Methyl Ferulate Research Articles

The Temperature Effect on Ultrasonic-assisted of Synthesis Methyl Ferulate and Its Antiplatelet Assay

<p>Ferulic acid (FA) has been reported to have antiplatelet activity through indirectly inhibiting P2Y12 receptor. In order to increase the activity of FA, by improving its lipophilicity, so that it is easier to enter the cell, synthesis methyl ferulate was conducted through Fisher esterification. Ultrasonic waves were utilized as source of energy emitted through water as the medium at two various of temperature, i.e., 55 °C and 65 °C. The purposes of this study are to produce methyl ferulate and to determine the reaction constant rate (<em>k</em>) and its energy activation (<em>E<sub>a</sub></em>), at temperature of 55 °C and 65 °C. Moreover, the biological activity as antiplatelet was investigated at dose 20 mg/kg BW. The antiplatelet assay was conducted by clotting time and bleeding time methods. The results were analyzed by one way ANOVA program (<em>P</em><0.05). The yield of methyl ferulate are 50.3% and 67.1% at 55 °C and 65 °C, respectively. The <em>k</em> value at 55°C is 4x10<sup>-5</sup> cons<sup>-1</sup>min<sup>-1</sup>, while that of at 65 °C is 9x10<sup>-5</sup> cons<sup>-1</sup>min<sup>-1</sup>. The clotting time and bleeding time of methyl ferulate obtained were 265 sec and 175 sec, respectively. The antiplatelet activity of methyl ferulate is better than ferulic acid.</p>

Optimized hydrolytic methods by response surface methodology to accurately estimate the phenols in cereal by HPLC-DAD: The case of millet

Extraction of free and bound phenols from millet in acidic and basic hydrolytic conditions were compared for the first time. Acidic hydrolysis was able to extract the highest amount of total phenolic compounds (up to 178 mg/100 g) while the basic hydrolysis underestimates the phenolic concentration. Our findings pointed out for the first time that methyl ferulate is naturally present as bound phenol in millet. Response Surface Methodology was then applied to both acidic and basic hydrolytic extractive conditions: the acidic procedure, optimized in terms of extractive time and temperature and concentration of the acidic mean, gave the best results, allowing definition of Method Operable Design Region and quantitation of the total amount of phenols in millet samples in a single extractive step. This optimized method is suitable for further accurate investigations of the typical phenols of the numerous varieties of this recently re-discovered minor cereal.

Molecular cloning, expression and characterization of a novel feruloyl esterase from a soil metagenomic library with phthalate-degrading activity.

To discover novel feruloyl esterases (FAEs) by the function-driven screening procedure from soil metagenome. A novel FAE gene bds4 was isolated from a soil metagenomic library and over-expressed in Escherichia coli. The recombinant enzyme BDS4 was purified to homogeneity with a predicted molecular weight of 38.8kDa. BDS4 exhibited strong activity (57.05 U/mg) toward methyl ferulate under the optimum pH and temperature of 8.0 and 37°C. Based on its amino acid sequence and model substrates specificity, BDS4 was classified as a type-C FAE. The quantity of the releasing ferulic acid can be enhanced significantly in the presence of xylanase compared with BDS4 alone from de-starched wheat bran. In addition, BDS4 can also hydrolyze several phthalates such as diethyl phthalate, dimethyl phthalate and dibutyl phthalate. The current investigation discovered a novel FAE with phthalate-degrading activity and highlighted the usefulness of metagenomic approaches as a powerful tool for discovery of novel FAEs.

Study on a Fermented Whole Wheat: Phenolic Content, Activity on PTP1B Enzyme and In Vitro Prebiotic Properties.

Fermented cereals, staple foods in Asia and Africa, are recently receiving a growing interest in Western countries. The object of this work is the characterization of a fermented wheat used as a food ingredient and dietary supplement. To this aim, the phenolic composition, the activity on protein tyrosine phosphatase 1B (PTP1B), an enzyme overexpressed in type-II diabetes, the in vitro prebiotic properties on Lactobacillus reuteri and the microbial composition were investigated. Basic and acidic hydrolysis were tested for an exhaustive recovery of bound phenols: the acidic hydrolysis gave best yields. Methyl ferulate and neocarlinoside were identified for the first time in wheat. The inhibitory power of the extracts of several batches were investigated on PTP1B enzyme. The product was not able to inhibit the enzyme, otherwise, for the first time, a complete inhibition was observed for schaftoside, a major C-flavonoid of wheat. The microbial composition was assessed identifying Lactobacillus, Enterococcus, and Pediococcus as the main bacterial species. The fermented wheat was a suitable substrate for the grown of L. reuteri, recognized for its health properties in the human gut. The proposed method for phenols is easier compared to those based on strong basic hydrolysis; our results assessed the bound phenols as the major fraction, differently from that suggested by the literature for fermented cereals.

