The use of degrading enzymes in polymer formulation is a very attractive strategy to manage the end-of-life of plastics. However, high temperatures cause the denaturation of enzymes and the loss of their catalytic activity; therefore, protection strategies are necessary. Once protected, the enzyme needs to be released in appropriate media to exert its catalytic activity. A successful protection strategy involves the use of layered double hydroxides: cutinase, selected as a highly degrading polyester hydrolytic enzyme, is thermally protected by immobilization in Mg/Al layered double hydroxide structures. Different triggering media are here evaluated in order to find the best releasing conditions of cutinase from LDH. In detail, phosphate and citrate-phosphate buffers, potassium carbonate, sodium chloride, and sodium sulfate solutions are studied. After the comparison of all media in terms of protein release and activity retained, phosphate buffer is selected as the best candidate for the release of cutinase from LDH, and the effect of pH and concentration is also evaluated. The amount of the enzyme released is determined with the Lowry method. Activity tests are performed via spectrophotometry.
As a new generation of green solvents, deep eutectic solvents (DESs) are considered a promising alternative to current harsh organic solvents and find application in many chemical processing methods such as extraction and synthesis. DESs, normally formed by two or more components via various hydrogen bond interactions, offer high potential as medium for biocatalysis reactions where they can improve efficiency by enhancing substrate solubility and the activity and stability of the enzymes. In the current study, the stabilization of Humicola insolens cutinase (HiC) in natural deep eutectic solvents (NADESs) was assessed. The best hydrogen bond donor among sorbitol, xylitol, erythritol, glycerol and ethylene glycol, and the best acceptor among betaine, choline chloride, choline acetate, choline dihydrogen citrate and tetramethylammonium chloride, were selected, evaluating binding energies and molecular orientations through molecular docking simulations, and finally used to prepare NADES aqueous solutions. The effects of component ratio and NADES concentration on HiC thermostability at 90 degreesC were also investigated. The choline dihydrogen citrate:xylitol, in a 1:1 ratio with a 20 wt% concentration, was selected as the best combination in stabilizing HiC, increasing its half-life three-fold.
OBJECTIVE: Consumption of energy-dense palatable "comfort" food can alleviate stress and negative emotions, while abrupt withdrawal from a palatable diet can worsen these symptoms, causing difficulties with adherence to weight-loss diets. Currently, no pharmacological treatment is effective for obesity-related anxiety, so we investigated the endocannabinoid system (ECS), and specifically the fatty acid amide hydrolase (FAAH), as an interesting emerging target in this context because of its key role in the regulation of both energy homeostasis and emotional behavior. METHODS: Rats were subjected to exposure and subsequent abstinence from a palatable cafeteria diet. During abstinence period, rats were treated with the selective FAAH inhibitor PF-3845 (10 mg/kg; intraperitoneal administration every other day). RESULTS: Abstinent rats displayed an anxiogenic-like behavior and changes in the proteins of ECS signaling machinery in brain areas involved both in anxiety and food intake regulation. In particular, withdrawal caused a reduction of the expression of cannabinoid receptors in the nucleus accumbens and of enzymes diacylglycerol lipase alpha and monoacylglycerol lipase (MAGL) in the amygdala. Pharmacological inhibition of FAAH exerted an anxiolytic-like effect in abstinent animals and increased both MAGL expression in amygdala and CB2 expression in prefrontal cortex. DISCUSSION: Overall, our results suggest that emotional disturbances associated with dieting are coupled with region-specific alterations in the cerebral expression of the ECS and that the enhancement of the endocannabinoid signaling by FAAH inhibition might represent a novel pharmacological strategy for the treatment of anxiety related to abstinence from palatable food. PUBLIC SIGNIFICANCE: The present study focused on evaluating the role of the endocannabinoid system in modulating withdrawal from naturally rewarding activities that have an impact on mood, such as feeding. The variations observed in the emotional behavior of abstinent rats was linked to neuroadaptations of the ECS in specific brain areas.
