Alginate-Chitosan Membranes for the Encapsulation of Lavender Essential Oil and Development of Biomedical Applications Related to Wound Healing.

Biopolymers such as chitosan (CHT) or alginate (ALG) are among the most prominent for health-related applications due to their broad bioactivity. Their combination for the preparation of membranes is hereby proposed as an application for wound healing with the incorporation of lavender essential oil (LEO), widely known for its antioxidant and antimicrobial properties. The preparation of CHT, CHT + LEO, ALG, ALG + LEO, and CHT/ALG + LEO membranes was accomplished, and its composition was analyzed using Fourier Transform Infrared Spectroscopy (FTIR). The water absorption capacity and oil release profile of the membranes revealed higher water uptake capacity when a lower LEO release was obtained. The combined CHT/ALG + LEO film showed a water uptake percentage of 638% after 48 h and a maximum LEO release concentration of 42 mg/L. Cytotoxicity and biocompatibility of the prepared membranes were studied using a HaCaT cell line, with an assessment of cell viability regarding film leachables, DNA quantification, and DAPI-phalloidin staining. The results revealed that the indirect contact of the prepared membranes via its leachables does not compromise cell viability, and upon direct contact, cells do not adhere or proliferate on the surface of the membranes. Moreover, the CHT/ALG + LEO membrane increases cell proliferation, making it suitable for applications in wound healing.

Cost Effective Use of a Thiosulfinate-Enriched Allium sativum Extract in Combination with Chemotherapy in Colon Cancer.

In this work, we sought to investigate the effects of a thiosulfinate-enriched garlic extract, co-administered with 5-fluorouracil (5-FU) or oxaliplatin chemotherapy, on the viability of colon cancer cells (Caco-2 and HT-29). We also addressed the economic feasibility of a new combined treatment of this thiosulfinate-enriched garlic extract, with oxaliplatin that could reduce the dosage and costs of a monotherapy. The thiosulfinate-enriched garlic extract not only enhanced the impact of 5-FU and oxaliplatin (500 µM) in decreasing Caco-2 and HT-29 viability, but also showed a higher effect than standard 5-FU and oxaliplatin chemotherapy as anti-cancer agents. These results provided evidences for the combination of lyophilized garlic extract and 5-FU or oxaliplatin as a novel chemotherapy regimen in colon cancer cells that may also reduce the clinical therapy costs.

Antibacterial Activity Assessment of Chitosan/Alginate Lavender Essential Oil Membranes for Biomedical Applications.

The demand for natural products in the treatment of dermatological pathologies has boosted the use of bioactive substances such as lavender essential oil (LEO), which stands out for its anti-inflammatory and antioxidant properties and its antimicrobial potential. Biopolymers such as chitosan (CHT) and alginate (ALG) are biodegradable and biocompatible and have proven their viability in biomedical applications such as skin regeneration. The inhibitory effect of LEO on the growth of skin-related bacterial species Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and the fungus Candida albicans was studied by incorporating 1% v/v LEO encapsulated in CHT, ALG, and CHT/ALG membranes. Despite the verification of the antimicrobial effect of all type of membranes, no synergistic effect was observed following the addition of LEO. S. aureus and P. aeruginosa showed the most growth on the different substrates and C. albicans demonstrated the highest inhibition. This is a first approach using microorganisms isolated from clinical samples or skin microbiota. Further investigation would be advisable using more clinical strains for each microorganism to validate their biomedical applicability.

Garlic Extracts: Effect of pH on Inhibition of Helicobacter pylori.

