Exploring in the classroom the relationship between alcohol intake and behavioral disorders through an animal model.

Alcohol consumption has profound effects on behavior, such as impaired judgment, addiction or even death. It is estimated that alcohol contributes to around three million deaths worldwide, 13.5% of them in young people with ages between 20 and 39 years. Consequently, it is necessary to raise awareness among college and high school students of the risk related to alcohol drinking. The small nematode Caenorhabditis elegans is an animal widely used as a model organism to study nearly all aspects of Biochemistry. It is a powerful tool to test the potential bioactivity and molecular mechanisms of natural compounds and drugs in vivo. Therefore, it is an interesting topic to include in an undergraduate course of Biotechnology, Biochemistry or Biology students among other scientific vocations. C. elegans is also used as a neurobiological model to evaluate substances´ neurotoxicity and behavioral effects. The proposed experiment introduces students to the handling of this preclinical model and to the evaluation of behavioral alterations induced by chemicals in scientific research. The effects of different doses of ethanol on C. elegans behavior are studied using a versatile chemotaxis assay. This laboratory experiment is suitable for an undergraduate course. The practical session can be used in the global strategies of information and awareness of educational centres to mitigate the impact of alcohol abuse among students, both in formal courses or in Science fairs or exhibitions.

Novel Re(I) Complexes as Potential Selective Theranostic Agents in Cancer Cells and In Vivo in Caenorhabditis elegans Tumoral Strains.

A series of rhenium(I) complexes of the type fac-[Re(CO)3(N^N)L]0/+, Re1-Re9, was synthesized, where N^N = benzimidazole-derived bidentate ligand with an ester functionality and L = chloride or pyridine-type ligand. The new compounds demonstrated potent activity toward ovarian A2780 cancer cells. The most active complexes, Re7-Re9, incorporating 4-NMe2py, exhibited remarkable activity in 3D HeLa spheroids. The emission in the red region of Re9, which contains an electron-deficient benzothiazole moiety, allowed its operability as a bioimaging tool for in vitro and in vivo visualization. Re9 effectivity was tested in two different C. elegans tumoral strains, JK1466 and MT2124, to broaden the oncogenic pathways studied. The results showed that Re9 was able to reduce the tumor growth in both strains by increasing the ROS production inside the cells. Moreover, the selectivity of the compound toward cancerous cells was remarkable as it did not affect neither the development nor the progeny of the nematodes.

Consumption of commonly used artificial food dyes increases activity and oxidative stress in the animal model Caenorhabditis elegans.

In recent decades, the consumption of artificial colorants in foods and beverages has increased despite of concerns in the general population raised by studies that have shown possible injurious effects. In this study, tartrazine, sunset yellow, quinoline yellow, ponceau 4R, carmoisine and allura red were employed as pure compounds to explore their effects in vivo in the animal model Caenorhabditis elegans. The exposition of C. elegans to these artificial dyes produced damage related with aging such as oxidative stress and lipofuscin accumulation, as well as a heavy shortening of lifespan, alterations in movement patterns and alterations in the production of dopamine receptors. Besides, microarray analysis performed with worms treated with tartrazine and ponceau 4R showed how the consumption of synthetic colorants is able to alter the expression of genes involved in resistance to oxidative stress and neurodegeneration.

Characterization of betalain-loaded liposomes and its bioactive potential in vivo after ingestion.

Betalains are plant pigments characterized by showing a wide range of beneficial properties for health. Its bioactive potential has been studied for the first time after its encapsulation in liposomes and subsequent administration to the animal model Caenorhabditis elegans. Phenylalanine-betaxanthin and indoline carboxylic acid-betacyanin encapsulated at concentrations of 25 and 500 µM managed to reduce lipid accumulation and oxidative stress in the nematodes. Highly antioxidant betalains dopaxanthin and betanidin were also included in the survival analyses. The results showed that phenylalanine-betaxanthin was the most effective betalain by increasing the lifespan of C. elegans by 21.8%. In addition, the administration of encapsulated natural betanidin increased the nematodes' survival rate by up to 13.8%. The preservation of the bioactive properties of betalains manifested in this study means that the stabilization of the plant pigments through encapsulation in liposomes can be postulated as a new way for administration in pharmacological and food applications.

