl alcohol blend hydrogels for the controlled release of nano-insulin

Chitosan (CS)-polyvinyl alcohol (PVA) blend hydrogels were prepared using glutaraldehyde as the cross-linking agent. The obtained hydrogels, which have the advantages of both PVA and CS, can be used as a material for the transdermal drug delivery (TDD) of insulin. The nano-insulin-loaded hydrogels were prepared under the following conditions: 1.2 g of polyethylene glycol, 1.5 g of CS, 1.2 g of PVA, 1.2 mL of 1% glutaraldehyde solution, 16 mL of water, and 40 mg of nano-insulin with 12 min of mixing time and 3 min of cross-linking time. The nano-insulin-loaded hydrogels were characterized using scanning electron microscopy, energy dispersive spectrometry, Fourier-transform infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis, X-ray diffraction, and its mechanical properties were analyzed. The results show that all molecules in the hydrogel have good compatibility and they formed a honeycomb-like structure. The hydrogel also showed good mechanical and thermal properties. The in vitro drug release of the hydrogel showed that the nano-insulin accorded with Fick's first law of diffusion and it has a high permeation rate (4.421 μg/(cm² h)). These results suggest that the nano-insulin-loaded hydrogels are a promising non-invasive TDD system for diabetes chemotherapy.     

Functionalization of cellulosic fibers by graft copolymerization of acrylonitrile and ethyl acrylate from their binary mixtures

Functionalization of Agave fibers was carried out by graft copolymerization of acrylonitrile (AN) and ethyl acrylate (EA) from their binary solutions in presence of Ce (IV) ions at a temperature of 45 ± 0.1 °C. An increase in the graft copolymerization was obtained with the increase in the feed molarity of the comonomers up to certain extent. Contrary to lesser affinity of acrylonitrile to grafting on Agave fibers, a synergistic effect of ethyl acrylate on acrylonitrile was observed when graft copolymers were prepared using different feed compositions (f_AN). The graft copolymers were characterized by various techniques such as FT-IR, TGA/DTA, X-RD and SEM analysis. Further swelling behavior of grafted fibers in different solvents, moisture absorption behavior and resistance to chemicals was investigated as a function of percent grafting to define their end uses in different environments.     

Horseradish peroxidase covalent grafting onto screen-printed carbon electrodes for levetiracetam chronoamperometric determination

A new enzymatic electrochemical biosensor based on disposable transducers, namely screen-printed carbon electrodes, has been developed for the determination of the antiepileptic drug levetiracetam. Horseradish peroxidase was immobilized onto the carbon working electrode previously modified by an aryl diazonium salt. The formation of amide bonds between the amino and carboxylic groups of the enzyme surface, catalyzed by hydroxysuccinimide and carbodiimide, leads to the electrode functionalization. This orientated enzymatic modification results in high reproducibility, with an associated relative standard deviation of 6.21% for the slopes of several calibration curves in the calibration range from 0.10 to 0.83 mM. Experimental variables that can affect levetiracetam chronoamperometric response, such as hydrogen peroxide concentration, pH, and applied potential, were optimized to perform a selective determination. An average limit of detection of 1.75 × 10⁻⁵ M (α = β = 0.05) was obtained. The biosensors were finally applied to the determination of levetiracetam in complex matrices such as pharmaceutical drugs, yielding successful results.     

Graft copolymerization of ethylacrylate onto xanthan gum, using potassium peroxydisulfate as an initiator

Graft copolymer of xanthan gum (XG) and ethylacrylate (EA) has been synthesized by free radical polymerization using potassium peroxydisulfate (KPS) as an initiator in an air atmosphere. The grafting parameters, i.e. grafting ratio and efficiency decrease with increase in concentration of xanthan gum from 0.050 mg/25 mL to 0.350 mg/25 mL, but these grafting parameters increase with increase in concentration of ethylacrylate from 9 × 10⁻² to 17 × 10⁻² ML⁻¹, and KPS from 15 × 10⁻³ to 35 × 10⁻³ ML⁻¹. The graft copolymer has been characterized by FTIR, XRD, TGA and SEM analysis. The grafted copolymer was also evaluated as efficient Zn²⁺ metal binder. The grafted copolymer shows improvement in the stability, solubility as well as their sorbing capacity. Thus graft copolymer formed could find applications in metal ion removal and in drug delivery.     