The present work studies the influence of pH on the stability of thiosulfinates, compounds responsible for the bacteriostatic properties shown by ethanolic and acetonic garlic extracts (EGE and AGE) against the in vitro growth of Helicobacter pylori (Hp), a bacterium which is implicated in the etiology of diverse gastrointestinal diseases. The influence of pH and time on the stability of thiosulfinates and the microbiological activities of EGE and AGE has been evaluated at human body temperature (37 °C) and in a pH range of 0.9-4.7. A marked decrease in thiosulfinate concentration was observed in a relatively short time at pH values below 2.0. However, at pH values over 2.0, the samples maintained 70% of thiosulfinate concentration for 12 h. The inhibition halo diameters showed a maximum value at pH 2.50, with an inhibition halo of 28.94 ± 0.61 mm. The reduction in the activity at pH values below 2.0 was particularly remarkable. These results suggest that, for medical application, the pH of the selected extracts must only be maintained above 2 to maintain a high level of antibacterial activity. This fact would overcome the need for proton pump inhibitors and/or antibiotics during the treatment of Hp infections in human patients.

In Vitro Antioxidant and Pancreatic Anticancer Activity of Novel 5-Fluorouracil-Coumarin Conjugates.

Molecular hybridization consists of the combination of two or more non-identical pharmacophores in a single molecule. It has emerged as a promising strategy that allows the design of molecular frameworks with enhanced activity and affinity compared to their parent drugs. In this work, two novel hybrids that combine the well-known anticancer chemotherapeutic agent 5-fluorouracil with antioxidant coumarin derivatives have been synthesized and characterized by means of a copper-catalyzed azide-alkyne cycloaddition (CuAAC). The conjugates showed good antioxidant properties and a high tendency to aggregate and form stable nanoparticles in aqueous media, with regular shape and uniform size. These materials have proven to be preferential cytotoxic agents in vitro against human pancreatic cancer cells PANC-1, with an activity superior to free 5-fluorouracil. These results open up the possibility of exploiting the synergistic combination between 5-fluorouracil and coumarin derivatives and warrant further investigation of these hybrids as promising pancreatic anticancer agents.

Black Garlic and Thiosulfinate-Enriched Extracts as Adjuvants to Ceftriaxone Treatment in a Rat Peritonitis Model of Sepsis.

To date, there have been no new drugs or adjuvants able to decrease both morbidity and mortality in the context of sepsis and septic shock. Our objective was to evaluate the use of thiosulfinate-enriched Allium sativum and black garlic extracts as adjuvants in the management of sepsis. An experimental in vivo study was carried out with male Sprague-Dawley® rats. Animals were randomized in four treatment groups: antibiotic (ceftriaxone) treatment (group I), ceftriaxone plus thiosulfinate-enriched extract (TASE, group II), ceftriaxone plus thiosulfinate-enriched extract and black garlic extracts (TASE + BGE, group III), and ceftriaxone plus black garlic extract (BGE, group IV). All animals were housed and inoculated with 1 × 1010 CFU/15 mL of intraperitoneal Escherichia coli ATCC 25922. Subsequently, they received a daily treatment according to each group for 7 days. Clinical, analytical, microbiological, and histopathological parameters were evaluated. Statistically significant clinical improvement was observed in rats receiving garlic extracts in weight (groups II and III), ocular secretions, and piloerection (group IV). Moreover, less liver edema, vacuolization, and inflammation were observed in groups receiving adjuvant support (groups II, III, and IV). When comparing interleukins 24 h after bacteria inoculum, we found statistically significant differences in TNF-alpha levels in groups receiving BGE (groups III and IV, p ≤ 0.05). Blood and peritoneal liquid cultures were also analyzed, and we detected a certain level of Enterococcus faecalis in peritoneal cultures from all treatment groups and less bacteria presence in blood cultures in rats receiving garlic extracts (groups II, III, and IV). In conclusion, TASE and BGE could be promising nutraceutical or medicinal agents as coadjuvants in the treatment of sepsis because of its effects in modulating the inflammatory response.

Synthesis and Operating Optimization of the PEG Conjugate via CuAAC in scCO2.