Design, Synthesis and Gene Modulation Insights into Pigments Derived from Tryptophan-Betaxanthin, Which Act against Tumor Development in Caenorhabditis elegans.

The use of betalains, which are nitrogenous plant pigments, by the food industry is widespread and reflects their safety after intake. The recent research showed outstanding results for L-tryptophan-betaxanthin, a phytochemical present in traditional Chinese medicine, as an antitumoral agent when the activity was evaluated in the animal model Caenorhabditis elegans. Thus, L-tryptophan-betaxanthin is now presented as a lead compound, from which eleven novel structurally related betaxanthins have been designed, biotechnologically produced, purified, and characterized. The antitumoral effect of the derived compounds was evaluated on the JK1466 tumoral strain of C. elegans. All the tested molecules significantly reduced the tumoral gonad sizes in a range between 31.4% and 43.0%. Among the novel compounds synthesized, tryptophan methyl ester-betaxanthin and tryptophan benzyl ester-betaxanthin, which are the first betalains to contain an ester group in their structures, caused tumor size reductions of 43.0% and 42.6%, respectively, after administration to the model animal. Since these were the two most effective molecules, their mechanism of action was investigated by microarray analysis. Differential gene expression analysis showed that tryptophan methyl ester-betaxanthin and tryptophan benzyl ester-betaxanthin were able to down-regulate the key genes of the mTOR pathway, such as daf-15 and rict-1.

Bioactive potential and spectroscopical characterization of a novel family of plant pigments betalains derived from dopamine.

With two compounds first discovered in quinoa, an entire novel family of betalain pigments derived from dopamine is obtained and characterized. Betalains are nitrogenous water-soluble pigments and bioactive molecules with health-promoting effects and nutraceutical potential. It was assumed that all betalains contained betalamic acid as a structural unit derived from l-dihydroxyphenylalanine (l-DOPA). However, hitherto ignored compounds derived from dopamine have recently been discovered in nature. Here an entire family of betalains is described as decarboxylated pigments where 6-decarboxy-betalamic acid is the chromophoric and structural unit. This paper shows for the first time the production, purification and characterization of color and fluorescent properties of this novel family of pigments. Antioxidant and anti-aging effects of the just discovered betalains were tested in vivo using the animal model Caenorhabditis elegans. Some of them presented extraordinary properties, being glutamic acid-6-decarboxy-betaxanthin the most fluorescent molecule among both families of betalains. Methionine sulfoxide-6-decarboxy-betaxanthin is described as the most potent betalain in the reduction of oxidative stress in vivo in C. elegans (99.5 % at 25 µM) and dopa-6-decarboxy-betaxanthin increased the lifespan of the animal model up to 7.0 % at 25 µM. These results open new research lines in the search for molecules from plants with health-promoting properties and bioactivities.

Potent anticancer activity of a novel iridium metallodrug via oncosis.

Oncosis (from Greek ónkos, meaning "swelling") is a non-apoptotic cell death process related to energy depletion. In contrast to apoptosis, which is the main form of cell death induced by anticancer drugs, oncosis has been relatively less explored but holds potential to overcome drug resistance phenomena. In this study, we report a novel rationally designed mitochondria-targeted iridium(III) complex (OncoIr3) with advantageous properties as a bioimaging agent. OncoIr3 exhibited potent anticancer activity in vitro against cancer cells and displayed low toxicity to normal dividing cells. Flow cytometry and fluorescence-based assays confirmed an apoptosis-independent mechanism involving energy depletion, mitochondrial dysfunction and cellular swelling that matched with the oncotic process. Furthermore, a Caenorhabditis elegans tumoral model was developed to test this compound in vivo, which allowed us to prove a strong oncosis-derived antitumor activity in animals (with a 41% reduction of tumor area). Indeed, OncoIr3 was non-toxic to the nematodes and extended their mean lifespan by 18%. Altogether, these findings might shed new light on the development of anticancer metallodrugs with non-conventional modes of action such as oncosis, which could be of particular interest for the treatment of apoptosis-resistant cancers.

