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.

Stilbenes: Characterization, bioactivity, encapsulation and structural modifications. A review of their current limitations and promising approaches.

Stilbenes are phenolic compounds naturally synthesized as secondary metabolites by the shikimate pathway in plants. Research on them has increased in recent years due to their therapeutic potential as antioxidant, antimicrobial, anti-inflammatory, anticancer, cardioprotective and anti-obesity agents. Amongst them, resveratrol has attracted the most attention, although there are other natural and synthesized stilbenes with enhanced properties. However, stilbenes have some physicochemical and pharmacokinetic problems that need to be overcome before considering their applications. Human clinical evidence of their bioactivity is still controversial due to this fact and hence, exhaustive basis science on stilbenes is needed before applied science. This review gathers the main physicochemical and biological properties of natural stilbenes, establishes structure-activity relationships among them, emphasizing the current problems that limit their applications and presenting some promising approaches to overcome these issues: the encapsulation in different agents and the structural modification to obtain novel stilbenes with better features. The bioactivity of stilbenes should move from promising to evident.

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.

Development if healthy milk and yogurt products for reducing metabolic diseases using cyclodextrin and omega-3 fatty acids from fish oil.

The food industry is constantly attempting to develop better products that will have a positive effect on health. Feiraco® and Clesa®, expressed their intention to create novel products using UNICLA® milk as a matrix to develop functional foods. In this respect, ß-cyclodextrin (ß-CD) at 1% was able to reduce the cholesterol concentration in Feiraco-UNICLA® milk products by around 87-85%. Products were fortified with omega-3 from fish oil with α- and ß-CD acting as carriers. It was possible to add around 50% of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) Recommended Dietary Allowances (RDA), with a high diet proportion of fibre and similar organoleptic properties to commercial omega-3 products. 80% of a sensory panel found our formulations satisfactory. The final product was stable, and the bioaccessibilty of the fatty acids added to the milk was around 74%. These results as a whole satisfy the aid of Feiraco® and Clesa® to develop improved products.

Formation of carboxylated and decarboxylated betalains in ripening grains of Chenopodium quinoa by a dual dioxygenase.

Chenopodium quinoa (quinoa) is a pseudo-cereal that forms part of the cultural heritage of Andean countries, and its grains have high nutritional value and potential health benefits. Betalains are nitrogenous water-soluble pigments and bioactive molecules that contribute to these health-promoting properties. Betalains are restricted to plants of the order Caryophyllales, to which quinoa belongs. A new family of betalains has been discovered in the form of unconventional decarboxylated pigments. Here, we show that these pigments accumulate in ripening quinoa grains of fluorescent nature, and are putatively based on a dopamine-cleaving activity. This study describes for the first time the purification and molecular and functional characterization of a 4,5-dopamine extradiol dioxygenase enzyme from plants. It is a monomeric protein with a molecular mass of 34.5 kDa characterized by chromatography, electrophoresis, and time-of-flight mass spectrometry. We demonstrate that this key enzyme has a dual function in a square-shaped biosynthetic pathway towards the formation of both carboxylated and decarboxylated pigments. Enzyme kinetic properties are characterized for the production of 6-decarboxy-betalamic acid and 3,4-dihydroxy-l-phenylalanine-derived betalamic acid, the two structural units of plant pigment in nature. The profile of multiple betalains present in quinoa grains has been reproduced in one-pot bioreactors containing the novel enzyme and two competing substrates.

The use of cyclodextrins as solubility enhancers in the ORAC method may cause interference in the measurement of antioxidant activity.

The ability of cyclodextrins to enhance the water solubility of lipophilic compounds is used to modify the water-based Oxygen Radical Absorbance Capacity (ORAC) method to measure antioxidant activity in vitro. However, the use of these solubility enhancers may alter fluorescent readings, which has led to contradictory results being described in the literature. The low specificity of these oligosaccharides and their controlled release effect can result in cyclodextrins forming inclusion complexes with other reagents in the assay, changing the kinetics. In this study, the cause of cyclodextrins' interference in the ORAC method is evaluated through a physicochemical and computational approach. Cyclodextrins showed a clear increase in the fluorescent signal both in the presence and absence of the antioxidant oxyresveratrol, the precise effect being dependent on the type and concentration of cyclodextrin. Although the glucidic nature of cyclodextrins could play a minimal role in this effect, it seems that the main cause was the encapsulation of other substrates in the reaction, fluorescein and AAPH.

Molecular encapsulation and bioactivity of gnetol, a resveratrol analogue, for use in foods.