Purification and partial biochemical characterization of polyphenol oxidase from mamey (Pouteria sapota)

While a long shelf life for fruit products is highly desired, enzymatic browning is the main cause of quality loss in fruits and is therefore a main problem for the food industry. In this study polyphenol oxidase (PPO), the main enzyme responsible for browning was isolated from mamey fruit (Pouteria sapota) and characterized biochemically. Two isoenzymes (PPO 1 and PPO 2) were obtained upon ammonium sulfate precipitation and hydrophobic and ion exchange chromatography; PPO 1 was purified up to 6.6-fold with 0.28% yield, while PPO 2 could not be characterized as enzyme activity was completely lost after 24 h of storage. PPO 1 molecular weight was estimated to be 16.1 and 18 kDa by gel filtration and SDS-PAGE, respectively, indicating that the native state of the PPO 1 is a monomer. The optimum pH for PPO 1 activity was 7. The PPO 1 was determined to be maximum thermally stable up to 35 °C. Kinetic constants for PPO 1 were K_m = 44 mM and K_m = 1.3 mM using catechol and pyrogallol as substrate, respectively. The best substrates for PPO 1 were pyrogallol, 4-methylcatechol and catechol, while ascorbic acid and sodium metabisulfite were the most effective inhibitors.     

Alginate beads as a tool to handle,cryopreserve and culture isolated human primordial/primary follicles

Background

One major concern of grafting cryopreserved ovarian tissue to restore fertility in cancer patients is the possibility of reintroducing tumor cells. Cryopreservation of isolated primordial/primary follicles (PFs) may circumvent this problem. The aim of our work was to compare dimethyl sulfoxide (ME₂SO) and ethylene glycol (EG) as cryoprotectants (CPAs) for slow-freezing of isolated human PFs in alginate.

Methods

Ovarian biopsies from four women were processed for follicle isolation. PFs were embedded in alginate (5–15 per group). Follicles were frozen-thawed using 1.4 M ME₂SO or 1.5 M EG as CPAs. Fresh and cryopreserved isolated follicles were in vitro cultured (IVC) for 7 days. At different time periods (after isolation, cryopreservation and IVC), follicles were evaluated with live/dead assay (using fluorescent probes) and diameter measurement. Follicle viability was calculated according to the percentage of dead follicular cells and the presence of a live/dead oocyte.

Results

A total of 841 PFs were isolated, embedded in alginate and cryopreserved with ME₂SO (n = 424) or EG (n = 259), or used as controls (n = 158). After 7 days of IVC, a significant increase in follicle size was observed in the fresh and ME₂SO groups, but not in the EG group. The percentage of totally viable PFs was not significantly different before or after seven days of culture in fresh (100% and 82%) or ME₂SO (93.2% and 85.1%) tissue. The EG group showed significantly lower viability before (63.9%) and after IVC (66.2%) than the fresh and ME₂SO groups.

Conclusions

Our results show that 1.4 M ME₂SO yields better preservation of isolated PF viability after thawing and 7 days of IVC than 1.5 M EG. Alginate constitutes an easy, safe hydrogel matrix to handle and cryopreserve isolated human follicles using ME₂SO as a CPA.     

A comparative study on the graft copolymerization of acrylic acid onto rayon fibre by a ceric ion redox system and a γ-radiation method

Functionalization of rayon fibre has been carried out by grafting acrylic acid (AAC) both by a chemical method using a Ce⁴⁺-HNO₃ redox initiator and by a mutual irradiation (γ-rays) method. The reaction conditions affecting the grafting percentage have been optimized for both methods, and the results are compared. The maximum percentage of grafting (50%) by the chemical method was obtained utilizing 18.24 × 10⁻³ moles/L of ceric ammonium nitrate (CAN), 39.68 × 10⁻² moles/L of HNO₃, and 104.08 × 10⁻² moles/L of AAc in 20 mL of water at 45 °C for120 min. For the radiation method, the maximum grafting percentage (60%) was higher, and the product was obtained under milder reaction conditions using a lower concentration of AAc (69.38 × 10⁻² moles/L) in 10 mL of water at an optimum total dose of 0.932 kGy. Swelling studies showed higher swelling for the grafted rayon fibre in water (854.54%) as compared to the pristine fibre (407%), while dye uptake studies revealed poor uptake of the dye (crystal violet) by the grafted fibre in comparison with the pristine fibre. The graft copolymers were characterized by IR, TGA, and scanning electron micrographic methods. Grafted fibre, prepared by the radiation-induced method, showed better thermal behaviour. Comparison of the two methods revealed that the radiation method of grafting of acrylic acid onto rayon fibre is a better method of grafting in comparison with the chemical method.     