Directed evolution of the type C feruloyl esterase from Fusarium oxysporum FoFaeC and molecular docking analysis of its improved variants

The need to develop competitive and eco-friendly processes in the cosmetic industry leads to the search for new enzymes with improved properties for industrial bioconversions in this sector. In the present study, a complete methodology to generate, express and screen diversity for the type C feruloyl esterase from Fusarium oxysporium FoFaeC was set up in a high-throughput fashion. A library of around 30,000 random mutants of FoFaeC was generated by error prone PCR of fofaec cDNA and expressed in Yarrowia lipolytica. Screening for enzymatic activity towards the substrates 5-bromo-4-chloroindol-3-yl and 4-nitrocatechol-1-yl ferulates allowed the selection of 96 enzyme variants endowed with improved enzymatic activity that were then characterized for thermo- and solvent- tolerance. The five best mutants in terms of higher activity, thermo- and solvent- tolerance were selected for analysis of substrate specificity. Variant L432I was shown to be able to hydrolyze all the tested substrates, except methyl sinapate, with higher activity than wild type FoFaeC towards methyl p-coumarate, methyl ferulate and methyl caffeate. Moreover, the E455D variant was found to maintain completely its hydrolytic activity after two hour incubation at 55 °C, whereas the L284Q/V405I variant showed both higher thermo- and solvent- tolerance than wild type FoFaeC. Small molecule docking simulations were applied to the five novel selected variants in order to examine the binding pattern of substrates used for enzyme characterization of wild type FoFaeC and the evolved variants.

Preliminary assessment for the synthesis of lignin-type molecules using crude onion peroxidase

The aim of the study concerned the production of lignan-type molecules using inexpensive unpurified peroxidase from onion bulbs. The crude peroxidase (POD) extracted from onion bulbs is an inexpensive tool for the synthesis of fine chemicals as well as converting food industry waste materials into high added-value bio-products. The experiments were focusing on the ability of a crude onion POD preparation to act as a biocatalyst for the oxidative dimerization–cyclization of hydroxycinnamic acids and their methyl esters. The favorable conditions (pH and temperature) of the enzymatic reaction have been assayed using response surface methodology (RSM). Subsequently, a kinetics assay has been performed to distinguish the affinity of the enzyme towards the different substrates. The results indicated a strong substrate specificity toward methyl ferulate. Nevertheless, these findings on the ability of crude POD extract to generate dehydrodimers merit additional investigation and should be regarded as a preliminary assessment.

Alkyl Ferulate Esters as Multifunctional Food Additives: Antibacterial Activity and Mode of Action against Escherichia coli in Vitro.

This work aims to prepare ferulic acid alkyl esters (FAEs) through the lipase-catalyzed reaction between methyl ferulate and various fatty alcohols in deep eutectic solvents and ascertain their antibacterial activities and mechanisms. Screens of antibacterial effects of FAEs against Escherichia coli ATCC 25922 ( E. coli) and Listeria monocytogenes ATCC 19115 ( L. monocytogenes) revealed that hexyl ferulate (FAC6) exerted excellent bacteriostatic and bactericidal effects on E. coli and L. monocytogenes (minimum inhibitory concentration (MIC): 1.6 and 0.1 mM, minimum bactericidal concentration (MBC): 25.6 and 0.2 mM, respectively). The antibacterial mechanism of FAC6 against E. coli was systematically studied to facilitate its practical use as a food additive with multifunctionalities. The growth and time-kill curves implied the partial cell lysis and inhibition of the growth of E. coli caused by FAC6. The result related to propidium iodide uptake and cell constituents' leakage (K+, proteins, nucleotides, and β-galactosidase) implied that bacterial cytomembranes were substantially compromised by FAC6. Variations on morphology and cardiolipin microdomains and membrane hyperpolarization of cells visually verified that FAC6 induced cell elongation and destructed the cell membrane with cell wall perforation. SDS-PAGE analysis and alterations of fluorescence spectra of bacterial membrane proteins manifested that FAC6 caused significant changes in constitutions and conformation of membrane proteins. Furthermore, it also could bind to minor grooves of E. coli DNA to form complexes. Meanwhile, FAC6 exhibited antibiofilm formation activity. These findings indicated that that FAC6 has promising potential to be developed as a multifunctional food additive.