Title: Enzymatic Degradation of the Most Common Aliphatic Bio-Polyesters and Evaluation of the Mechanisms Involved: An Extended Study Rosato A, Romano A, Totaro G, Celli A, Fava F, Zanaroli G, Sisti L Ref: Polymers (Basel), 14:, 2022 : PubMed
Commercial hydrolytic enzymes belonging to different subclasses (several lipases, proteinase k, cutinase) were investigated for their ability to degrade different aliphatic polyesters, i.e., poly(butylene succinate) (PBS), poly(butylene succinate-co-adipate) (PBSA), two poly(caprolactone), having two different molecular weights, poly(lactic acid) (PLA) and poly(propylene carbonate) (PPC). The enzyme screening was first carried out by investigating the capacity of fully degrading the target polymers in 24 h, then weight loss measurements of selected polyesters and target enzymes were performed. Solid residues after enzyme degradation were characterized by proton nuclear magnetic resonance ((1)H NMR), gel permeation chromatography (GPC), infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and thermogravimetry (TGA). Liquid fractions were studied via GPC, (1)H NMR and high-performance liquid chromatography (HPLC). PCL and PBSA were found to be the most biodegradable polyesters, under the conditions used in this study. PBS was fully degraded only by cutinase, whereas none of the tested enzymes were able to completely degrade PLA and PPC, in the conditions assessed here. Cutinase exhibited the highest hydrolytic activity on PBSA, while lipase from Candida sp. (CALB) on low molecular weight PCL. Chemical analyses on residual solids showed that the enzymatic degradation occurred homogeneously from the surface through an erosion mechanism and did not significantly affect the macromolecular structure and thermal stability. Cleaving action mode for each enzyme (endo- and/or exo-type) on the different polyesters were also proposed based on the evaluation of the degradation products in the liquid fraction.
Title: Impact of Metallic Nanoparticles on In Vitro Culture, Phenolic Profile and Biological Activity of Two Mediterranean Lamiaceae Species: Lavandula viridis L'Her and Thymus lotocephalus G. Lopez and R. Morales Goncalves S, Mansinhos I, Rodriguez-Solana R, Pereira-Caro G, Moreno-Rojas JM, Romano A Ref: Molecules, 26:, 2021 : PubMed
Nanoparticles (NPs) recently emerged as new chemical elicitors acting as signaling agents affecting several processes in plant metabolism. The aim of this work was to investigate the impact of the addition of copper oxide (CuO), zinc oxide (ZnO) and iron oxide (Fe(3)O(4)) NPs (<100 nm) at different concentrations (1, 5 and 10 mg/L) to the culture media on several morphological, physiological and -biochemical parameters of in vitro shoot cultures of Lavandula viridis L'Her and Thymus lotocephalus G. Lopez and R. Morales (Lamiaceae), as well as on phenolic profile and bioactivity (antioxidant and enzyme inhibition capacities). Although some decreases in shoot number and length were observed in response to NPs, biomass production was not affected or was improved in both species. Most NPs treatments decreased total chlorophyll and carotenoid contents and increased malondialdehyde levels, an indicator of lipid peroxidation, in both species. HPLC-HR-MS analysis led to the identification of thirteen and twelve phenolic compounds, respectively, in L. viridis and T. lotocephalus extracts, being rosmarinic acid the major compound found in all the extracts. ZnO and Fe(3)O(4) NPs induced an increase in total phenolic and rosmarinic acid contents in T. lotocephalus extracts. Additionally, some NPs treatments also increased antioxidant activity in extracts from this species and the opposite was observed for L. viridis. The capacity of the extracts to inhibit tyrosinase, acetylcholinesterase and butyrylcholinesterase enzymes was not considerably affected. Overall, NPs had a significant impact on different parameters of L. viridis and T. lotocephalus in vitro shoot cultures, although the results varied with the species and NPs type.
Title: Ultrasonic-Assisted Extraction and Natural Deep Eutectic Solvents Combination: A Green Strategy to Improve the Recovery of Phenolic Compounds from Lavandula pedunculata subsp. lusitanica (Chaytor) Franco Mansinhos I, Goncalves S, Rodriguez-Solana R, Ordonez-Diaz JL, Moreno-Rojas JM, Romano A Ref: Antioxidants (Basel), 10:, 2021 : PubMed
The present study aimed at evaluating the effectiveness of different natural deep eutectic solvents (NADES) on the extraction of phenolic compounds from Lavandula pedunculata subsp. lusitanica (Chaytor) Franco, on the antioxidant activity, and acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and tyrosinase (Tyr) inhibitory capacities. Ten different NADES were used in this research and compared with conventional solvents. Ultrasound-assisted extraction (UAE) for 60 min proved to be the best extraction condition, and proline:lactic acid (1:1) and choline chloride:urea (1:2) extracts showed the highest total phenolic contents (56.00 +/- 0.77 mg(GAE)/g(dw)) and antioxidant activity [64.35 +/- 1.74 mg(TE)/g(dw) and 72.13 +/- 0.97 mg(TE)/g(dw) in 2.2-diphenyl-1-picrylhydrazyl (DPPH) and 2.2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) methods, respectively]. These extracts also exhibited enzymes inhibitory capacity particularly against Tyr and AChE. Even so, organic acid-based NADES showed to be the best extractants producing extracts with considerable ability to inhibit enzymes. Twenty-four phenolic compounds were identified by HPLC-HRMS, being rosmarinic acid, ferulic acid and salvianolic acid B the major compounds. The results confirmed that the combination of UAE and NADES provide an excellent alternative to organic solvents for sustainable and green extraction, and have huge potential for use in industrial applications involving the extraction of bioactive compounds from plants.