BACKGROUND: Gnetol is a stilbene whose characterization and bioactivity have been poorly studied. It shares some bioactivities with its analogue resveratrol, such as anti-inflammatory, anti-thrombotic, cardioprotective and anti-cancer activities. However, the low solubility of stilbenes may limit their potential applications in functional foods. Encapsulation in cyclodextrins could be a solution. RESULTS: The antioxidant activity of gnetol was evaluated by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation and ferric reducing antioxidant power methods (Trolox equivalents 13.48 µmol L-1 and 37.08 µmol L-1 respectively at the highest concentration) and it was higher than that of resveratrol, and depending on the method, similar or higher to that of oxyresveratrol. Spectrophotometric and spectrofluorimetric characterization of gnetol is published for the first time. Moreover, its water solubility was determined and improved almost threefold after its molecular encapsulation in cyclodextrins, as well as its stability after storage for a week. A physicochemical and computational study revealed that cyclodextrins complex gnetol in a 1:1 stoichiometry, with better affinity for like 2-hydroxypropyl-ß-cyclodextrin (KF  = 4542.90 ± 227.15 mol-1  L). Temperature and pH affected the encapsulation constants. CONCLUSION: These results could increase interest of gnetol as an alternative to the most studied stilbene, resveratrol, as well as aid in the development of more stable inclusion complexes that improve its aqueous solubility and stability so that it can be incorporated into functional foods. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

Enhanced production of 2-phenylethanol by salicylic acid and cyclodextrins in cell suspension cultures of the unexplored filamentous fungus Monochaetinula geoffroeana.

BACKGROUND: 2-Phenylethanol (PEA) is a higher aromatic alcohol with a rose-like odor, which is used in several industries. Although PEA can be synthesized, consumers are increasingly concerned about the toxicity of chemically synthesized products, and prefer natural aroma compound. PEA occurs naturally in the environment but concentrations are too low to justify extraction. RESULTS: The present study offers a novel biological source of PEA: the filamentous fungi Monochaetinula geoffroeana. We report the highest recorded yield of PEA of fungal origin to date: 6.52 g L-1 . The volatility and low water solubility of PEA can affect its use in many industries, for which reason complexation studies of PEA and cyclodextrins were carried out using the phase solubility technique. PEA formed 1:1 stoichiometric inclusion complexes with natural and modified CDs, the highest encapsulation constant being obtained with MßCD (K1:1  = 299.88 L mol-1 ). The complexation process significantly increased the water solubility of PEA. A computational study showed a high degree of correlation between computed scores and experimental values. Furthermore, this study reports the role of salicylic acid as an effective elicitor for improved PEA production by the studied fungi. Supplementation with 10 µmol L-1 salicylic acid increased PEA production from 6.52 to 10.54 g L-1 . CONCLUSION: The best treatment to enhance PEA production by M. geoffroeana under laboratory conditions was to use salicylic acid 10 µmol L-1 . Due to the commercial importance of PEA, further investigation is needed to improve PEA production by M. geoffroeana and to optimize culture conditions in order to standardize yields. © 2021 Society of Chemical Industry.

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.

A dopamine-based biosynthetic pathway produces decarboxylated betalains in Chenopodium quinoa.

Betalains are the nitrogenous pigments that replace anthocyanins in the plant order Caryophyllales. Here, we describe unconventional decarboxylated betalains in quinoa (Chenopodium quinoa) grains. Decarboxylated betalains are derived from a previously unconsidered activity of the 4,5-DOPA-extradiol-dioxygenase enzyme (DODA), which has been identified as the key enzymatic step in the established biosynthetic pathway of betalains. Here, dopamine is fully characterized as an alternative substrate of the DODA enzyme able to yield an intermediate and structural unit of plant pigments: 6-decarboxy-betalamic acid, which is proposed and described. To characterize this activity, quinoa grains of different colors were analyzed in depth by chromatography, time-of-flight mass spectrometry, and reactions were performed in enzymatic assays and bioreactors. The enzymatic-chemical scheme proposed leads to an uncharacterized family of 6-decarboxylated betalains produced by a hitherto unknown enzymatic activity. All intermediate compounds as well as the final products of the dopamine-based biosynthetic pathway of pigments have been unambiguously determined and the reactions have been characterized from the enzymatic and functional perspectives. Results evidence a palette of molecules in quinoa grains of physiological relevance and which explain minor betalains described in plants of the Caryophyllales order. An entire family of betalains is anticipated.

Study of the fluorescence and interaction between cyclodextrins and neochlorogenic acid, in comparison with chlorogenic acid.

Neochlorogenic acid, a less-studied isomer of chlorogenic acid, has been seen to posses antioxidant, antifungal, anti-inflammatory and anticarcinogenic effects, which makes it an interesting candidate for incorporation in functional foods. However, its poor solubility in water and susceptibility to oxidation make such a task difficult. To overcome that, its encapsulation in cyclodextrins (CDs) is proposed. The fluorescence of neochlorogenic acid in different pH conditions was analyzed, and caffeic acid was proved to be the fluorescent moiety in the molecule. An encapsulation model whereby the ligand poses two potential complexation sites (caffeic and D-(-)-quinic moieties), showed that α-CD and HP-ß-CD formed the best inclusion complexes with neochlorogenic acid, followed by M-ß-CD, ß-CD and γ-CD. Molecular docking with the two best CDs gave better scores for α-CD, despite HP-ß-CD providing stabilization through H-bonds. The encapsulation of chlorogenic acid led to a similar CD order and scores, although constants were higher for α-CD, ß-CD and M-ß-CD, lower for HP-ß-CD, and negligible for γ-CD. The protonation state affected these results leading to a different order of CD preference. The solubility and the susceptibility to oxidation of neochlorogenic acid improved after complexation with α-CD and HP-ß-CD, while the antioxidant activity of both isomers was maintained.

