Enzymatic Oxidation of Ferulic Acid as a Way of Preparing New Derivatives.

The ferulic acid (FA)-oxidation by Myceliophthora thermophila laccase was performed in phosphate buffer at 30 °C and pH 7.5 as an eco-friendly procedure. LC-MS analysis showed that oxidation products were four dehydrodimers (P1, P2, P3, P5) at MM = 386 g/mol, two dehydrotetramers (P6, P7) at MM = 770 g/mol and one decarboxylated dehydrodimer (P4) at MM = 340 g/mol. Structural characterization showed that FA-dehydrodimers were symmetric for P1 and P5 while asymmetric for P2, P3 and P4. Physicochemical characterization showed that oxidation products presented a higher lipophilicity than that of FA. Moreover, symmetric dimers and tetra dimers had a higher melting point compared to FA and its asymmetric dimers. Antioxidant and anti-proliferative assessments indicated that enzymatic oligomerization increased antioxidant and anti-proliferative properties of oxidation products for P2, P3 and P6 compared to FA. Finally, this enzymatic process in water could produce new molecules, having good antiradical and anti-proliferative activities.

Green synthesis of glyco-phenol by enzymatic coupling between ferulic acid and glucosamine: An ecofriendly procedure.

A new glyco-phenol was produced by the coupling between glucosamine (Glu) and ferulic acid (FA) using Myceliophthora thermophila laccase as biocatalyst in mild conditions (distilled water and 30°C) as an environmentally friendly process. Results indicated that the enzymatic reaction created a new derivative (FA-Glu), produced from coupling between Glu and FA by covalent bonds. By the high production of (FA-Glu) derivative and its stability, the optimal ratio of (FA:Glu) was of (1:1) at optimal time reaction of 6 h. Under these optimal conditions, almost 55% of -NH2 groups on Glu were bound with FA oxidation products. The new derivative showed higher hydrophobic character than Glu due to the presence of FA in its structure. Liquid chromatography-mass spectrometry analysis showed that (FA-Glu) derivative exhibited a molecular mass at MM 713 g/mol containing one Glu molecule and three FA molecules after decarboxylation. Furthermore, the new derivative presented good antioxidant and antiproliferative activities in comparison with Glu and FA. These results suggest that the enzymatic conjugation between Glu and FA is a promising process to produce a new glyco-phenol having good functional properties for potential applications.

Genotoxic effects of food contact recycled paperboard extracts on two human hepatic cell lines.

Food contact paperboards may be a potential source of food contamination as they can release chemicals (intentionally added or not), especially recycled paperboards. This study assessed the in vitro genotoxicity of food contact paperboard samples from a manufacturer, collected at the beginning and at the end of a recycling production chain. Samples were extracted in water to mimic a wet food contact. Different genotoxic endpoints were evaluated in two human hepatic cell lines (HepG2 and HepaRG) using bioassays: γH2AX and p53 activation, primary DNA damage with the comet assay and micronucleus formation. It was found that the samples from the beginning and the end of the production chain induced, with the same potency, γH2AX and p53-ser15 activation and DNA damage with the comet assay. The micronucleus assay was negative with the paperboard extract from the beginning of the chain, whereas positive data were observed for the end paperboard extract. These results indicate that samples from recycled food contact paperboard can induce in vitro genotoxic effects in this study's experimental conditions.

Chitosan nanoparticles plus quercetin suppress the oxidative stress, modulate DNA fragmentation and gene expression in the kidney of rats fed ochratoxin A-contaminated diet.

This study aimed to evaluate the protective role of chitosan nanoparticles (COS-NPs) singly or plus quercetin (Q) against OTA-induced oxidative stress and renal genotoxicity. Twelve groups of male Sprague-Dawley rats were treated orally for 3 weeks included the control group, animals fed OTA-contaminated diet (3 mg/kg diet); COS-NPs-treated groups at low (140 mg/kg b.w.) or high (280 mg/kg b.w.) dose, Q-treated group (50 mg/kg b.w.), Q plus low or high dose of COS-NPs-treated groups and OTA plus Q and/or COS-NPs at the two tested doses-treated groups. The results indicated that COS-NPs were roughly rod in shape with average particle size of 200 nm and zeta potential 31.4 ± 2.8 mV. Animals fed OTA-contaminated diet showed significant changes in serum biochemical parameters, increase kidney MDA and DNA fragmentation and decreased GPx and SOD gene expression accompanied with severe histological changes. Q and/or COS-NPs at the two tested doses induced significant improvements in all tested parameters and succeeded to overcome these effects especially in the animals treated with Q plus the high dose of COS-NPs. It could be concluded that COS-NPs are promise candidate to enhance the antioxidant effect of Q and protect against the nephrotoxicity of OTA in high endemic areas.

Enzymatic modification of polysaccharides: Mechanisms, properties, and potential applications: A review.