A new sustainable green protocol for obtaining polyethylene glycol (PEG) conjugates, with a prototype molecule, which in this work was coumarin, by means of click chemistry is presented. The organic solvents commonly used for this type of reaction were replaced by supercritical carbon dioxide (scCO2). The synthesis and characterization of PEG-coumarin were successfully reported using FTIR, 1H NMR, and MALDI TOF. Subsequently, a preliminary study was carried out using the response surface methodology to examine the variables that most affect the use of scCO2 as a reaction medium. The main effects caused by these variables, individually and their binary interaction, have been estimated. The response surface methodology has been used in this work to screen variables using a factorial design 23. The p-values of temperature and pressure were 0.006 and 0.0117, being therefore the most significant variables of the response surface methodology study. Subsequently, a more intensive study has been carried out on the variables that have shown the greatest significant effect on reaction performance where an 82.32% synthesis success was achieved, which broadens the scope of the use of scCO2 as a reaction medium. The conjugated coumarin with mPEG-alkyne and coumarin were evaluated for their in vitro antioxidant activities by the DPPH radical scavenging assay and were found to exhibit substantial activities. The click product showed comparable or even better efficacy than the initial coumarin.

Study of the solubility and stability of polystyrene wastes in a dissolution recycling process.

Dissolution with suitable solvents is one of the cheapest and more efficient processes for polystyrene waste management. In this work the solubility of polystyrene foams in several solvents benzene, toluene, xylene, tetrahydrofuran, chloroform, 1,3-butanediol, 2-butanol, linalool, geraniol, d-limonene, p-cymene, terpinene, phellandrene, terpineol, menthol, eucalyptol, cinnamaldheyde, nitrobenzene, N,N-dimethylformamide and water has been determined. Experimental results have shown that to develop a "green process" the constituents of essential oils, d-limonene, p-cymene, terpinene, phellandrene, are the most appropriate solvents. The action of these solvent does not produce any degradation of polymer chains. The solubility of the polymer in the mentioned solvents at different temperatures has been investigated. The solvent can be easily recycled by distillation.

Impregnation of Curcumin into a Biodegradable (Poly-lactic-co-glycolic acid, PLGA) Support, to Transfer Its Well Known In Vitro Effect to an In Vivo Prostate Cancer Model.

Prostate cancer (PCa) is one of the most common cancers in older men and is associated with high mortality. Despite advances in screening for early detection of PCa, a large proportion of patients continue to be diagnosed with metastatic disease, with ~20% of men showing a high tumor grade and stage. Medicinal plant extracts have a great potential to prevent/treat PCa, as well as to reduce its incidence/prevalence and improve survival rates. One of the most promising extracts is curcumin, which is a major, nontoxic, bioactive compound of Curcuma longa. Curcumin has strong antitumor activity in vitro. However, its potential beneficial in vivo affects are limited by its low intestinal absorption and rapid metabolism. In this study, curcumin was impregnated into a biodegradable poly(lactic-co-glycolic) acid (PLGA) support and characterized by FTIR and DSC, and its release by UV spectrophotometry. PLGA-curcumin was tested in different subcutaneous PCa xenograft models (PC3, 22rv1, and DU145 PCa cell-lines), and its effects evaluated by tumor progression an immuno-histochemical analysis (Trichromic, Ki67 and TUNEL stainings), were compared with those of a commercial curcumin preparation. Our results indicate that curcumin-impregnated PLGA is significantly more active (~2-fold increase) with respect to oral curcumin, which supports its use for subcutaneous administration.

Biomonitoring of trace metals in a mine-polluted estuarine system (Spain).