Polyphenols from traditional Chinese medicine and Mediterranean diet are effective against Aß toxicity in vitro and in vivo in Caenorhabditis elegans.

The potential of naturally occurring polyphenols as nutraceuticals to prevent and/or treat Alzheimer's disease is studied. Five structurally related flavones and four tyrosols were tested in vitro in human amyloid-ß peptide aggregation assays. The most promising compounds were two flavones, scutellarein and baicalein, and two tyrosols hydroxytyrosol and hydroxytyrosol acetate. These compounds caused a dose-dependent reduction of Aß-peptide aggregation up to 90% for the flavones and 100% for the tyrosols, at concentrations of 83.3 µM and 33.3 mM, respectively. The IC50 value obtained for scutellarein was 22.5 µM, and was slightly higher for baicalein, 25.9 µM, while for hydroxytyrosol and hydroxytyrosol acetate they were 0.57 mM and 0.62 mM. Given these results, the compounds were selected to conduct in vivo assays with the Caenorhabditis elegans animal model of Alzheimer's disease. The amyloid anti-aggregation ability of these polyphenols was demonstrated in in vivo aggregation assays in which 1 mM hydroxytyrosol reduced the amyloid plaques in the mutant strain CL2331 by 43%. The neuroprotective effect was evaluated in chemotaxis experiments carried out with transgenic strain CL2355 that expresses the human amyloid-ß peptide in the neurons. The chemotaxis index was improved by 240% when the neuron-impaired animals were treated with 1 mM hydroxytyrosol. The results indicate that the four molecules would be viable candidates to develop nutraceuticals that interfere in amyloid-ß peptide aggregation and, consequently, prevent and/or treat Alzheimer's disease.

Flavonoids' Effects on Caenorhabditis elegans' Longevity, Fat Accumulation, Stress Resistance and Gene Modulation Involve mTOR, SKN-1 and DAF-16.

Flavonoids are potential nutraceutical compounds present in diary food. They are considered health-promoting compounds and promising drugs for different diseases, such as neurological and inflammatory diseases, diabetes and cancer. Therefore, toxicological and mechanistic studies should be done to assert the biological effects and identify the molecular targets of these compounds. In this work we describe the effects of six structurally-related flavonoids-baicalein, chrysin, scutellarein, 6-hydroxyflavone, 6,7-dihydroxyflavone and 7,8-dihydroxyflavone-on Caenorhabditis elegans' lifespan and stress resistance. The results showed that chrysin, 6-hydroxyflavone and baicalein prolonged C. elegans' lifespan by up to 8.5%, 11.8% and 18.6%, respectively. The lifespan extensions caused by these flavonoids are dependent on different signaling pathways. The results suggested that chrysin's effects are dependent on the insulin signaling pathway via DAF-16/FOXO. Baicalein and 6-hydroxyflavone's effects are dependent on the SKN-1/Nfr2 pathway. In addition, microarray analysis showed that baicalein downregulates important age-related genes, such as mTOR and PARP.

Biosynthesis of a novel polymeric chitosan-betaxanthin and characterization of the first sugar-derived betalains and their effects in the in vivo model Caenorhabditis elegans.

Betaxanthins are nitrogenous plant pigments belonging to the family of betalains and they are known for their health-promoting effects and fluorescent properties. A novel biotechnological approach in the synthesis of these compounds has allowed the synthesis of high amounts of known betalains and of novel, tailor-made betalains through the condensation of the structural unit - betalamic acid - with amine groups of different compounds. Here we describe the synthesis and characterization of chitosan-betaxanthin, the first fluorescent polymeric betaxanthin which forms nanoparticles and that might combine the fluorescent properties of betalains and the properties of chitosan, a sugar polymer widely used with medical purposes. In addition, glucosamine, the structural unit of chitosan, and its stereoisomer galactosamine were shown to condense in solution with betalamic acid. This produced novel molecules with spectral and in vivo antioxidant and anti-aging properties similar to those of biological betaxanthins, which are the first sugar-derived betaxanthins described.

