Chitosan and Kappa-Carrageenan Vaginal Acyclovir Formulations for Prevention of Genital Herpes. In Vitro and Ex Vivo Evaluation

Vaginal formulations for the prevention of sexually transmitted infections are currently gaining importance in drug development. Polysaccharides, such as chitosan and carrageenan, which have good binding capacity with mucosal tissues, are now included in vaginal delivery systems. Marine polymer-based vaginal mucoadhesive solid formulations have been developed for the controlled release of acyclovir, which may prevent the sexual transmission of the herpes simplex virus. Drug release studies were carried out in two media: simulated vaginal fluid and simulated vaginal fluid/simulated seminal fluid mixture. The bioadhesive capacity and permanence time of the bioadhesion, the prepared compacts, and compacted granules were determined ex vivo using bovine vaginal mucosa as substrate. Swelling processes were quantified to confirm the release data. Biocompatibility was evaluated through in vitro cellular toxicity assays, and the results showed that acyclovir and the rest of the materials had no cytotoxicity at the maximum concentration tested. The mixture of hydroxyl-propyl-methyl-cellulose with chitosan- or kappa-carrageenan-originated mucoadhesive systems that presented a complete and sustained release of acyclovir for a period of 8–9 days in both media. Swelling data revealed the formation of optimal mixed chitosan/hydroxyl-propyl-methyl-cellulose gels which could be appropriated for the prevention of sexual transmission of HSV.     

Cytotoxic Effects of Fucoidan Nanoparticles against Osteosarcoma

In this study, we analyzed the size-dependent bioactivities of fucoidan by comparing the cytotoxic effects of native fucoidan and fucoidan lipid nanoparticles on osteosarcoma in vitro and in vivo. In vitro experiments indicated that nanoparticle fucoidan induced apoptosis of an osteosarcoma cell line more efficiently than native fucoidan. The more potent effects of nanoparticle fucoidan, relative to native fucoidan, were confirmed in vivo using a xenograft osteosarcoma model. Caco-2 cell transport studies showed that permeation of nanoparticle fucoidan was higher than native fucoidan. The higher bioactivity and superior bioavailability of nanoparticle fucoidan could potentially be utilized to develop novel therapies for osteosarcoma.     

Control of postharvest anthracnose of banana using a new edible composite coating

Anthracnose is a postharvest disease of banana caused by the fungus Colletotrichum musae that results in major economic losses during transportation and storage. For the management of banana anthracnose, antifungal effects of Arabic gum (AG) (5, 10, 15 and 20%), chitosan (CH) (1.0%), and the combination of AG with CH were investigated in vitro as well as in vivo. CH at 1.0% and 1.5% had fungicidal effects on C. musae. AG alone did not show any fungicidal effects while the combination of 1.0% CH with all tested AG concentrations had fungicidal effects. However, the potato dextrose agar (PDA) medium amended with 10% AG incorporated with 1.0% CH showed the most promising results among all treatments in suppressing the mycelial growth (100%) and conidial germination inhibition (92.5%). In vivo analysis also revealed that 10% AG incorporated with 1.0% CH was the optimal concentration in controlling decay (80%), showing a synergistic effect in the reduction of C. musae in artificially inoculated bananas. The 10% AG incorporated with 1.0% CH coatings significantly delayed ripening as in terms of percentage weight loss, fruit firmness, soluble solids concentration and titratable acidity. The results showed the possibility of using 10% Arabic gum incorporated with 1.0% chitosan as a biofungicide for controlling postharvest anthracnose in banana.     

Chitosan-Genipin Microspheres for the Controlled Release of Drugs: Clarithromycin,Tramadol and Heparin

