Artesunate Dry Emulsion Formulation Combined with Antibiotics for Treatment of Helicobacter pylori Infections: In Vitro/In Vivo Evaluation.

Helicobacter pylori is the primary pathogen responsible for causing gastroduodenal ulcers and stomach cancer. The standard treatment for H. pylori typically involves a combination of antibiotics and acid-reducing medications. However, the recurrence of ulcers is closely linked to the emergence of antibiotic resistance in H. pylori, necessitating the development of alternative drugs. This report focuses on the investigation of artesunate as a potential alternative to reduce antibiotic use and enhance effectiveness against H. pylori. Unfortunately, commercial artesunate is available in an acid form, which has poor solubility, especially in gastric acid fluid. The aim of this study is to utilize a water-soluble formulation of artesunate called dry emulsion formulation (ADEF) and combine it with amoxicillin to eradicate H. pylori. In vitro studies were conducted to evaluate the activity of ADEF against H. pylori and determine its inhibitory concentrations. In addition, pharmacokinetic parameters of orally administered ADEF and native artesunate were investigated in rats for in vivo studies. The results showed that when combined with amoxicillin and pantoprazole, ADEF exhibited effectiveness against H. pylori. It is worth noting that the solubility of ADEF in gastric acid appears to be a critical factor for achieving successful treatment. Consequently, ADEF could be considered a promising candidate for H. pylori therapy.

Anthocyanins Formulated with Carboxymethyl Starch for Gastric and Intestinal Delivery.

Anthocyanins obtained from jambolan have been used as active agents in different carboxymethyl starch-based tablet formulations and their release profiles evaluated in simulated gastric fluids (SGF) and simulated intestinal (SIF) fluids. Structural analysis highlighted a strong interaction between anthocyanins and carboxymethyl starch, evidenced by scanning electron microscopy and infrared analysis. Tablet dissolution behavior varied according to the pH of the media, being controlled by the swelling and/or erosion of the polymeric matrix. Various formulations for immediate, fast, and sustained release of anthocyanins for 30 min, 2 h and 12 h of dissolution have been developed. It was found that monolithic carboxymethyl starch tablets loaded with powdered jambolan extract efficiently afforded the complete delivery (100% of anthocyanins) to different sites of the simulated gastrointestinal tract and ensured the stability of these pigments, which maintained their antioxidant activity.

Antiplasmodial Combined Formulation of Artemisinin with Peschiera fuchsiaefolia Bis-Indole Alkaloids.

Antimalarial agents used as monotherapy are increasingly ineffective due to the emergence of Plasmodium resistant strains. Artemisinin (Arte), extracted from Artemisia annua, presents a good efficiency against the Plasmodium strains and is currently used to treat malaria. To avoid the appearance of new resistant strains to artemisinin, the use of Artemisinin-based Combination Therapy (ACT) with another antimalaria agent was recommended by WHO to provide an effective cure and delayed resistance. Although combined formulations of various drugs with Artemisinin have been developed, their release is immediate, and they require multiple doses with side detrimental effects and effectiveness still desired. To improve its efficiency, controlled release formulations were developed to ensure long-term antiplasmodial activity by associating Artemisinin with a natural antimalarial agent extracted from Peschiera fuchsiaefolia (Pf). The Pf extract (containing mostly low soluble alkaloids) was complexed with carboxymethylcellulose to improve its solubility and stability. Two formulation types are reported. As bilayer tablet dosage form, the kinetic release pattern was an immediate release of Artemisinin, followed by a slow sustained release of Pf for 12 h. As monolithic tablet, the release profile shows a simultaneous sustained release of the two active agents, about of 10 h for Arte and 12 h for Pf.

Enhanced Solubility of Alkaloids by Complexation with Polycarboxylic Materials for Controlled Release Formulations: Case of Peschiera fuchsiaefolia.