Loss of muscle strength in patients under hemodialysis evaluated by dynamometry in the Mexican population / Pérdida de fuerza muscular en pacientes en hemodiálisis evaluados mediante dinamometría en la población mexicana

INTRODUCTION: chronic kidney disease contributes to decreased muscle strength and physical function through a decrease in muscle mass. Current evidence suggests that hemodialysis can accentuate this complication, as well as lead to deterioration of the patient's overall health. The aim of this study is to compare muscle strength in a group of Mexican patients undergoing hemodialysis, evaluated by dynamometry, with available reference values. MATERIALS AND METHODS: a cross-sectional study was conducted in male and female patients between 20 and 81 years of age, with stage-5 chronic kidney disease, from the outpatient Hospital General Regional No 46 of the Mexican Social Security Institute. Muscle strength was assessed by means of a mechanical dynamometer. The average value classified by age and gender was compared with the 50th percentile of a reference study. Inter-group differences were calculated with the nonparametric Mann-Whitney U-test, and correlation using Pearson's test, logistic regression, and chi-squared test. All patients signed an informed consent form. RESULTS: a total of 150 patients, 97 (64.7 %) men and 53 (35.3 %) women, were included in the study. The mean dynamometric value for muscle strength was 21.5 ± 10.1 kg, and a significant correlation was found with age, weight, and hemoglobin concentration. CONCLUSION: patients undergoing hemodialysis treatment for chronic kidney disease were found to be at the 10th percentile for muscle strength, as measured by dynamometry, thus demonstrating a marked decrease in muscle strength. This result could, however, also have been affected by different variables such as patient age, height, weight, glomerular filtration rate (GFR), hemoglobin concentration, serum creatinine, serum glucose, and the subjective global assessment, given that a significant association was also found between these and muscle strength

INTRODUCCIÓN: la enfermedad renal crónica contribuye a disminuir la fuerza muscular y la función física a través de una disminución de la masa muscular. De acuerdo con la evidencia, la hemodiálisis puede acentuar esta complicación, así como llevar al paciente a un deterioro del estado general de salud. El objetivo de la investigación fue comparar la fuerza muscular de pacientes con hemodiálisis, evaluada mediante dinamometría en una población mexicana, con los valores de referencia. MATERIAL Y MÉTODOS: se realizó un estudio transversal en pacientes masculinos y femeninos de 20 a 81 años, con enfermedad renal crónica en estadio 5, del área de consulta externa del Hospital General Regional No 46 del Instituto Mexicano del Seguro Social. La fuerza muscular se evaluó por medio de un dinamómetro mecánico. El valor promedio clasificado por rango de edad y género se comparó con el percentil 50 de un estudio de referencia. Las diferencias intergrupales se calcularon con la prueba no paramétrica de la U de Mann-Whitney y la correlación mediante la prueba de Pearson. Todos los pacientes firmaron la carta de consentimiento informado. RESULTADOS: la muestra del estudio fue de 150 pacientes, 97 (64,7 %) hombres y 53 (35,3 %) mujeres. De acuerdo con la dinamometría, la media fue de 21,5 ± 10,1 kg; se demostró una correlación significativa entre la edad, el peso y la hemoglobina. CONCLUSIÓN: se encontró que los pacientes con enfermedad renal crónica sometidos a hemodiálisis se encontraban en el percentil 10 de fuerza muscular, medido por dinamometría, lo que demuestra una disminución marcada de dicha fuerza muscular. Sin embargo, este resultado también podría verse afectado por diferentes variables, como la edad del paciente, la altura, el peso, la tasa de filtración glomerular (TFG), la concentración de hemoglobina, la creatinina sérica, la glucosa sérica y la evaluación global subjetiva, dado que se encontró una asociación significativa entre estos factores y la fuerza muscular

Loss of muscle strength in patients under hemodialysis evaluated by dynamometry in the Mexican population.