The aim of this study was to first evaluate whether the chitosan hydrochloride-genipin crosslinking reaction is influenced by factors such as time, and polymer/genipin concentration, and second, to develop crosslinked drug loaded microspheres to improve the control over drug release. Once the crosslinking process was characterized as a function of the factors mentioned above, drug loaded hydrochloride chitosan microspheres with different degrees of crosslinking were obtained. Microspheres were characterized in terms of size, morphology, drug content, surface charge and capacity to control in vitro drug release. Clarithromycin, tramadol hydrochloride, and low molecular weight heparin (LMWH) were used as model drugs. The obtained particles were spherical, positively charged, with a diameter of 1–10 μm. X-Ray diffraction showed that there was an interaction of genipin and each drug with chitosan in the microspheres. In relation to the release profiles, a higher degree of crosslinking led to more control of drug release in the case of clarithromycin and tramadol. For these drugs, optimal release profiles were obtained for microspheres crosslinked with 1 mM genipin at 50 ºC for 5 h and with 5 mM genipin at 50 ºC for 5 h, respectively. In LMWH microspheres, the best release profile corresponded to 0.5 mM genipin, 50 ºC, 5 h. In conclusion, genipin showed to be eligible as a chemical-crosslinking agent delaying the outflow of drugs from the microspheres. However, more studies in vitro and in vivo must be carried out to determine adequate crosslinking conditions for different drugs.     

Comparative Study of Lipophilic and Hydrophilic Antioxidants from <Emphasis Type="Italic">In vivo</Emphasis> and <Emphasis Type="Italic">In vitro</Emphasis> Grown <Emphasis Type="Italic">Coriandrum sativum</Emphasis>

Coriander is commonly used for medicinal purposes, food applications, cosmetics and perfumes. Herein, the production of antioxidants in vegetative parts (leaves and stems) of in vivo and in vitro grown samples was compared. In vitro samples were clone A- with notorious purple pigmentation in stems and leaves and clone B- green. Seeds were also studied as they are used to obtain in vivo and in vitro vegetative parts. Lipophilic (tocopherols, carotenoids and chlorophylls) and hydrophilic (sugars, ascorbic acid, phenolics, flavonols and anthocyanins) compounds were quantified. The antioxidant activity was evaluated by radical scavenging activity, reducing power and lipid peroxidation inhibition. The in vivo sample showed the highest antioxidant activity mainly due to its highest levels of hydrophilic compounds. Otherwise, in vitro samples, mainly clone A, gave the highest concentration in lipophilic compounds but a different profile when compared to the in vivo sample. Clones A and B revealed a lack of β-carotene, β- and δ-tocopherols, a decrease in α-tocopherol, and an increase in γ-tocopherol and clorophylls in comparison to the in vivo sample. In vitro culture might be useful to explore the plants potentialities for industrial applications, controlling environmental conditions to produce higher amounts of some bioactive products.     

Bioavailability of ferulic acid is determined by its bioaccessibility

Epidemiological studies have linked whole grain consumption to prevention of several chronic diseases. Whole grain is a source of important phytochemicals, such as ferulic acid (FA). FA is the most abundant phenolic and major contributor to the in vitro antioxidant capacity of wheat grain. Several studies have reported highly variable results on FA bioavailability (0.4–98%). The binding of FA to polysaccharides may limit its bioavailability. Therefore, our study aimed at monitoring release features of FA during gastrointestinal (GI) transit. This was termed bioaccessibility. The bioaccessibility of FA was studied from different wheat fractions and breads with the use of a dynamic in vitro system that simulates the upper GI transit and digestion. The results showed low bioaccessibility of FA from the wheat fractions and breads (<1%). However, the bioaccessibility was high when free FA was added to flour (∼60%). The bioaccessibility of FA appeared to be determined by the percentage of free FA. In wheat grain, most of FA is bound to arabinoxylans and other indigestible polysaccharides restricting its release in the small intestine. New processing developments should be considered to increase free FA in the cereal matrix in order to improve its bioavailability and systemic health effect.     

Alginate Hydrogels Coated with Chitosan for Wound Dressing

In this work, a coating of chitosan onto alginate hydrogels was realized using the water-soluble hydrochloride form of chitosan (CH-Cl), with the dual purpose of imparting antibacterial activity and delaying the release of hydrophilic molecules from the alginate matrix. Alginate hydrogels with different calcium contents were prepared by the internal setting method and coated by immersion in a CH-Cl solution. Structural analysis by cryo-scanning electron microscopy was carried out to highlight morphological alterations due to the coating layer. Tests in vitro with human mesenchymal stromal cells (MSC) were assessed to check the absence of toxicity of CH-Cl. Swelling, stability in physiological solution and release characteristics using rhodamine B as the hydrophilic model drug were compared to those of relative uncoated hydrogels. Finally, antibacterial activity against Escherichia coli was tested. Results show that alginate hydrogels coated with chitosan hydrochloride described here can be proposed as a novel medicated dressing by associating intrinsic antimicrobial activity with improved sustained release characteristics.     