Malaria is a major public health problem with hundreds of thousands of deaths yearly. Extracts of Peschiera fuchsiaefolia (Pf), an Apocynaceae family plant, are used as malaria treatment by several populations. Artemisinin is another effective largely used antimalarial agent but susceptible to generate resistant forms of Plasmodium. To reduce the risk of new resistant strains' appearance, the WHO recommended artemisinin-based combination therapy (ACT) with another bioactive agent, ensuring a long duration of antiplasmodial activity. Pf alkaloids are good candidates for ACT, but their solubility is very low. This research was aimed to improve the solubility of Pf alkaloids by complexation via their amine groups with carboxylate groups of carboxymethylstarch (CMS), an excipient used to formulate oral dosage forms for controlled drug release. It was found that when complexed as CMS-Pf, the solubility of Pf is increased (four to five times in function of dissolution medium). A new specific and faster approach to evaluate the solubility was proposed, measuring the effective saturation concentration of the compound of interest via one of its specific capacities, i.e., absorption capacity at a specific wavelength or antioxidant properties. This approach is more convenient for solubility evaluation of various active agents from complexes or crude extracts, or in heterogeneous samples. Also, the storage stability was markedly improved from 1 week for Pf co-processed with maltodextrin (MD/Pf) to several months for CMS-Pf (in similar controlled temperature and humidity conditions). The co-processing as MD/Pf or complexation as CMS-Pf affected physical properties but not the biological (i.e., antioxidant) activity of Pf.

Zymographic Determination of Intrinsic Specific Activity of Oxidases in the Presence of Interfering Proteins.

Zymography on peroxidase-entrapped gels enables the determination of the intrinsic specific activity (ISA) of H2O2-producing oxidases in the presence of interfering enzymes including catalase (decomposing hydrogen peroxide). To reveal the searched oxidase, the zymography gel is incubated first in the developing solution containing the appropriate substrate of the oxidase and an oxidizable chromogen such as o-phenylenediamine (o-PDA) peroxidase substrate. The zymographic gel, after image analysis, can be restained by Coomassie Blue for molecular weights, for the whole electrophoretic pattern and for the protein concentration, thus allowing for the determination of oxidase ISA.

Zymographic approach to determine the intrinsic enzyme specific activity of diamine oxidase in presence of interfering enzymes.

The purpose of this investigation was to elaborate a fast zymographic assay of oxidase enzymes in the presence of interfering enzymes as catalase (which disturbs current dosages based on H2O2 detection). This method also allows the determination of intrinsic specific activity (ISA) of oxidases, such as diamine oxidase (DAO) or glucose oxidase (GOD). The SDS-PAGE gels with entrapped peroxidase have been obtained by polymerization of acrylamide and bis-acrylamide in the presence of horse-radish peroxidase. The entrapped peroxidase was uniformly distributed in the PolyacrylAmide (PAA) material and did not migrate during electrophoresis. The obtained PAA gels allow the electrophoretic separation of various oxidases from contaminating proteins. As an example, to reveal DAO, the resulting PAA-gel should be incubated after the electrophoretic run in the developing solution containing putrescine (a DAO substrate) and o-phenylenediamine (a HRP substrate) to give coloured bands on the gel in the presence of DAO-generated H2O2. The results showed that is possible to determine the DAO in the presence of interfering catalase because they migrate differently. Thus, the H2O2 released in situ by DAO is no more decomposed by catalase because of its different mobility. It was also found that the same electrophoretic gel, after zymography, can be restained by Coomassie Blue for quantitation of proteins corresponding to the zymographic bands. With the obtained enzyme units and protein concentration it is also possible to calculate the intrinsic specific activity of DAO directly from the intensities of enzyme bands in zymography and from those of protein bands (Coomassie Blue staining), quantified by densitometry.

Two release rates from monolithic carboxymethyl starch tablets: formulation, characterization, and in vitro/in vivo evaluation.

Most of non-steroidal anti-inflammatory drugs (NSAIDs) including ibuprofen at more than 1200 mg/day may generate gastrointestinal and cardiovascular side effects. Bilayer or multiparticulate devices have been developed for controlled release in order to prevent undesired side effects. A new "two release rate (2RR) monolithic tablets" approach is now proposed for controlled release of poorly soluble drugs, particularly NSAIDs. Ibuprofen was used as model drug. This concept is based on a calcium carboxymethyl-starch (CaCMS) complex as a novel, low-cost excipient for monolithic dosage forms easy to manufacture by direct compaction. The in vitro dissolution from CaCMS formulations (tablets containing 400 or 600 mg active principle) showed two distinct release rates: (i) an initial fast release (for 30 min in simulated gastric fluid) of about 200 mg ibuprofen, an amount similar to the dosage of conventional immediate-release form (Motrin® 200 mg), and (ii) a slow release of remaining about 200 or 400 mg for a period of 12 h. A preliminary in vivo study (beagle dogs) showed pharmacokinetic parameters of one single controlled-release dosage of ibuprofen (400 mg) formulated with CaCMS, near equivalence with multiple doses (three tablets of 200 mg ibuprofen) of conventional Motrin®. A marked reduction (with 33%) of administered dose (400 instead 600 mg) was achieved by the new formulation with equivalent therapeutic effects. This dose reduction may be beneficial and is expected to minimize side damage risks. Although the present study was limited to NSAIDs, the 2RR concept can be applied for other drugs, particularly for subjects unable to follow frequent administrations.