In this paper, we examine metal concentrations in the water and in the crustacean Balanus balanoides from the Huelva estuary, one of the most polluted estuaries in Europe. Metal levels in waters are very high, especially those of Zn, Fe and Cu. Zn presents the highest concentrations, with a mean value of 690 microg l-1 in 2001 and 301 microg l-1 in 2002. As the water flows down through the estuary toward the sea, the metal concentrations drop sharply and the pH rises. The metal concentrations in balanoides tissues are, in general, very high, undoubtedly due to the high metal pollution of the water where it lives. Metal concentrations in Balanus balanoides tissues behave similarly to those in the water, reaching maximums in the upper part of the estuary and diminishing as we approach the sea. The element that reaches the highest levels in Balanus balanoides is Zn, with a mean value of 54.6 g kg-1 in 2001 and 29.9 g kg-1 in 2002, followed by Cu and Fe. There is a significant correlation (p<0.01) for concentrations of Cd, Cu, Fe, Mn, Ni and Zn in Balanus balanoides relative to their concentration in waters. Barnacles showed a great capacity to accumulate metals, especially Zn, Cu and Fe. Based on the results obtained, we can conclude that Balanus balanoides is a good tool for monitoring trace metals in the Huelva estuary.

Heavy metal concentrations in molluscs from the Atlantic coast of southern Spain.

Trace metals were determined in the two most abundant species of bivalve molluscs along the Atlantic coast of southern Spain (Donax trunculus and Chamelea gallina) and in the sediments where they live. The results show that the area near the mouth of the Huelva estuary is where the highest metal concentrations are found in sediments and in the two bivalve species. This is not surprising, considering that the Huelva estuary is the mouth of the Tinto and Odiel rivers, which have one of the highest levels of metal pollution of all the rivers of Europe. The two species of bivalves have different amounts of metals in their tissues. The concentrations of Cr, Cu, Pb, Zn, As and Hg in D. trunculus were significantly higher (p < 0.05) than in C. gallina; however, C. gallina contained more Ni and Cd. In both species the most abundant elements were Cu and Zn, while Hg showed the lowest values. There is a significant correlation (p < 0.05) for concentrations of Cu, Pb, Zn and Hg in D. trunculus and C. gallina relative to their concentrations in surface sediments.

Optimization of Allium sativum solvent extraction for the inhibition of in vitro growth of Helicobacter pylori.

Helicobacter pylori (Hp) is the bacterium responsible for serious gastric diseases such as ulcers and cancer. The work described here involved the study of the inhibitory power of Allium sativum extracts against the in vitro growth of Hp (Hp ivg). We used purple garlic of the "Las Pedroñeras" variety for this study. The effects of two different extraction methods (Soxhlet, stirred tank extractor) and four solvents with different characteristics (water, acetone, ethanol, and hexane) were investigated in terms of the efficiency of the extraction process. Satisfactory results were obtained in most cases in the activity tests, indicating that different extracts gave rise to good inhibitory activity against Hp ivg. The extracts that showed the highest bacteriostatic activities were selected to evaluate the influence of the most important operation variables on the extraction yield: stirring speed, operation time, garlic conditioning, and garlic storage time. The best results were obtained using ethanol and acetone as solvents in a stirred tank. The inhibitory powers of these extracts were compared to those shown by some commercial antibiotics used in the medical treatment of Hp infections. The results of this study show that garlic extracts produce levels of inhibition similar to those of the commercial materials. These extracts were also tested against other common bacteria, and equally satisfactory results were obtained. The research described here represents an important starting point in the fight against and/or prevention of peptic ulcers, as well as other pathologies associated with Hp infections such us gastric cancer. The extracted material can be used by direct application and involves a simple and economical extraction procedure that avoids isolation or purification techniques.

Thermal degradation of allicin in garlic extracts and its implication on the inhibition of the in-vitro growth of Helicobacter pylori.