Biochemical characterization of L-DOPA 2,3-dioxygenase, a single-domain type I extradiol dioxygenase from lincomycin biosynthesis

l-DOPA-2,3-dioxygenase from Streptomyces lincolnensis is a single-domain type I extradiol dioxygenase of the vicinal oxygen chelate superfamily and catalyzes the second step in the metabolism of tyrosine to the propylhygric acid moiety of the antibiotic, lincomycin. S. lincolnensisl-DOPA-2,3-dioxygenase was overexpressed, purified and reconstituted with Fe(II). The activity of l-DOPA-2,3-dioxygenase was kinetically characterized with l-DOPA (K_M = 38 μM, k_cat = 4.2 min⁻¹) and additional catecholic substrates including dopamine, 3,4-dihydroxyhydrocinnamic acid, catechol and d-DOPA. 3,4-Dihydroxyphenylacetic acid was characterized as a competitive inhibitor of the enzyme (K_i = 2.2 mM). Site-directed mutagenesis and its effects on enzymatic activity were used to identify His14 and His70 as iron ligands.     

Biochemical characterization of pea ornithine-δ-aminotransferase: Substrate specificity and inhibition by di- and polyamines

Ornithine-δ-aminotransferase (OAT, EC 2.6.1.13) catalyzes the transamination of l-ornithine to l-glutamate-γ-semialdehyde. The physiological role of OAT in plants is not yet well understood. It is probably related to arginine catabolism resulting in glutamate but the enzyme has also been associated with stress-induced proline biosynthesis. We investigated the enzyme from pea (PsOAT) to assess whether diamines and polyamines may serve as substrates or they show inhibitory properties. First, a cDNA coding for PsOAT was cloned and expressed in Escherichia coli to obtain a recombinant protein with a C-terminal 6xHis tag. Recombinant PsOAT was purified under native conditions by immobilized metal affinity chromatography and its molecular and kinetic properties were characterized. Protein identity was confirmed by peptide mass fingerprinting after proteolytic digestion. The purified PsOAT existed as a monomer of 50 kDa and showed typical spectral properties of enzymes containing pyridoxal-5′-phosphate as a prosthetic group. The cofactor content of PsOAT was estimated to be 0.9 mol per mol of the monomer by a spectrophotometric analysis with phenylhydrazine. l-Ornithine was the best substrate (K_m = 15 mM) but PsOAT also slowly converted N_α-acetyl-l-ornithine. In these reactions, 2-oxoglutarate was the exclusive amino group acceptor (K_m = 2 mM). The enzyme had a basic optimal pH of 8.8 and displayed relatively high temperature optimum. Diamines and polyamines were not accepted as substrates. On the other hand, putrescine, spermidine and others represented weak non-competitive inhibitors. A model of the molecular structure of PsOAT was obtained using the crystal structure of human OAT as a template.     

Characterization of Ca and phosphocholine interactions with C-reactive protein using a surface plasmon resonance biosensor

The interactions between Ca²⁺ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca²⁺ or phosphocholine were injected. Binding of Ca²⁺ induced a 10-fold higher signal than expected from the molecular weight of Ca²⁺. It was interpreted to result from the conformational change that occurs on binding of Ca²⁺. Two sites with different characteristics were distinguished: a high-affinity site with K_D = 0.03 mM and a low-affinity site with K_D = 5.45 mM. The pH dependencies of the two Ca²⁺ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had K_D = 5 μM at 10 mM Ca²⁺. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and suggest that Ca²⁺ is an efficient regulator under physiological conditions.     