Carboxymethyl starch/alginate microspheres containing diamine oxidase for intestinal targeting.

The association of carboxymethyl starch (CMS) and alginate is proposed as a novel matrix for the entrapment of bioactive agents in microspheres affording their protection against gastrointestinal degradation. In this study, the enzyme diamine oxidase (DAO) from white pea (Lathyrus sativus) was immobilized by inclusion in microspheres formed by ionotropic gelation of CMS/alginate by complexation with Ca(2+) . The association of CMS to alginate generated a more compact structure presenting a lesser porosity, thus decreasing the access of gastric fluid inside the microspheres and preventing the loss of entrapped enzyme. Moreover, the immobilized enzyme remained active and was able to oxidize the polyamine substrates even in the presence of degrading proteases of pancreatin. The inclusion yield in terms of entrapped protein was of about 82%-95%. The DAO entrapped in calcium CMS/alginate beads retained up to 70% of its initial activity in simulated gastric fluid (pH 2.0). In simulated intestinal fluid (pH 7.2) with pancreatin, an overall retention of 65% of activity for the immobilized DAO was observed over 24 H, whereas in similar conditions the free enzyme was totally inactivated. Our project proposes the vegetal DAO as an antihistaminic agent orally administered to treat food histaminosis and colon inflammation.

The effect of ethylene oxide sterilization on the surface chemistry and in vitro cytotoxicity of several kinds of chitosan.

The surfaces of three chitosan samples, differing only in their degrees of deacetylation and of carboxyethyl chitosan were chemically characterized by X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectroscopy, X-ray diffraction, and Fourier transform infrared, both before and after sterilization with ethylene oxide. Unexpected elemental ratios suggest that surface chemical modification occurred during the processing of the original chitin, with further surface modification on subsequent sterilization, despite previous reports to the contrary. Cell viability was evaluated by direct contact methyl thiazole tetrazolium and lactate dehydrogenase assays between the chitosan particles and A549 human epithelial cells, which demonstrated that the modifications incurred on sterilization are reflected in biocompatibility changes. All the samples were found to be biocompatible and nontoxic before sterilization and remained so subsequently. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2013.

The effect of ethylene oxide sterilization on the surface chemistry and in vitro cytotoxicity of several kinds of chitosan.

The surfaces of three chitosan samples, differing only in their degrees of deacetylation and of carboxyethyl chitosan were chemically characterized by X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectroscopy, X-ray diffraction, and Fourier transform infrared, both before and after sterilization with ethylene oxide. Unexpected elemental ratios suggest that surface chemical modification occurred during the processing of the original chitin, with further surface modification on subsequent sterilization, despite previous reports to the contrary. Cell viability was evaluated by direct contact methyl thiazole tetrazolium and lactate dehydrogenase assays between the chitosan particles and A549 human epithelial cells, which demonstrated that the modifications incurred on sterilization are reflected in biocompatibility changes. All the samples were found to be biocompatible and nontoxic before sterilization and remained so subsequently.

Mechanical and barrier properties of nanocrystalline cellulose reinforced chitosan based nanocomposite films.