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.

Characterization of Resveratrol, Oxyresveratrol, Piceatannol and Roflumilast as Modulators of Phosphodiesterase Activity. Study of Yeast Lifespan.

Our desire to live longer has led to an ever-increasing number of novel antiaging products. However, few molecules have any real effect and new ones need to be studied before they can be used commercially. In this contribution, activation of the caloric restriction (CR) pathway was studied using different three (resveratrol, oxyresveratrol and piceatannol)-a family with demonstrated bioactivity on phosphodiesterase activity. The high-affinity phosphodiesterase type 2 (PDE2) of Saccharomyces cerevisiae was expressed in Escherichia coli, purified and characterized. The activity and the inhibitory activity of each stilbene was studied, and the findings were compared in vitro and in silico with those obtained with roflumilast-a human PDE4 inhibitor widely used in chronic obstructive pulmonary diseases. Finally, an in vivo chronological lifespan assay using WT S. cerevisiae and ΔPDE2 S. cerevisiae strains was carried out. It was demonstrated that stilbenes can modulate yPDE2 activity, increasing the lifespan of the yeast by 18% over a control (in combination with other pathways). In addition, roflumilast increased the lifespan in the WT strain. The findings as a whole would increase the range of lifespan products available and suggest novel uses for approved drugs.

Lifespan extension in Caenorhabditis elegans by oxyresveratrol supplementation in hyper-branched cyclodextrin-based nanosponges.

In this work, the increase of the Caenorhabditis elegans (C. elegans) lifespan extension using hyper-branched cyclodextrin-based nanosponges (CD-NS) complexing oxyresveratrol (OXY), and the possible inhibition of C. elegans phosphodiesterase type 4 (PDE4) were evaluated. The titration displacement of fluorescein was used to calculate the apparent complexation constant (KF) between CD-NS and OXY. Moreover, PDE4 was expressed in E. coli, purified and refolded in presence of cyclodextrins (CDs) to study its possible inhibition as pharmacological target of OXY. The apparent activity was characterized and the inhibitory effect of OXY on PDE4 displayed a competitive in vitro inhibition corroborated in silico. A maximum increase of the in vivo life expectancy of about 9.6% of using OXY/CD-NS complexes in comparison with the control was obtained, in contrast to the 6.5% obtained with free OXY. No effect on lifespan or toxicity with CD-NS alone was found. These results as a whole represent new opportunities to use OXY and CD-NS in lifespan products.

First description of betalains biosynthesis in an aquatic organism: characterization of 4,5-DOPA-extradiol-dioxygenase activity in the cyanobacteria Anabaena cylindrica.

The biosynthesis of betalamic acid, the structural unit of pigments betalains, is performed by enzymes with 4,5-DOPA-extradiol-dioxygenase activity. These enzymes were believed to be limited to plants of the order Caryophyllales and to some fungi. However, the discovery of Gluconacetobacter diazotrophicus as the first betalain-forming bacterium opened a new field in the search for novel biological systems able to produce betalains. This paper describes molecular and functional characterization of a novel dioxygenase enzyme from the aquatic cyanobacterium Anabaena cylindrica. The enzyme was found to be a homodimer of a polypeptide of 17.8 kDa that, opposite to previous related enzymes, showed a strong inhibition by excess of the precursor L-DOPA. However, its heterologous expression has allowed detecting the formation of the main compounds in the biosynthetic pathway of betalains. In addition, phylogenetic analysis has shown that this enzyme is not close related to enzymes from plants, fungi or proteobacteria such as G. diazotrophicus. The presence of enzymes that produce these health-promoting compounds is more diverse than expected. The discovery of this novel dioxygenase in the phylum cyanobacteria expands the presence of betalamic acid-forming enzymes in organisms of different nature with no apparent relationship among them.

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.

Recent advances in the treatment of Niemann pick disease type C: A mini-review.

Niemann Pick disease Type C (NPC) is a recessive rare disease caused by the mutation on NPC1 and/or NPC2 genes changing the processing of the Low-density proteins (LDL) resulting in an accumulation of lipids in the cells. Until today there is not a cure, the current treatment is based on palliative affairs to reduce the symptoms and prevent its appearance. Among all the treatments proposed the use of cyclodextrins (CDs), nanocarriers which can complex cholesterol, is one of the most useful alternatives. Indeed, for several years 2-hydroxypropyl-ß-CD (HPß-CD) is approved as orphan drug for FDA and EMA to the treatment. However, different CDs based materials are created each year to improve the cholesterol uptake. This review is focused on the novelty of CD based materials for NPC treatment.