Seaweed Components as Potential Modulators of the Gut Microbiota

Macroalgae, or seaweeds, are a rich source of components which may exert beneficial effects on the mammalian gut microbiota through the enhancement of bacterial diversity and abundance. An imbalance of gut bacteria has been linked to the development of disorders such as inflammatory bowel disease, immunodeficiency, hypertension, type-2-diabetes, obesity, and cancer. This review outlines current knowledge from in vitro and in vivo studies concerning the potential therapeutic application of seaweed-derived polysaccharides, polyphenols and peptides to modulate the gut microbiota through diet. Polysaccharides such as fucoidan, laminarin, alginate, ulvan and porphyran are unique to seaweeds. Several studies have shown their potential to act as prebiotics and to positively modulate the gut microbiota. Prebiotics enhance bacterial populations and often their production of short chain fatty acids, which are the energy source for gastrointestinal epithelial cells, provide protection against pathogens, influence immunomodulation, and induce apoptosis of colon cancer cells. The oral bioaccessibility and bioavailability of seaweed components is also discussed, including the advantages and limitations of static and dynamic in vitro gastrointestinal models versus ex vivo and in vivo methods. Seaweed bioactives show potential for use in prevention and, in some instances, treatment of human disease. However, it is also necessary to confirm these potential, therapeutic effects in large-scale clinical trials. Where possible, we have cited information concerning these trials.     

The validity of in vitro techniques using rumen fluid or cellulase for predicting changes in the dry matter digestibility of grasses caused by fertilizer calcium, sulphur, phosphorus and nitrogen

Sixty-four samples were used to measure the accuracy of in vitro techniques using rumen fluid-pepsin or pepsin-cellulase for predicting digestibility of grasses grown with different levels of fertilizer calcium(Ca), sulphur(S), phosphorus (P) and nitrogen (N). In each case, fertilizers changed in vivo dry matter digestibility (DMD) and the in vitro techniques accurately predicted the changes in in vivo DMD caused by fertilizer Ca, P and N. The in vitro techniques gave biased estimates of in vivo DMD in studies with fertilizer S. This bias was caused by a decrease in the digestive efficiency of the sheep when fed the unfertilized feed. It was concluded that biased estimates of DMD may be associated with low fertilizer levels and these cannot be eliminated by improving the in vitro techniques and would also apply to estimates of DMD based on chemical analysis of the feed.     

A comparison of methods of predicting the in vivo digestibility of heather by sheep

Methods of predicting the in vivo dry matter digestibility (DMD) of heather were examined in studies on ten samples of heather which had been fed to sheep in standard digestibility trials. DMD was predicted with similar accuracy from estimates of in vitro dry matter digestibility (in vitro DMD) and from the concentration of cell-wall constituents. In vitro DMD was found to be between 10 and 18 percentage units lower than DMD. Freeze drying the samples was the only modification to the technique which both increased in vitro DMD and the accuracy of the prediction of DMD. Other modifications attempted were the use of different liquor sources, changes in first-stage incubation time and dilution rate, and nitrogen supplementation of the inocula. The concentration of nitrogen or undigested dietary nitrogen in the faeces could not be used to predict the digestibility of heather with precision. It was concluded that the DMD of a heather diet could be satisfactorily predicted from the in vitro DMD of samples collected from sheep fistulated at the oesophagus.     

Relationships between antithrombogenicity and surface free energy of regenerated silk fibroin films

Silk fibroin (SF) was dissolved in calcium chloride/ethanol/water mixture (1/2/8 in mole ratio) at 70°C for 4 h. The dissolved silk fibroin was regenerated by casting the dialyzed solution into the films. The films were treated with 50% aqueous solution of methanol for different times, and their antithrombogenicity was evaluated byin vitro andin vivo tests.In vivo blood tests were made by a method of peripheral vein indwelling suture. It was found that the silk fibroin had a good anti-thrombogenicity and an absorbability even though the polymer showed foreign body reaction. Finally, the blood compatibilty of silk fibroin films which were subjected to structural change by the methanol treatment, was examined in connection with their interfacial surface energy, and a correlation between these properties was found to be present.     