Reversed-phase high-performance liquid chromatography method for simultaneous analysis of two liposome-formulated short interfering RNA duplexes

Gene silencing induced by short interfering RNA (siRNA) has proven to be useful in genomic research and has great potential for therapeutic applications; however, siRNAs are not readily bioavailable. Cationic liposomes offer effective protection of drug product from nucleases and enable distribution to desired target organs. The amount of siRNA in the formulation must be determined accurately. We have developed a stability-indicating, ion-pair, reversed-phase high-performance liquid chromatography method to separate and accurately quantitate two siRNA duplexes in a liposome without sample pretreatment. The gradient mobile phase system consisted of 385 mM hexafluoro-2-propanol, 14.5 mM triethylamine, and 5% methanol (mobile phase A) and 385 mM hexafluoro-2-propanol, 14.5 mM triethylamine, and 90% methanol (mobile phase B). The column used was an XBridge C18 column (50 × 2.1 mm i.d., 2.5 μm particle size), and separation was performed at 60 °C. Quantitation was achieved with ultraviolet (UV) detection at 260 nm. Linearity was established for the single strands of both siRNA duplexes for concentrations ranging from 10 to 110 μg/ml. Accuracy of the method was determined by replicate analysis (n = 5) at four concentrations (R² > 0.996 and relative standard deviations [RSDs] of 1-4%). The use of an ion-pairing reagent that is compatible with mass spectrometry detection makes this method amenable to liquid chromatography-mass spectrometry (LC-MS) impurity profiling.     

Preparation and characterization of a novel pH-sensitive chitosan-g-poly (acrylic acid)/attapulgite/sodium alginate composite hydrogel bead for controlled release of diclofenac sodium

A series of pH-sensitive composite hydrogel beads composed of chitosan-g-poly (acrylic acid)/attapulgite/sodium alginate (CTS-g-PAA/APT/SA) was prepared as drug delivery matrices crosslinked by Ca²⁺ owing to the ionic gelation of SA. The structure and surface morphology of the composite hydrogel beads were characterized by FTIR and SEM, respectively. pH-sensitivity of these composite hydrogels beads and the release behaviors of drug from them were investigated. The results showed that the composite hydrogel beads had good pH-sensitivity. The cumulative release ratios of diclofenac sodium (DS) from the composite hydrogel beads were 3.76% in pH 2.1 solution and 100% in pH 6.8 solutions within 24 h, respectively. However, the cumulative release ratio of DS in pH 7.4 solution reached 100% within 2 h. The DS cumulative release ratio reduced with increasing APT content from 0 to 50 wt%. The drug release was swelling-controlled at pH 6.8.     

Application of screen-printed microband biosensors to end-point measurements of glucose and cell numbers in HepG2 cell culture

Microband glucose biosensors were produced by insulating and sectioning through a screen-printed, water-based carbon electrode containing cobalt phthalocyanine redox mediator and glucose oxidase enzyme. Under quiescent conditions at 37 °C, at an operating potential of +0.4 V, they produced an amperometric response to glucose in buffer solutions with a sensitivity of 26.4 nA/mM and a linear range of 0.45 to 9.0 mM. An optimal pH value of 8.5 was obtained under these conditions, and a value for activation energy of 40.55 kJ mol⁻¹ was calculated. In culture medium (pH 7.3), a sensitivity of 13 nA/mM was obtained and the response was linear up to 5 mM with a detection limit of 0.5 mM. The working concentration was up to 20 mM glucose with a precision of 11.3% for replicate biosensors (n = 4). The microband biosensors were applied to determine end-point glucose concentrations in culture medium by monitoring steady-state current responses 400 s after transfer of the biosensors into different sample solutions. In conjunction with cultures of HepG2 (human Caucasian hepatocyte carcinoma) cells, current responses obtained in 24-h supernatants showed an inverse correlation (R² = 0.98) with cell number, indicating that the biosensors were applicable for monitoring glucose metabolism by cells and of quantifying cell number. Glucose concentrations determined using the biosensor assay were in good agreement, for concentrations up to 20 mM, with those determined spectrophotometrically (R² = 0.99). This method of end-point glucose determination was used to provide an estimated rate of glucose uptake for HepG2 cells of 7.9 nmol/(10⁶ cells min) based on a 24-h period in culture.     