INTRODUCTION: Introduction: chronic kidney disease contributes to decreased muscle strength and physical function through a decrease in muscle mass. Current evidence suggests that hemodialysis can accentuate this complication, as well as lead to deterioration of the patient's overall health. The aim of this study is to compare muscle strength in a group of Mexican patients undergoing hemodialysis, evaluated by dynamometry, with available reference values. Materials and methods: a cross-sectional study was conducted in male and female patients between 20 and 81 years of age, with stage-5 chronic kidney disease, from the outpatient Hospital General Regional No 46 of the Mexican Social Security Institute. Muscle strength was assessed by means of a mechanical dynamometer. The average value classified by age and gender was compared with the 50th percentile of a reference study. Inter-group differences were calculated with the nonparametric Mann-Whitney U-test, and correlation using Pearson's test, logistic regression, and chi-squared test. All patients signed an informed consent form. Results: a total of 150 patients, 97 (64.7 %) men and 53 (35.3 %) women, were included in the study. The mean dynamometric value for muscle strength was 21.5 ± 10.1 kg, and a significant correlation was found with age, weight, and hemoglobin concentration. Conclusion: patients undergoing hemodialysis treatment for chronic kidney disease were found to be at the 10th percentile for muscle strength, as measured by dynamometry, thus demonstrating a marked decrease in muscle strength. This result could, however, also have been affected by different variables such as patient age, height, weight, glomerular filtration rate (GFR), hemoglobin concentration, serum creatinine, serum glucose, and the subjective global assessment, given that a significant association was also found between these and muscle strength.

INTRODUCCIÓN: Introducción: la enfermedad renal crónica contribuye a disminuir la fuerza muscular y la función física a través de una disminución de la masa muscular. De acuerdo con la evidencia, la hemodiálisis puede acentuar esta complicación, así como llevar al paciente a un deterioro del estado general de salud. El objetivo de la investigación fue comparar la fuerza muscular de pacientes con hemodiálisis, evaluada mediante dinamometría en una población mexicana, con los valores de referencia. Material y métodos: se realizó un estudio transversal en pacientes masculinos y femeninos de 20 a 81 años, con enfermedad renal crónica en estadio 5, del área de consulta externa del Hospital General Regional No 46 del Instituto Mexicano del Seguro Social. La fuerza muscular se evaluó por medio de un dinamómetro mecánico. El valor promedio clasificado por rango de edad y género se comparó con el percentil 50 de un estudio de referencia. Las diferencias intergrupales se calcularon con la prueba no paramétrica de la U de Mann-Whitney y la correlación mediante la prueba de Pearson. Todos los pacientes firmaron la carta de consentimiento informado. Resultados: la muestra del estudio fue de 150 pacientes, 97 (64,7 %) hombres y 53 (35,3 %) mujeres. De acuerdo con la dinamometría, la media fue de 21,5 ± 10,1 kg; se demostró una correlación significativa entre la edad, el peso y la hemoglobina. Conclusión: se encontró que los pacientes con enfermedad renal crónica sometidos a hemodiálisis se encontraban en el percentil 10 de fuerza muscular, medido por dinamometría, lo que demuestra una disminución marcada de dicha fuerza muscular. Sin embargo, este resultado también podría verse afectado por diferentes variables, como la edad del paciente, la altura, el peso, la tasa de filtración glomerular (TFG), la concentración de hemoglobina, la creatinina sérica, la glucosa sérica y la evaluación global subjetiva, dado que se encontró una asociación significativa entre estos factores y la fuerza muscular.

Antitumoral Drug Potential of Tryptophan-Betaxanthin and Related Plant Betalains in the Caenorhabditis elegans Tumoral Model.

Betalains are plants pigments identified as potent antioxidant molecules, naturally present in foods like beetroot and prickly pears. Although activities described for betalain-containing formulations include cancer prevention and treatment, the use of extracts instead of purified pigments has avoided the investigation of the real chemopreventive and chemotherapeutic potential of these phytochemicals. Three betalain-rich extracts and six individual pure betalains were used in this work to characterize the activity and to explore possible molecular mechanisms. The animal model Caenorhabditis elegans (tumoral strain JK1466) was used to evaluate the effect of betalains as chemotherapeutics drugs. An objective evaluation method of tumor growth in C. elegans has been developed to assess the possible antitumoral activity of the different treatments. This protocol allowed a fast and reliable screening of possible antitumoral drugs. Among the betalains tested, tryptophan-betaxanthin reduced tumor size by 56.4% and prolonged the animal's lifespan by 9.3%, indicating high effectiveness and low toxicity. Structure-activity relationships are considered. Assays with mutant strains of C. elegans showed that the mechanism underlying these effects was the modulation of the DAF-16 transcription factor and the insulin signaling pathway. Our results indicate that tryptophan-betaxanthin and related betalains are strong candidates as antitumoral molecules in cancer treatment.