BACKGROUND: Mentha pulegium is an aromatic and medicinal plant used in different Mediterranean herb-based liqueurs, whose phytochemical composition depends on the elaboration technique used. The aim of this work was to investigate differences in the sensory, chemical, and biological characteristics of pennyroyal liqueurs elaborated by hydro-alcoholic (HI) and aqueous (AI) infusions, percolation (P), maceration (M), and distillation (D) techniques, and by the mixture of spirit with pennyroyal essential oil (EO). The volatile composition determined by gas chromatography-mass spectrometry (GC-MS) and gas chromatography coupled to flame ionization detection (GC-FID), the total phenolic (TPC) and flavonoid (TFC) contents, the antioxidant (AC), and enzyme (alpha-glucosidase, tyrosinase, and acetylcholinesterase) inhibitory capacities, and the sensory analysis by napping, were evaluated in the liqueurs obtained. RESULTS: Monoterpenes and sesquiterpenes were the main volatiles present in liqueurs. The M, P, HI, and AI liqueurs showed the highest TPC, TFC, and AC values; and pulegone, the main hepatotoxic chemotype, was found in concentrations that gave rise to concern. The D and EO liqueurs stand out for their acceptable pulegone content and greater tyrosinase inhibition. Similar liqueur classifications were achieved by sensory analyses, and physico-chemical and biological analyses using multiple factor and principal component analyses, respectively. CONCLUSION: These results are an important contribution to the identification of the most appropriate elaboration conditions for herbal liqueurs with the healthiest chemical composition and functional properties. 2020 Society of Chemical Industry.
Title: Extraction of Antioxidants from Winemaking Byproducts: Effect of the Solvent on Phenolic Composition, Antioxidant and Anti-Cholinesterase Activities, and Electrochemical Behaviour Jara-Palacios MJ, Gonalves S, Heredia FJ, Hernanz D, Romano A Ref: Antioxidants (Basel), 9:, 2020 : PubMed
Extraction solvent is a very important factor in the recovery of antioxidants from natural matrices. In this study, the effect of three solvents (ethanol, ethanol/water and water) on the phenolic composition, antioxidant and anti-cholinesterase activities and electrochemical behaviour of four winemaking byproducts (seeds, skins, stems, and pomace) was evaluated. Phenolic composition was determined by the Folin-Ciocalteu method and ultra-high-performance liquid chromatography (UHPLC), antioxidant activity by the capacity to scavenge 2,2-diphenyl-1-picrylhydrazyl and hydroxyl radicals, anti-cholinesterase activity by the Ellman's method, and electrochemical behaviour by cyclic voltammetry. Eight phenolic compounds were quantified with higher content in water/ethanol extracts (e.g., epicatechin in pomace: 17 mg/100 g vs. 7 and 6 mg/100 g in ethanol and water extracts, respectively), although there were some exceptions (e.g., gallic acid in seeds was most abundant in water extracts). Moreover, the highest total phenolic content (TPC) and antioxidant activity were found in ethanol/water extracts (between 2 and 30-fold the values of the other extracts). Overall, the most active extracts in inhibiting both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes were ethanol/water and ethanol extracts from seeds (between 31.11 and 53.90%). The electrochemical behaviour allowed for differentiating the extracts depending on the solvent and the byproduct. Our findings indicate that winemaking byproducts represent a source of phenolic compounds with antioxidant and anti-cholinesterase activities and suggest that cyclic voltammetry is a promising technique to evaluate the phenolic extraction process from these byproducts.