Polysaccharides are natural biopolymers found in almost all living organisms. They are used extensively in various industrial applications, such as food, adhesives, pharmaceuticals, and cosmetics. In many cases, their practical use is limited because of their weak solubility in neutral pH, their unsuitable hydrophilic/hydrophobic balance. In this context, chemical or enzymatic modification of their structure appears as a relevant way, to improve their properties, and thus to enlarge the field of their potential applications. Taking into account the reduction of the input energy and the environmental impact, and due to high specificity and selectivity properties, enzymatic bioprocesses have been investigated as attractive alternatives to toxic and non-specific chemical approaches. This review discusses the methods of enzymatic functionalization of four well-known polysaccharides, chitosan, cellulose, pectin and starch. Particular emphasis was placed on the methods, the reaction types and the enzymes implicated in the modification such as laccases, peroxidases lipases, tyrosinases, and transglutaminases. The impact of functionalization on the properties and the applications of polysaccharide derivatives were described.

Laccase mediated-synthesis of hydroxycinnamoyl-peptide from ferulic acid and carnosine.

Carnosine (CAR) dipeptide was functionalized with ferulic acid (FA) as substrate using laccase from Myceliophtora thermophila as biocatalyst. The enzymatic reaction was performed in aqueous medium under mild conditions (pH 7.5, 30°C) as an eco-friendly procedure. Results showed that this enzymatic process led to the synthesis of two new derivatives (P1, P2), from the coupling between CAR and FA derived products. Conditions allowing a high production of P1, P2 derivatives were determined with an optimal ratio of (FA: CAR) of (1:1.6) at optimal time reaction of 8h. Under these optimal conditions, the coupling between CAR and FA-products was demonstrated, resulting in the decrease of -NH2 groups (almost 50%) as quantified via derivatization. Due to the presence of FA in the structure of these new derivatives, they exhibited higher hydrophobic property than carnosine. Structural analyses by mass spectrometry showed that P1 and P2 (FA-CAR) derivatives exhibited the same molecular mass (MM 770g/mol) containing one CAR-molecule and three FA-molecules but with different chemical structures. Furthermore, these derivatives presented improved antioxidant (almost 10 times) and anti-proliferative (almost 18 times) properties in comparison with CAR. Moreover, P1 derivative exhibited higher antioxidant and anti-proliferative activities than P2 derivative, which confirmed the different structures of P1 and P2. These results suggested that the oxidized phenols coupling with carnosine is a promising process to enhance the CAR-properties.

Grafting of gallic acid onto chitosan nano particles enhances antioxidant activities in vitro and protects against ochratoxin A toxicity in catfish (Clarias gariepinus).

This study aimed to prepare and characterize enzymatic modified chitosan nanoparticles (CSNPs) with gallic acid (GA) or octyl gallate (OG) to optimize its potential in human application and to evaluate their protective role against ochrtoxin A (OTA) toxicity in catfish. The modified CSNPs have average size around 90 nm with positive charge and high scavenging activity especially GA-CSNPs. In the in vivo study, catfish were divided into 8 groups and treated for 3 weeks as follow: the control group, OTA-treated group (1 mg/kg b.w.), the groups treated with CSNPs, GA-CSNPs or OG-CSNPs (280 mg/kg b.w.) anole or in combination with OTA. Blood, liver and kidney samples were collected for different analyses. OTA induced a significant biochemical disturbances accompanied with oxidative stress in liver and kidney, histological changes and increase DNA fragmentation in the kidney. Co-treatment with OTA plus the different CSNPs resulted in a significant improvement in all tested parameters and histological picture of the kidney. This improvement was more pronounced in the group treated with GA-CSNPs. It could be concluded that grafting of GA or its ester improved the properties of CSNPs. Moreover, GA-CSNPs showed strong scavenging properties than OG-CSNPs due to the blocking of carboxyl groups responsible of the scavenging activity in OG.

Growth of human mesenchymal stem cells (MSCs) on films of enzymatically modified chitosan.

Mesenchymal stem cells (MSCs) are known to be an attractive cell source for tissue engineering and regenerative medicine. One of the main limiting steps for clinical use or biotechnological purposes is the expansion step. The research of compatible biomaterials for MSCs expansion is recently regarded as an attractive topic. The aim of this study was to create new functional biomaterial for MSCs expansion by evaluating the impact of chitosan derivative films modified by enzymatic approach. First, chitosan particles were enzymatically modified with ferulic acid (FA) or ethyl ferulate (EF) under an eco-friendly procedure. Then, films of chitosan and its modified derivatives were prepared and evaluated by physicochemical and biological properties. Results showed that the enzymatic grafting of FA or EF onto chitosan significantly increased hydrophobic and antioxidant properties of chitosan films. The MSCs cell viability on chitosan derivative films also increased depending on the film thickness and the quantity of grafted phenols. Furthermore, the cytotoxicity test showed the absence of toxic effect of chitosan derivative films towards MSCs cells. Cell morphology showed a well attached and spread phenotype of MSCs cells on chitosan derivative films. On the other hand, due to the higher phenol content of FA-chitosan films, their hydrophobic, antioxidant properties and cell adhesion were improved in comparison with those of EF-chitosan films. Finally, this enzymatic process can be considered as a promising process to favor MSCs cell growth as well as to create useful biomaterials for biomedical applications especially for tissue engineering. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:491-500, 2016.