Betalain health-promoting effects after ingestion in Caenorhabditis elegans are mediated by DAF-16/FOXO and SKN-1/Nrf2 transcription factors.

Betalain-rich extracts have been used for many years by their nutraceutical potential. However, the study of their bioactivities has always been hampered by their difficult obtention. To explain their mode of action, seventeen pure betalains were tested in vivo using the animal model C. elegans. Four betalains, named indicaxanthin, indoline carboxylic acid-betacyanin, phenylalanine-betaxanthin, and dopaxanthin, behaved as extraordinary in vivo antioxidants and anti-aging compounds, by increasing the lifespan of C. elegans up to 16.82%, 16.65%, 16.53%, and 12.93%, respectively. The first microarrays performed with betalains and biological confirmation with different mutant strains showed that this life extension is due to a reduction of oxidative stress and the activation of the transcription factors DAF-16/FOXO and SKN-1/Nrf2. They are involved in longevity and oxidative stress resistance pathways and lead to overexpression of HSPs genes, involved in resistance to cancer and Alzheimer's, opening novel research lines in the search for effective plant-based treatments.

A Way to Increase the Bioaccesibility and Photostability of Roflumilast, a COPD Treatment, by Cyclodextrin Monomers.

Roflumilast is an orally available inhibitor of phosphodiesterase (PDE) type 4, which is widely used in chronic obstructive pulmonary diseases. However, it has low solubility and adverse effects include diarrhea and nausea. Since its solubilization may improve treatment and, dismissing any adverse effects, its interaction with cyclodextrins (CDs) was studied. The Higuchi-Connors method was used to determine the complexation constant with different CDs, pH values and temperatures. Molecular docking was used to predict interaction between the complexes. An in vitro digestion experiment was carried out to test roflumilast protection. Finally, the photostability of the complex was evaluated. The complex formed with ß-CD had the highest K11 value (646 ± 34 M-1), although this value decreased with increasing temperature. Similarly, K11 decreased as the pH increased. In vitro digestion showed that CDs protect the drug during digestion and even improve its bioaccessibility. Finally, CDs reduced the drug's extreme photosensitivity, originating a fluorescence signal, which is described for first time. The kinetic parameters of the reaction were obtained. This study not only completes the complexation study of roflumilast-CD, but also points to the need to protect roflumilast from light, suggesting that tablets containing the drug might be reformulated.

Extension of life-span using a RNAi model and in vivo antioxidant effect of Opuntia fruit extracts and pure betalains in Caenorhabditis elegans.

Betalains are nitrogenous plant pigments known for their high antioxidant capacity. For the first time, this antioxidant nature has been studied in an in vivo system using the model organism Caenorhabditis elegans. The oxidative stress caused in the fluorescent strain TJ375 (hsp-16.2::GFP) was reversed by the presence of both natural and semi-synthetic betalains, with an ED50 value around 25 µM for betacyanins and up to 10 µM for betaxanthins, with indicaxanthin, the major pigments in prickly pear fruits, as the most effective betalain. The effect of model betalains on the lifespan of the wild-type N2 strain was carefully studied using the automatic platform "Lifespan Machine". In a search for different approaches to suppress progeny, pop-1 RNAi was used to avoid FUdR use. The presence of betalains in the medium, both as pure compounds and as enriched Opuntia extracts significantly increased the lifespan of C. elegans.

Improving the production, activity, and stability of CLEAs with diepoxides.