In this work we evaluated methanolic extracts from different parts (leaves, seeds, fruit peel and pulp) of Chamaerops humilis L. for antioxidant activity and the ability to inhibit enzymes linked with neurodegenerative diseases: acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and tyrosinase (TYR). The total content of phenolics, flavonoids and condensed tannins was also determined. The antioxidant and inhibitory activities of the extracts varied significantly according to the tissue. Seed extracts showed the greatest ability to scavenge DPPH (IC50 = 81.28 microg mL(-1)) and ABTS (1440.42 micromolTE [Formula: see text]) and to reduce iron (1142.46 micromolAAE [Formula: see text]). Seed and peel extracts strongly inhibited AChE (IC50 = 660.16 and 653.68 microg mL(-1), respectively) and BChE (IC50 = 304.86 and 701.54 microg mL(-1), respectively). The strongest inhibition of TYR was achieved by the seed and pulp extracts (268.97 and 279.99 microg mL(-1), respectively). The highest levels of phenolics and condensed tannins were found in the seed extract (1564.88 micromolGAE [Formula: see text] and 170.00 micromolcE [Formula: see text], respectively) whereas the leaf extract was the richest in flavonoids (139.88 micromolQE [Formula: see text]). HPLC-DAD analysis indicated the presence of flavonoids and phenolic acids (hydroxycinnamic acids) in the leaf and pulp extracts. A high correlation was found between the total condensed tannins content and the antioxidant and enzyme inhibition activities, suggesting these compounds are responsible for the biological activity of the extracts. Overall, our results indicate that C. humilis extracts may provide a new and alternative source of agents for medical and industrial applications.
Title: Phenolic profile, antioxidant activity and enzyme inhibitory activities of extracts from aromatic plants used in Mediterranean diet Goncalves S, Moreira E, Grosso C, Andrade PB, Valentao P, Romano A Ref: J Food Sci Technol, 54:219, 2017 : PubMed
The antioxidant and enzyme inhibitory properties of methanolic extracts from four aromatic plants used in traditional medicine and food [Calamintha nepeta (L.) Savi subsp. nepeta, Helichrysum italicum subsp. picardii Franco, Mentha spicata L. and Origanum vulgare subsp. virens (Hoffmanns. & Link) Bonnier & Layens] were evaluated. The extract from O. vulgare exhibited the strongest DPPH (IC50 of 4.65 +/- 0.12 microg/ml) and ABTS (1479.56 +/- 12.29 micromolTE/gextract) scavenging capacities, as well as the largest ferric reducing ability (1746.76 +/- 45.11 micromolAAE/gextract). This extract also showed the highest total phenolic content (1597.20 +/- 24.10 micromolGAE/gextract) and although HPLC-DAD analysis revealed rosmarinic acid as the main compound of the extract, other compounds seem to be involved in the antioxidant activity. Furthermore, the extract from H. italicum, which was found to be rich in caffeoylquinic and dicaffeoylquinic acids and in pinocembrin, showed the highest inhibitory potential against acetylcholinesterase, tyrosinase and alpha-glucosidase. Overall, the results obtained validate the usefulness of the studied plants as valuable sources of natural agents beneficial for human health.
Title: In vitro antioxidant and inhibitory activity of water decoctions of carob tree (Ceratonia siliqua L.) on cholinesterases, alpha-amylase and alpha-glucosidase Custodio L, Patarra J, Albericio F, Neng NR, Nogueira JM, Romano A Ref: Nat Prod Res, 29:2155, 2015 : PubMed
This work reports the in vitro inhibitory activity of water decoctions of leaves, germ flour, pulp, locust bean gum and stem bark of carob tree on alpha-amylase, alpha-glucosidase, acetylcholinesterase and butyrylcholinesterase. The antioxidant activity and the chemical characterisation of the extracts made by spectrophotometric assays and by high-performance liquid chromatography are also reported. Leaves and stem bark decoctions strongly inhibited all the enzymes tested, had significant antioxidant activity and the highest total phenolics content. The major compounds were identified as gallic acid in the leaves and gentisic acid in the stem bark.
We investigated the metabolic profile and biological activities of the essential oil and polar extracts of Lavandula pedunculata subsp. lusitanica (Chaytor) Franco collected in south Portugal. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that oxygen-containing monoterpenes was the principal group of compounds identified in the essential oil. Camphor (40.6%) and fenchone (38.0%) were found as the major constituents. High-performance liquid chromatography with diode array detection (HPLC-DAD) analysis allowed the identification of hydroxycinnamic acids (3-O-caffeoylquinic, 4-O-caffeoylquinic, 5-O-caffeoylquinic and rosmarinic acids) and flavones (luteolin and apigenin) in the polar extracts, with rosmarinic acid being the main compound in most of them. The bioactive compounds from L. pedunculata polar extracts were the most efficient free-radical scavengers, Fe(2+) chelators and inhibitors of malondialdehyde production, while the essential oil was the most active against acetylcholinesterase. Our results reveal that the subspecies of L. pedunculata studied is a potential source of active metabolites with a positive effect on human health.