Chitosan nanoparticles and quercetin modulate gene expression and prevent the genotoxicity of aflatoxin B1 in rat liver.

The aims of the current study were to prepare chitosan nanoparticles (CNPs) and to evaluate its protective role alone or in combination with quercetin (Q) against AFB1-induce cytotoxicity in rats. Male Sprague-Dawley rats were divided into 12 groups and treated orally for 4 weeks as follow: the control group, the group treated with AFB1 (80 µg/kg b.w.) in corn oil, the groups treated with low (140 mg/kg b.w.) or high (280 mg/kg b.w.) dose of CNPs, the group treated with Q (50 mg/kg b.w.), the groups treated with Q plus the low or the high dose of CNPs and the groups treated with AFB1 plus Q and/or CNPs at the two tested doses. The results also revealed that administration of AFB1 resulted in a significant increase in serum cytokines, Procollagen III, Nitric Oxide, lipid peroxidation and DNA fragmentation accompanied with a significant decrease in GPx I and Cu-Zn SOD-mRNA gene expression. Q and/or CNPs at the two tested doses overcome these effects especially in the group treated with the high dose of CNPs plus Q. It could be concluded that CNPs is a promise candidate as drug delivery enhances the protective effect of Q against the cytogenetic effects of AFB1 in high endemic areas.

Laccase-catalysed functionalisation of chitosan by ferulic acid and ethyl ferulate: evaluation of physicochemical and biofunctional properties.

Chitosan and its derivatives functionalized by laccase-catalyzed oxidation of ferulic acid (FA) and ethyl ferulate (EF) were characterised for their physico-chemical, antioxidant and antibacterial properties. The enzymatic grafting of oxidised phenols led to FA-coloured and EF-colourless chitosan derivatives with good stability of colour and grafted phenols towards the chemical treatment by organic solvents. The efficiency of FA-products grafting onto chitosan was higher than that of EF-products. Moreover, the enzymatic grafting of phenols onto chitosan changed its morphological surface, increased its molecular weight and its viscosity. Furthermore, the chitosan derivatives presented improved antioxidant properties especially for FA-chitosan derivative when compared with chitosan with good antioxidant stability towards thermal treatment (100°C/1h). Chitosan and its derivatives showed also similar antibacterial activities and more precisely bactericidal activities. This enzymatic procedure provided chitosan derivatives with improved properties such as antioxidant activity, thermal antioxidant stability as well as the preservation of initial antibacterial activity of chitosan.

Laccase-catalysed oxidation of ferulic acid and ethyl ferulate in aqueous medium: a green procedure for the synthesis of new compounds.

The enzymatic oxidation of ferulic acid (FA) and ethyl ferulate (EF) with Myceliophthora thermophila laccase, as biocatalyst, was performed in aqueous medium using an eco-friendly procedure to synthesize new active molecules. First, the commercial laccase was ultrafiltrated allowing for the elimination of phenolic contaminants and increasing the specific activity by a factor of 2. Then, kinetic parameters of this laccase were determined for both substrates (FA, EF), indicating a higher substrate affinity for ethyl ferulate. Additionally, enzymatic oxidation led to the synthesis of a FA-major product, exhibiting a molecular mass of 386 g/mol and a EF-major product with a molecular mass of 442 g/mol. Structural analyses by mass spectrometry allowed the identification of dimeric derivatives. The optical properties of the oxidation products showed the increase of red and yellow colours, with FA-products compared to EF-products. Additionally, enzymatic oxidation led to a decrease of antioxidant and cytotoxic activities compared to initial substrates. Consequently, this enzymatic procedure in aqueous medium could provide new compounds presenting optical, antioxidant and cytotoxic interest.

Functionalization of chitosan by laccase-catalyzed oxidation of ferulic acid and ethyl ferulate under heterogeneous reaction conditions.

Chitosan particles were functionalized with ferulic acid (FA) and ethyl ferulate (EF) as substrates using laccase from Myceliophtora thermophyla as biocatalyst. The reactions were performed with chitosan particles under an eco-friendly procedure, in a heterogeneous system at 30°C, in phosphate buffer (50mM, pH 7.5). The FA-chitosan derivative presented an intense yellow-orange color stable while the EF-chitosan derivative was colorless. The spectroscopic analyses indicated that the reaction products bound covalently to the free amino groups of chitosan exhibiting a novel absorbance band in the UV/Vis spectra between 300 and 350nm, at C-2 region by the duplication of C-2 signal in the 13C NMR spectrum, via Schiff base bond (NC) exhibiting novel bands in the FT-IR spectrum at 1640 and 1620cm-1. Additionally, antioxidant capacities of chitosan derivatives showed that the chitosan derivatives presented improved antioxidant properties, especially for FA-chitosan derivative (EC50 were 0.52±0.04, 0.20±0.02mg/ml for DPPH and ABTS+ scavenging, respectively).