Nanocrystalline cellulose (NCC) reinforced chitosan-based biodegradable films were prepared by solution casting. The NCC content in the films was varied from 1 to 10% (dry wt. basis). It was found that the tensile strength (TS) of the nanocomposite films with 5% (w/w) NCC content was optimum with an improvement of 26% compared to the control chitosan films. Incorporation of NCC also significantly improved barrier properties. Water vapor permeability (WVP) of the chitosan/NCC films was decreased by 27% for the optimum 5% (w/w) NCC content. Swelling studies revealed a decrease in water uptake of the NCC-reinforced chitosan films. Analyses of thermal properties showed no significant effect of NCC whereas X-ray diffraction studies confirmed the appearance of crystalline peaks in the nanocomposite films. Surface morphology of the films was investigated by scanning electron microscopy and it was found that NCC was dispersed homogenously into chitosan matrix.

Nanocrystalline cellulose (NCC) reinforced alginate based biodegradable nanocomposite film.

Nanocrystalline cellulose (NCC) reinforced alginate-based nanocomposite film was prepared by solution casting. The NCC content in the matrix was varied from 1 to 8% ((w/w) % dry matrix). It was found that the nanocomposite reinforced with 5 wt% NCC content exhibits the highest tensile strength which was increased by 37% compared to the control. Incorporation of NCC also significantly improved water vapor permeability (WVP) of the nanocomposite showing a 31% decrease due to 5 wt% NCC loading. Molecular interactions between alginate and NCC were supported by Fourier Transform Infrared Spectroscopy. The X-ray diffraction studies also confirmed the appearance of crystalline peaks due to the presence of NCC inside the films. Thermal stability of alginate-based nanocomposite films was improved after incorporation of NCC.

Zymographic assay of oxidases using peroxidase or hemin entrapped in polyacrylamide gel.

This chapter describes a zymographic assay of oxidases which is based on a coupled peroxidase or hemin reaction. The enzymatic activity of oxidases (i.e., diamine oxidase/DAO, glucose oxidase, galactose oxidase) can be directly monitored on polyacrylamide gels containing horseradish peroxidase or hemin, in the presence of their specific substrates and ortho-phenylenediamine (OPDA), an oxidizable chromogen. In the presence of hydrogen peroxide, OPDA is oxidized to azo-aniline, which led to well-defined yellow-brown bands on gels, with intensities corresponding to the enzymatic activity of oxidases.

Improvement of water barrier property of paperboard by coating application with biodegradable polymers.

Biopolymeric coatings were prepared and applied onto paperboard to improve its water barrier property. To prepare whey protein isolate (WPI)/cellulose-based films, WPI and glycerol were dissolved in water with glutaraldehyde (cross-linking agent) and cellulose xanthate. The solution was cast, dried, and insolubilized by entrapment of WPI in regenerated cellulose. Films were combined with beeswax (BW) into a bilayer coating system and then applied onto paperboard by heating compression. Another coating solution consisting of poly(vinyl butyral) (PVB)/zein was prepared by dissolving poly(vinyl alcohol) (PVA) and zein in 70% ethanol with glutaraldehyde and butyraldehyde (functionalization agent). The PVB/zein solution was applied onto paperboard after BW was sprayed. The structure of the PVB/zein-based coatings was analyzed by Fourier transform infrared spectroscopy (FTIR). The water vapor barrier property of coated paperboards was evaluated by water vapor transmission rate (WVTR) measurements. From the FTIR spectra, PVA functionalization after cross-linking and efficient acetalization into PVB were confirmed. WPI/cellulose and PVB/zein coating treatments improved the water barrier properties of paperboard by decreasing the WVTR by 77-78%. Although the BW coating was more efficient (decrease of WVTR by 89%), bilayer coatings composed of BW and polymer coatings had a stronger barrier effect with a decrease of WVTR to 92-95%, hence approaching commercial attributes required to ensure water vapor barrier in paperboard-based food containers (10 g/m(2).day). These results suggest that surface coating by biodegradable polymers may be utilized for the manufacture of paperboard containers in industrial applications.

DNA analysis of a radiotolerant bacterium Pantoea agglomerans by FT-IR spectroscopy.