Allicin, the main active principle related to Allium sativum chemistry, is considered to be responsible for the bacteriostatic properties of garlic. The work described here has demonstrated the direct implication of the allicin present in solvent-free garlic extracts obtained with ethanol (ethanolic garlic extract, EGE) and acetone (acetonic garlic extract, AGE) in the inhibition of the in-vitro growth of Helicobacter pylori (Hp), the bacterium responsible for serious gastric diseases such as ulcers and even gastric cancer. The evolution of allicin concentration as a function of time and temperature has been the subject of a kinetic study. The reaction order, activation energy, and preexponential factor (in accordance with Arrhenius theory) have been determined for the decomposition process of allicin in these organic media. First-order decomposition, an activation energy of 97.4 kJ/mol, and an Arrhenius preexponential factor of 8.9 x 10(10) s(-1) have been determined for allicin in EGE. For allicin in AGE the kinetic order determined was 1.5, the activation energy 184.5 kJ/mol, and the preexponential factor 3.1 x 10(24) s(-1) (mg/L)(-0.5). The presence or absence of allicin in these garlic products was found to be crucial for the inhibition of the in-vitro growth of Hp, as demonstrated by microbiological analysis for AGE. A relationship has been identified between the effectiveness and durability of the anti-Hp properties shown by AGE and the allicin content of these products. The bacteriostatic properties were active for up to 10 months if the samples were maintained at 6 degrees C.

Allyl-thiosulfinates, the bacteriostatic compounds of garlic against Helicobacter pylori.

Allicin and allyl-methyl plus methyl-allyl thiosulfinate from acetonic garlic extracts (AGE) have been isolated by high-performance liquid chromatography. These compounds have shown inhibition of the in vitro growth of Helicobacter pylori (Hp), the bacterium responsible for serious gastric diseases such as ulcers and even gastric cancer. A chromatographic method was optimized and used to isolate these thiosulfinates. The method developed has allowed the isolation of natural thiosulfinates extracted from garlic by organic solvents and is an easy and cheap methodology that avoids complex synthesis and purification procedures. The capacity and effectiveness of isolated natural thiosulfinates have been tested, and this has enabled the identification of the main compounds responsible for the bacteriostatic activity shown by AGE origin of these kinds of organosulfur compounds along with ethanolic garlic extracts (EGE). Additionally, microbiological analyses have suggested that these compounds show a synergic effect on the inhibition of the in vitro growth of Hp. The results described here facilitate the process of obtaining garlic extracts with optimal bacteriostatic properties. The product is obtained in a way that avoids expensive purification methods and will allow the design of live tests with the aim of investigating the potential for the use of these garlic derivatives in the treatment of patients with Hp infections.

Partitioning of metals in sediments from the Odiel River (Spain).

The Odiel River runs through an area known as the Iberian pyrite belt in the province of Huelva in the southwestern corner of the Iberian Peninsula. Its waters are acidic and contain large amounts of heavy metals from erosion and mining activity. In addition, the Odiel River estuary is one of the most industrialized areas in southern Spain and, consequently, receives the discharge of industrial and urban waste. Seventeen sediment samples from the Odiel and its main tributaries were analyzed for this study. The chemical partitioning of metals (Cu, Zn, Cd, Pb, Fe, Ni, Cr and Co) in each sample was determined in four fractions (acid-soluble, reducible, oxidizable and residual). The total content of each of the metals was also determined. The results showed high concentrations of Fe, Cu, Zn, Pb and Cd, undoubtedly as a result of contamination from the mining and industrial activity mentioned above. However, the concentrations of the other metals analyzed were low since there are no sources of pollution by them in the Odiel River. Based on the chemical distribution of metals, we found that Cd, Zn and Cu are the most mobile metals (i.e., metals that can pass easily into the water under changing environmental conditions). Cd is the metal that showed the highest percentages in the acid-soluble fraction (the most labile) and the lowest in the residual fraction. However, Pb, Fe, Cr and Ni are present in the greatest percentages in the residual fraction, which implies that these metals are strongly linked to the sediments.

Heavy metals in fish (Solea vulgaris, Anguilla anguilla and Liza aurata) from salt marshes on the southern Atlantic coast of Spain.