Lactobacillus sp. strain 30a (Lactobacillus saerimneri) produces the biogenic amines histamine, putrescine, and cadaverine by decarboxylating their amino acid precursors. We report its draft genome sequence (1,634,278 bases, 42.6% G+C content) and the principal findings from its annotation, which might shed light onto the enzymatic machineries that are involved in its production of biogenic amines.
Title: Inhibitory effect of Lavandula viridis on Fe(2+)-induced lipid peroxidation, antioxidant and anti-cholinesterase properties Costa P, Goncalves S, Andrade PB, Valentao P, Romano A Ref: Food Chem, 126:1779, 2011 : PubMed
In this research, the total phenolic content of a Lavandula viridis methanol extract was evaluated and the phenolic identification and quantification was assessed. Rosmarinic acid and luteolin-7-O-glucoside were the two major compounds identified by HPLC-DAD (ca. 39 and 13g/kg, respectively). This extract showed a strong antioxidant activity in ORAC (2858.39+/-70.97micromolTE/gextract) and TEAC (967.18+/-22.57micromolTE/gextract) assays, as well as Fe(2+) chelating and OH scavenging abilities. Furthermore, the extract prevented Fe(2+)-induced lipid peroxidation, by reducing MDA content in mouse brains (in vitro), and inhibited AChE and BChE activities both in vitro and in vivo. These findings demonstrate that the methanol extract from L. viridis is a potential source of natural antioxidants and cholinesterase inhibitors.
Observational studies in humans suggest that exposure to marijuana and other cannabis-derived drugs produces a wide range of subjective effects on mood tone and emotionality. These observations have their counterpart in animal studies, showing that cannabinoid agonists strongly affect emotional reactivity in directions that vary depending on dose and context. Based on these evidence, the activation of central CB(1) receptor has emerged as potential target for the development of antianxiety and antidepressant therapies. However, the variable effects of exogenous cannabinoid agonists have gradually shifted the interest to the alternative approach of amplifying the effects of endogenous cannabinoids (EC), namely anandamide (AEA) and 2-arachidonoylglycerol (2-AG), by preventing their deactivation. The enzyme fatty acid amide hydrolase (FAAH) has been the target of intense research efforts aimed at developing potent and selective inhibitors that might prolong AEA actions in vivo. Among the inhibitors developed, the compound URB597 was found to potently inhibit FAAH activity in vivo and cause brain AEA levels to increase. Interestingly, the enhanced AEA tone produced by URB597 does not result in the behavioral effects typical of a direct-acting cannabinoid agonist. Though URB597 does not elicit a full-fledged cannabinoid profile of behavioral responses, it does elicit marked anxiolytic-like and antidepressant-like effects in rats and mice. Such effects involve the downstream activation of CB(1) receptors, since they are attenuated by the CB(1) antagonist SR141716 (rimonabant). Parallel to FAAH inhibition, similar results can also be observed by pharmacologically blocking the AEA transport system, which is responsible of the intracellular uptake of AEA from the synaptic cleft. The reason why FAAH inhibition approach produces a smaller set of cannabimimetic effects might depend on the mechanism of EC synthesis and release upon neuronal activation and on the target selectivity of the drug. The mechanism of EC release is commonly referred to as "on request", since they are not synthesized and stored in synaptic vesicles, such as classical neurotransmitters, but are synthesized from membrane precursors and immediately released in the synaptic cleft following neuronal activation. The neural stimulation in specific brain areas, for example, those involved in the regulation of mood tone and/or emotional reactivity, would result in an increased EC tone in these same areas, but not necessarily in others. Therefore, inhibition of AEA metabolism activity could amplify CB(1) activation mainly where AEA release is higher. Furthermore, the inhibition of FAAH causes an accumulation of AEA but not 2-AG, which, being 200-fold more abundant than AEA in the brain, might differently modulate CB(1)-mediated behavioral responses. The evidence outlined above supports the hypothesis that the EC system plays an important role in anxiety and mood disorders and suggests that modulation of FAAH activity might be a pharmacological target for novel anxiolytic and antidepressant therapies.
The hydrolysis of (RS)-isopropylideneglycerol acetate with whole cells of the yeast Kluyveromyces marxianus is reported. The biotransformation furnished (R)-isopropylideneglycerol as major enantiomer with good enantioselectivity (E=28) under optimised conditions. The reaction can be performed in an ultrafiltration-membrane reactor allowing for the obtainment of 19.2 g/L of enantiomerically pure (R)-isopropylideneglycerol acetate starting from 60 g/L of racemic mixture.