Soluble arabinoxylans extracted from soft and hard wheat show a differential prebiotic effect in vitro and in vivo

Arabinoxylans (AX) are part of dietary fiber. They are currently under study due to their potential prebiotic effect. Wheat whole grain flours contain all the grain layers and, therefore, present a higher arabinoxylan content than white flour. It is known that the chemical structure of these compounds varies with the type of wheat cultivar and the tissue from which they are extracted. In this work, water soluble extractable arabinoxylans (WE-AX) from two types of wheat whole flours (hard and soft) were extracted. We characterized the molecular size distribution and the potential prebiotic effect of those extracts. The prebiotic effect was evaluated in vitro and confirmed in vivo. Bacterial group abundance (Lactobacillus, Bifidobacterium, Clostridium, Enterococcus, Bacteriodes and total bacteria) was determined by quantitative RT-PCR. The molecular size of AX from hard wheats was significantly higher than AX from soft wheats. Both extracts showed potential prebiotic activity by increasing the growth of beneficial bacteria in vitro and in vivo, decreasing the pathogens in the profile of intestinal microorganisms and increasing the amount of short chain fatty acids in the intestine. WE-AX from hard wheat showed a higher prebiotic activity. Prebiotic effect assessed in vitro and in vivo assays showed a significant correlation between both types of analysis. This finding suggests that the in vitro indices performed allow predicting the potential prebiotic effect in vivo.     

Bioaccessibility of Phenolic Compounds and Antioxidant Capacity of Chia (<Emphasis Type="Italic">Salvia hispanica</Emphasis> L.) Seeds

The aim of this work was to evaluate (i) the phenol and flavonoid recovery and bioaccessibility indexes and (ii) the antioxidant activity of both types of non-defatted and defatted chia seeds during the in vitro gastrointestinal digestion. The ground samples were subjected to in vitro simulated gastrointestinal digestion, and the resultant fractions were extracted and subjected to spectrophotometric assays. The results pointed to increasing concentrations of polyphenolic compounds during digestion, although only a low-medium percentage of phenols and a low percentage of flavonoids were available for absorption in the intestinal tract. In addition, the high level of fats seemed to have a negative effect on the bioaccessibility of flavonoids. Further studies should be undertaken to better understand the stabilization of the bioactive compounds of chia and to improve their bioaccessibility. Meanwhile, the present study represents a solid base for studying the bioavailability of bioactive compounds of chia seeds.     

Anti-Inflammatory Activity of Chitooligosaccharides in Vivo

All the reports to date on the anti-inflammatory activity of chitooligosaccharides (COS) are mostly based on in vitro methods. In this work, the anti-inflammatory activity of two COS mixtures is characterized in vivo (using balb/c mice), following the carrageenan-induced paw edema method. This is a widely accepted animal model of acute inflammation to evaluate the anti-inflammatory effect of drugs. Our data suggest that COS possess anti-inflammatory activity, which is dependent on dose and, at higher doses, also on the molecular weight. A single dose of 500 mg/kg b.w. weight may be suitable to treat acute inflammation cases; however, further studies are needed to ascertain the effect upon longer inflammation periods as well as studies upon the bioavailability of these compounds.     

Producing Gelatin Nanoparticles as Delivery System for Bovine Serum Albumin

Background: Progress in the field of biology and biochemistry has led to the discovery of numerous bioactive peptides and proteins in the last few decades. Delivery of therapeutic proteins/peptides has received a considerable amount of attention in recent years. Methods: In this study, a two-step desolvation method was used to produce biodegradable hydrophilic gelatin nanoparticles (GNP) as a delivery system of protein model (BSA). The size and shape of the nanoparticles were examined by dynamic light scattering and scanning electron microscopy. Results: Particles with a mean diameter of 200-300 nm were produced and the percentage of entrapment efficiency was found to be 87.4. The optimum amount of theoretical BSA loading was obtained, the release of BSA was monitored in vitro, and the mechanism of release was studied. The BSA release profile showed a biphasic modulation characterized by an initial, relatively rapid release period, followed by a slower release phase. Conclusion: Results show that the two-step desolvation is an appropriate method for preparing GNP as a delivery vehicle for BSA. Key Words: Gelatin, Nanoparticles, Bovine serum albumin     