A radiation tolerance strain, Pantoea agglomerans was isolated from gamma-irradiated carrot samples (Daucus carota). D(10) determination showed that the radioresistance of this bacterium is five-fold higher than Escherichia coli, both belonging to the family of Enterobacteriaceae. DNA isolated from untreated and irradiated bacterial cells was analyzed by FT-IR spectroscopy to investigate the radiotolerance of this bacterium. At doses <5kGy, an alteration of the interbase hydrogen networks was observed and characterized mainly by an increase of bands assigned to the carbonyl non-pairing and the free amine groups. Moderate breakage of the DNA backbone and damage of the osidic structure were also observed. Similar spectral profiles were noticed at doses > or =5kGy, but additional increase of the band intensity of C=C and C=N suggests damages of nucleobases. High number of asymmetric PO(2)(-) and upper shift of symmetric PO(2)(-) are indicative of DNA strand breaks. Osidic damages were evidenced by decrease of the absorption bands ascribed to deoxyribosyl moieties and by appearance of C-OH band. DNA degradation at high irradiation doses was also noticed by electrophoresis using agarose gel. It appeared that DNA underwent covalent cross-linking, as revealed by agglomeration of DNA in the wells of agarose gel.

Modified alginate and chitosan for lactic acid bacteria immobilization.

Beads with enhanced-stability acid media, which were based on alginate and chitosan functionalized by succinylation (increasing the anionic charges able to retain protons) or by acylation (improving matrix hydrophobicity), were developed for immobilization of bacterial cells. Beads (3 mm diameter) formed by ionotropic gelation with CaCl(2) presented good mechanical characteristics. After 30 min incubation of viable free Lactobacillus rhamnosus cells in simulated gastric fluid (pH 1.5), we noticed that the level of viable bacteria was undetectable. Bacterial immobilization in native-alginate-based beads generated a viable-cell count of 22-26%, whereas, when entrapped in succinylated alginate and chitosan beads, the percentage of viable cells was of 60 and 66%, respectively. Best viability (87%) was found for bacteria immobilized in N -palmitoylaminoethyl alginate, which affords a high protective effect, probably due to long alkyl pendants that improve the beads' hydrophobicity, limiting hydration in the acidic environment.

Modified alginate matrices for the immobilization of bioactive agents.

Bioactive agents (catalase - an enzyme, and nisin - a bacteriocin) were covalently immobilized on alginate activated with sodium periodate (oxidatively converting 2,3-dihydroxy groups into dialdehyde residues), followed or preceded by ionotropic gelation. For the same protein coupling yield, the retained enzyme activity of the immobilized enzyme (ImE) can be markedly increased by diminishing the bead diameter, a phenomenon that illustrates the role of substrate/product diffusion through the bead gel layer. When the amount of enzyme introduced for coupling was about 15 mg/100 mg of support and the bead diameter was about 100 microm, a high retained specific activity (95-98%) was obtained. Diffusion phenomena can be markedly decreased by enzyme immobilization on the surface of microbeads (obtained by gelation of activated alginate prior to immobilization). In this case, the retained activity was approx. 75% of that of the free enzyme. A slightly higher K (m) value of ImE suggested that the enzyme-substrate affinity was almost maintained. The profiles of ImE activities at various pH values, at various temperatures and when undergoing proteolysis showed a overall higher stability for the immbolized than that for the free enzyme. Nisin immobilized on the microbead surface, when submitted to proteolysis, conserved its bacteriocin activity, strongly inhibiting the growth of Lactobacillus sake when subjected to an agar spot test, whereas free nisin totally lost its activity.

N-acylated chitosan: hydrophobic matrices for controlled drug release.

N-acylation of chitosan with various fatty acid (C(6)-C(16)) chlorides increased its hydrophobic character and made important changes in its structural features. Unmodified chitosan exhibited a low degree of order (DO) and a weak tablet crushing strength. Chitosan acylated with a short chain length (C(6)) possessed similar properties, but exhibited significant swelling. Acylation with longer side chains (C(8)-C(16)) resulted in a higher DO and crushing strength but lower swelling. The best mechanical characteristics and drug release properties were found for palmitoyl chitosan (substitution degree 40-50%) tablets with 20% acetaminophen as a tracer. The high stability of these monolithic tablets appears to be due to hydrophobic interactions between side chains, as shown by a more organized structure. Infrared spectroscopy, X-ray diffractometry and proton nuclear magnetic resonance analyses of palmitoyl chitosan were consistent with a hydrophobic self-assembling model. Drug dissolution kinetics showed longer release times for higher degrees of functionalization, i.e. 30 h (for 47% substitution) and 90 h (for 69% substitution), suggesting palmitoyl chitosan excipients as interesting candidates for oral and subdermal pharmaceutical applications.