Comparisons were made of metal concentrations in water and sediment with those in the muscle and livers of three species of fish caught in four seawater reservoirs (two in the Odiel estuary and two in the Bay of Cádiz). The concentrations of a large number of metals in the water, sediment and fish were higher in the Odiel reservoirs than in those in Cádiz. We found high metal-enrichment factors in the livers as compared to muscle (over 100 for Fe and Cu and around 5 for Zn, Ni, Cd and Pb). The three fish species showed differences in metal content. For example, eels accumulated more metals in muscle and grey mullet in the liver. The metal levels found in muscle in the three species were below the legal limits for human consumption, although Cu in the liver was above the limit in the three species taken from the Odiel reservoirs, and Zn levels in liver were over the maximum in eels from one of the Odiel marshes. Significant correlations (p<0.05) were obtained for the levels of numerous metals in water, sediment and fish.

Heavy metal distribution in marine sediments from the southwest coast of Spain.

The latest version (1999) of the BCR-sequential extraction technique was used to determine the distribution of metals (Cu, Zn, Cd, Pb, Fe, Ni, Cr, and Mn) with major sedimentary phases (acid-soluble, reducible, oxidisable and residual) in samples from the southwest coast of Spain. The total metal content was also determined. The results showed that an extensive area along the coast (35 km long), near the joint mouth of the Tinto and Odiel Rivers, contains sediments with high concentrations of metals, with maximums of 649 mg/kg of Zn, 336 mg/kg of Cu, 197 mg/kg of Pb and 2.5 mg/kg of Cd. The values found for Cr, Ni and Mn are comparable to those in unpolluted areas. Based on the chemical distribution of metals, we found that Zn is the most mobile (i.e., it can pass easily into the water under changing environmental conditions). This metal showed the highest percentages in the acid-soluble fraction (the most labile), especially in the central coastal area, where the samples contained over 50% of this element associated with this fraction. This area close to the Tinto and Odiel river mouths also shows a significant increase in the mobility of Cd and Cu. In both cases the amount present in the residual fraction is lower, and the acid-soluble fraction is increased for Cd and the oxidisable fraction for Cu. However, the highest percentages of Fe, Cr and Ni are found in the residual fraction (84%, 89% and 75%, respectively), which implies that these metals are strongly bound to the sediments.

Copolymerization of D,L-lactide and glycolide in supercritical carbon dioxide with zinc octoate as catalyst.

The objective of the study is the development of a new technique based on supercritical technology for the production of bioabsorbable polymeric microparticles containing pharmaceutical principles, for their use in the controlled release of medicines. For this purpose, the ring-opening copolymerization of D,L-lactide and glycolide in supercritical carbon dioxide, using zinc(II) 2-ethylhexanoate (ZnOct2) as catalyst, was studied. The polymer obtained with ZnOct2 has similar characteristics to that obtained with stannous octoate (SnOct2), the conventional catalyst used up to now for this kind of polymerization process. Experiments were performed at various reaction times, pressures, and stirring rates. The most outstanding result was found by varying the stirring rate, where particles forming agglomerates seem to be obtained at the greater agitation levels.

Study of fractionation and potential mobility of metal from the Guadalquivir estuary: changes in mobility with time and influence of the aznalcollar mining spill.

This study analyzed the evolution of metal levels in surface sediments from the Guadalquivir estuary between 1997 and 2002 and assessed the impact of the April 1998 Aznalcollar tailings dam failure and subsequent cleanup on metal concentrations in surface sediments. We also analyzed metal distribution with time in five chemical fractions of the surface sediments: exchangable, carbonate, reducible, oxidizable, and residual. This work showed that the April 1998 Aznalcollar tailings dam failure caused a considerable increase in the concentrations of Cd, Zn, Cu, and Pb in the surface sediments of the Guadalquivir estuary. It was also found there was a change in the distribution of Cd, Zn, Cu, and Pb between the chemical fractions of the sediment after the failure of the dam. There were increases in the percentages of Cd in the carbonate fraction, of Zn and Pb in the reducible fraction, and of Cu in the oxidizable fraction, whereas the percentages of these elements associated with the residual fraction decreased. In 1999, there was a decrease in the metal concentrations in the surface sediments from the Guadalquivir estuary, and by 2000 the chemical distribution and metal levels had returned to levels similar those before the dam failure.