Fucoidan as a Marine Anticancer Agent in Preclinical Development

Fucoidan is a fucose-containing sulfated polysaccharide derived from brown seaweeds, crude extracts of which are commercially available as nutritional supplements. Recent studies have demonstrated antiproliferative, antiangiogenic, and anticancer properties of fucoidan in vitro. Accordingly, the anticancer effects of fucoidan have been shown to vary depending on its structure, while it can target multiple receptors or signaling molecules in various cell types, including tumor cells and immune cells. Low toxicity and the in vitro effects of fucoidan mentioned above make it a suitable agent for cancer prevention or treatment. However, preclinical development of natural marine products requires in vivo examination of purified compounds in animal tumor models. This review discusses the effects of systemic and local administration of fucoidan on tumor growth, angiogenesis, and immune reaction and whether in vivo and in vitro results are likely applicable to the development of fucoidan as a marine anticancer drug.     

Immunomodulatory Effects of Domoic Acid Differ Between In vivo and In vitro Exposure in Mice

The immunotoxic potential of domoic acid (DA), a well-characterized neurotoxin, has not been fully investigated. Phagocytosis and lymphocyte proliferation were evaluated following in vitro and in vivo exposure to assay direct vs indirect effects. Mice were injected intraperitoneally with a single dose of DA (2.5 μg/g b.w.) and sampled after 12, 24, or 48 hr. In a separate experiment, leukocytes and splenocytes were exposed in vitro to 0, 1, 10, or 100 μM DA. In vivo exposure resulted in a significant increase in monocyte phagocytosis (12-hr), a significant decrease in neutrophil phagocytosis (24-hr), a significant decrease in monocyte phagocytosis (48-hr), and a significant reduction in T-cell mitogen-induced lymphocyte proliferation (24-hr). In vitro exposure significantly reduced neutrophil and monocyte phagocytosis at 1 μM. B- and T-cell mitogen-induced lymphocyte proliferation were both significantly increased at 1 and 10 μM, and significantly decreased at 100 μM. Differences between in vitro and in vivo results suggest that DA may exert its immunotoxic effects both directly and indirectly. Modulation of cytosolic calcium suggests that DA exerts its effects through ionotropic glutamate subtype surface receptors at least on monocytes. This study is the first to identify DA as an immunotoxic chemical in a mammalian species.     

Identification and Biological Characterization of Angiogenic and Tumor Growth Inhibitors derived from Sinica cetorhinus maximum Cartilage

Shark (Sinica cetorhinus maximum) cartilage was extracted in 1 mol/L Gu- HCl guanidine. Two purified active proteins with apparent molecular weights of 15.2×10³ Da and 8.0×10³ Da (designated as Sp15 and Sp8, respectively) were obtained through ultrafiltration and Superdex 75 chromatography. The activities of the samples were studied in terms of their potential inhibition of vascular endothelial cell growth in vitro, of angiogenesis both in rabbit cornea and chick embryo chorioallantoic membrane (CAM) assay models in vivo, and of growth of transplanted S180 sarcoma in mice in vivo. The results showed that Sp15 expressed a typical lysozymatic activity up to 223,000 U/mg and its N-terminus was highly homologous to lysozymes of various mammalian origins. Sp15 exhibited a strong anti-angiogenic activity only in vitro, whereas Sp8 shared this effect both in vitro and in vivo. Both Sp15 and Sp8 provided an effective anti-tumor activity in mice bearing transplanted S180 sarcoma. These results suggest that Sp15 is a shark cartilage-derived lysozyme that participates in the defense to bacterial invasion to the body, while Sp8 is an angiogenic inhibitor that mediates at least part of the anti-tumor activity associated with shark cartilage probably through the inhibition of tumor-induced angiogenesis.     

An effective method for culturing pollen tubes of potato

In vitro culture of pollen tubes is a convenient way to distinguish inviable pollen from stylar incompatibilities which prevent pollen tube growthin vivo. This usefulness ofin vitro pollen tube culture depends on how successful the technique is in promoting optimal pollen tube growth. Experiments were conducted to compare the effects of the type of sugar and method of aeration of cultures ofSolanum species (potato) pollen. Lactose was superior to sucrose in promoting pollen germination and pollen tube growth on hanging drop cultures. Germination and growth were greatly enhanced when pollen was cultured in actively aerated lactose medium. Since pollen growth comparable to that occurringin vivo was observed using lactose medium with active aeration, this technique may be useful in various types of research related to reproductive biology.