Measurement of cortisol,cortisone, prednisolone,dexamethasone and 11-deoxycortisol with ultra high performance liquid chromatography–tandem mass spectrometry: Application for plasma,plasma ultrafiltrate,urine and saliva in a routine laboratory

We describe an ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC MS/MS) method suitable for a routine laboratory to determine endogenous and exogenous glucocorticoids in plasma, plasma ultrafiltrate, urine and saliva in a single analytical run. After addition of a multi-analyte internal standard, a standardised sample preparation procedure with solid phase extraction followed, before injecting into a tandem mass spectrometer with positive mode electron spray ionisation and multiple reactant monitoring acquisition. The chromatography time was 3 min. The limit of quantitation for cortisol and cortisone in plasma was 3.75 nmol/L and linearity extended to 2000 nmol/L. The limit of quantitation for cortisol in plasma ultrafiltrate and saliva was 0.6 nmol/L. The limit of quantitation for 11-deoxycortisol and prednisolone was 5 nmol/L and for dexamethasone 1 nmol/L. The intra-assay CV was <5% and the inter-assay CV <10% for all analytes in all matrices. Comparison with an immuno-assay (IA) plasma cortisol method resulted in a regression equation of UHPLC = 0.79 × IA + 31.12 with R² = 0.960 (p < 0.0001). Comparison with a high performance liquid chromatography (HPLC) cortisol method yielded a regression equation of UHPLC = 1.06 × HPLC + 9.82, R² = 0.992 (p < 0.0001). The simultaneous measurement of endogenous and exogenous glucocorticoids contributed to patient care in cases with dexamethasone and metyrapone dynamic tests and unsuspected therapeutic glucocorticoid use.     

Effect of post-thaw dilution with caffeine, pentoxifylline, 2’-deoxyadenosine and prostatic fluid on motility of frozen-thawed dog semen

The aim of the experiment was to evaluate the motility pattern of frozen-thawed canine semen to which pentoxifyilline (PTX), caffeine (CAF), 2’-deoxyadenosine (DX), and prostatic fluid (PROST) were added after thawing. Semen evaluations were performed using computer-assisted sperm analysis (CASA) at thawing and during 120 min of incubation at 37 °C. Three experiments were conducted: 1) to establish which concentrations of stimulants work best; 2) to investigate the interaction between thawing rate and addition of CAF 5 mM, PTX 2.5 mM and PROST; 3) to evaluate the effect of PTX 7.5 mM and DX 5 mM on semen motility after thawing. In experiment 1, ALH and VCL were enhanced at thawing by CAF 7.5 mM (CAF 7.5: 9.1 ± 0.5 μm; control: 6.7 ± 0.4 μm) and DX 5 and 7.5 mM (DX 5: 199.1 ± 12.8 μm/s; DX 7.5: 197.3 ± 13.9 μm/s; control: 162.5 ± 8.4 μm/s), while PTX 2.5-5-7.5 mM improved TOT after 120 min of incubation. In experiment 2, PROST lowered ALH values throughout incubation (P < 0.05) with respect to the other treatments, in particular when compared to CAF at Time = 30 and at Time = 60. In experiment 3, PTX 7.5 mM improved VAP (PTX: 101.6 ± 6.8 μm/s; control: 81.9 ± 10.5 μm/s), VSL (PTX: 82.9 ± 6.4 μm/s; control: 65.9 ± 9.8 μm/s), VCL (PTX: 214.3 ± 13.3 μm/s; control:167 ± 15.7 μm/s), ALH (PTX: 10.5 ± 0.3; control: 7.3 ± 1.4 μm), PM (PTX: 11.3 ± 4.2%; control: 7.7 ± 3.9%) and TOT (PTX: 20.1 ± 5.3%; control:15.6 ± 5.6%) at Time = 120, while DX 5 mM influenced VCL at Time = 60 (DX: 218.3 ± 14.3 μm/s; control: 188.5 ± 7.5 μm/s, P < 0.05). Motility stimulants may be useful for enhancing motility of canine frozen-thawed spermatozoa without affecting sperm longevity.     