Among enzyme immobilization techniques, the preparation of cross-linked enzyme aggregates has shown promising results in biocatalysis, because they are easy to prepare, versatile, and cheap. The method involves the precipitation of enzymes with ammonium sulfate or an organic solvent and subsequent cross-linking with glutaraldehyde. However, the Schiff base produced with glutaraldehyde is reversible and can be broken with acids or bases, releasing proteins to the reaction medium. To solve this problem, we propose replacing glutaraldehyde with diepoxide compounds to obtain an irreversible secondary amine bond. Such a substitution avoids protein leakage during the biocatalytic process, contamination of the final products, and loss of enzyme. It also improves the synthesis of the biocatalyst, because, while the Schiff base is favored at mildly acidic pH, the epoxide reaction can be made at the optimal enzyme pH, assuring its structural stability and catalytic performance. The proposed method has been successfully used in the production and optimization of aldolase epoxy-cross-linked aggregates, which retain 98% activity. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1425-1429, 2017.

An improved method to measure lipase activity in aqueous media.

An improved method based on the p-nitrophenyl long chain esters method is proposed for measuring lipase hydrolytic activity in aqueous media. Using ethylene glycol as co-solvent for hydrophobic p-nitrophenyl substrates in aqueous buffer, lipase activity is measured by following the release of p-nitrophenol. This fast and easy to handle method improves the solubility of both substrate and product, and also the stability of the substrate. It avoids the use of solvents such as ethanol or propanol, permits the comparison of all the p-nitrophenol acyl ester substrates and allows the influence of ions like Ca+2 to be studied, while avoiding turbidity in the reaction medium.

An integral study of cyclodextrins as solubility enhancers of α-methylstilbene, a resveratrol analogue.

trans-α-Methylstilbene (tMS), a resveratrol analogue, has recently been studied in a search for new bioactivities. However, such studies do not take into account that the poor solubility of tMS in aqueous solutions could affect its bioactivity. For this reason, we propose, for the first time, using cyclodextrins (CDs) as solubilizers to increase tMS solubility, in aqueous solutions. The HPLC-RP results obtained, point to a 1 : 1 stoichiometry for all the natural (α-, ß- and γ-CD) and modified (HPßCD and MßCD) CDs tested. The KFapp (apparent formation constant) for the tMS-CD complexes was seen to be closely dependent on several factors, including the temperature and type of CD. Indeed, the highest KFapp value was obtained for MßCD, while the KFapp decreased with increasing temperature. In addition, the results showed negative entropy (-8.86 × 10-3 ± 0.40 kJ mol-1 K-1) and enthalpy (-16.70 ± 0.98 kJ mol-1) changes and a negative Gibbs free energy value at 25 °C (-14.00 ± 0.55 kJ mol-1) for the encapsulation process. A computational study carried out using molecular docking calculations showed a high degree of correlation between the computed scores and experimental values. Finally, the complexation of trans-stilbene and pinosylvin with HPßCD was compared with tMS.

Kinetic study of hydroxytyrosol oxidation and its related compounds by Red Globe grape polyphenol oxidase.

Red Globe grape polyphenol oxidase, partially purified using phase partitioning with Triton-X114, was used to study the oxidation of hydroxytytosol (HT) and its related compounds tyrosol (TS), tyrosol acetate (TSA), and hydroxytyrosol acetate (HTA). The enzyme showed activity toward both monophenols (monophenolase activity) and o-diphenols (diphenolase activity) with a pH optimum (pH 6.5) that was independent of the phenol used. However, the optimal temperature for diphenolase activity was substrate-dependent, with a broad optimum of 25-65 °C for HT, compared with the maximum obtained for HTA (40 °C). Monophenolase activity showed the typical lag period, which was modulated by pH, substrate and enzyme concentrations, and the presence of catalytic amounts of o-diphenols. When the catalytic power (Vmax/K(M)) was determined for both activities, higher values were observed for o-diphenols than for monophenols: 9-fold higher for the HT/TS pair and 4-fold higher for HTA/TSA pair. Surprisingly, this ratio was equally higher for TSA (2.2-fold) compared with that of TS, whereas no such effect was observed for o-diphenols. This higher efficiency of TSA could be related to its greater hydrophobicity. Acetyl modification of these phenols not only changes the kinetic parameters of the enzyme but also affects their antioxidant activity (ORAC-FL assays), which is lower in HTA than in HT.