Kinetics of the Ca, H, and Mg Interaction with the Ion-Binding Sites of the SR Ca-ATPase

Electrochromic styryl dyes were used to investigate mutually antagonistic effects of Ca²⁺ and H⁺ on binding of the other ion in the E₁ and P-E₂ states of the SR Ca-ATPase. On the cytoplasmic side of the protein in the absence of Mg²⁺ a strictly competitive binding sequence, H₂E₁?HE₁?E₁?CaE₁?Ca₂E₁, was found with two Ca²⁺ ions bound cooperatively. The apparent equilibrium dissociation constants were in the order of K_1/2(2 Ca) = 34 nM, K_1/2(H) = 1 nM and K_1/2(H₂) = 1.32 μM. Up to 2 Mg²⁺ ions were also able to enter the binding sites electrogenically and to compete with the transported substrate ions (K_1/2(Mg) = 165 μM, K_1/2(Mg₂) = 7.4 mM). In the P-E₂ state, with binding sites facing the lumen of the sarcoplasmatic reticulum, the measured concentration dependence of Ca²⁺ and H⁺ binding could be described satisfactorily only with a branched reaction scheme in which a mixed state, P-E₂CaH, exists. From numerical simulations, equilibrium dissociation constants could be determined for Ca²⁺ (0.4 mM and 25 mM) and H⁺ (2 μM and 10 μM). These simulations reproduced all observed antagonistic concentration dependences. The comparison of the dielectric ion binding in the E₁ and P-E₂ conformations indicates that the transition between both conformations is accompanied by a shift of their (dielectric) position.     

The release of active substances from selected carbohydrate biopolymer membranes

As a biopolymer application to control release systems is increasing at present, flat matrices (transdermal systems) should be highlighted. They constitute one of the most friendly form of drug administration to the patient. The present results concern investigations on the active substance release (ibuprofen and salicylic acid) from film matrices made from biopolymers: polylactid acid (PLA), dibutyrylchitin (DBC) and chitosan (CH). The amount of released active substance was examined with UV-VIS spectrophotometer. The release process was conducted in the medium of pH = 5.6 as the transdermal systems are applied to human skin surface of pH value approximating 5.6. Swelling of polymer samples was confirmed in the buffer of pH = 5.6 as well.The paper comprises the analysis of obtained release results according to the proposed two stage complex diffusion model.     

Endothelium-independent vasorelaxation effects of 16-O-acetyldihydroisosteviol on isolated rat thoracic aorta

Aims

The aim of this study is to investigate the vasorelaxant effect of 16-O-acetyldihydroisosteviol (ADIS) and its underlying mechanisms in isolated rat aorta.

Main methods

Rat aortic rings were isolated, suspended in organ baths containing Kreb's solution, maintained at 37 °C, and mounted on tungsten wire and continuously bubbled with a mixture of 95% O₂ and 5% CO₂ under a resting tension of 1 g. The vasorelaxant effects of ADIS were investigated by means of isometric tension recording experiment.

Key findings

ADIS (0.1 μM–3 mM) induced relaxation of aortic rings pre-contracted by phenylephrine (PE, 10 μM) and KCl (80 mM) with intact-endothelium (E_max = 79.26 ± 3.74 and 79.88 ± 3.79, respectively) or denuded-endothelium (E_max = 88.05 ± 3.69 and 78.22 ± 6.86, respectively). In depolarization Ca²⁺-free solution, ADIS inhibits calcium chloride (CaCl₂)-induced contraction in endothelium-denuded rings in a concentration-dependent manner. In addition, ADIS attenuates transient contractions in Ca²⁺-free medium containing EGTA (1 mM) induced by PE (10 μM) and caffeine (20 mM). By contrast, relaxation was not affected by tetraethylammonium (TEA, 5 mM), 4-aminopyridine (4-AP, 1 mM), glibenclamide (10 μM), barium chloride (BaCl₂, 1 mM), and 1H-[1,2,3]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, 1 μM).

Significance

These findings reveal the vasorelaxant effect of ADIS, through endothelium-independent pathway. It acts as a Ca^2 + channel blocker through both intracellular and extracellular Ca^2 + release.