Type C feruloyl esterase from Aspergillus ochraceus: A butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system

Aspergillus ochraceus was isolated from coffee pulp and selected as an interesting hydroxycinnamoyl esterase strain producer, using an activity microplate high-throughput screening method. In this work, we purified and characterized a new type C A. ochraceus feruloyl esterase (AocFaeC), which synthesized specifically butyl hydroxycinnamates in a ternary solvent system. AocFaeC was produced by solid state fermentation, reaching its maximal activity (1.1 U/g) after 48 h of culture. After purification, the monomeric protein (34 kDa) showed a specific activity of 57.9 U/mg towards methyl ferulate. AocFaeC biochemical characterization confirmed its identity as a type C feruloyl esterase and suggested the presence of a catalytic serine in the active site. Its maximum hydrolytic activity was achieved at 40°C and pH 6.5 and increased by 109 and 77% with Ca2 + and Mg2 +, but decreased by 90 and 45% with Hg2 + and Cu2 +, respectively. The initial butyl ferulate synthesis rate increased from 0.8 to 23.7 nmol/min after transesterification condition improvement, using an isooctane:butanol:water ternary solvent system, surprisingly the synthesis activity using other alcohols was negligible. At these conditions, the synthesis specific activities for butyl p-coumarate, sinapinate, ferulate, and caffeate were 87.3, 97.6, 168.2, and 234 U/μmol, respectively. Remarkably, AocFaeC showed 5 folds higher butyl caffeate synthesis rate compared to type B Aspergillus niger feruloyl esterase, a well-known enzyme for its elevated activity towards caffeic acid esters. Type C feruloyl esterase from A. ochraceus is a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system. How to cite: Romero E, Grajales D, Armendáriz M, et al. Type C feruloyl esterase from Aspergillus ochraceus: A butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system. Electron J Biotechnol 2018;35. https://doi.org/10.1016/j.ejbt.2018.06.004.

Identification of a Novel Feruloyl Esterase by Functional Screening of a Soil Metagenomic Library

A cosmid metagenomic library containing 1.3 × 105 clones was created from a soil sample. A novel gene (fae-xuan) encoding a feruloyl esterase was identified through functional screening. Primary sequence analysis showed that the gene consisted of 759 base pairs and encoded a protein of 252 amino acids. The gene was expressed in Escherichia coli BL21 (DE3) and the corresponding purified recombinant enzyme exhibited a molecular weight of 29kDa. The FAE-Xuan showed high activity (40.0U/mg) toward methyl ferulate with an optimal temperature and pH of 30°C and 5.0, respectively. Besides methyl ferulate, FAE-Xuan can also hydrolyze methyl sinapate and methyl p-coumarate. The substrate utilization preferences and phylogenetic analysis indicated that FAE-Xuan belongs to type A FAE. FAE-Xuan was quite stable over a broad pH range from 3.0 to 10.0. The activity reduced remarkably in presence of Cu2+. FAE-Xuan can enhance the quantity of ferulic acid from de-starched wheat bran in presence of xylanase. The work presented here highlighted the effectiveness of metagenomic strategy in identifying novel FAEs with diverse properties for potential use in industrial production.

Selective Fragmentation of Biorefinery Corncob Lignin into p-Hydroxycinnamic Esters with a Supported Zinc Molybdate Catalyst.

Lignin is the largest renewable resource of bioaromatics, and the catalytic fragmentation of lignin into phenolic monomers is increasingly recognized as an important starting point for lignin valorization. Herein, we report that ZnMoO4 supported on MCM-41 can catalyze the fragmentation of biorefinery technical lignin, enzymatic mild acidolysis lignin, and native lignin derived from corncob to yield lignin oily products that contain 15-37.8 wt % phenolic monomers, in which the high selectivities towards methyl coumarate (1) and methyl ferulate (2) were obtained (up to 78 %). The effects of some key parameters such as the influence of the solvent, reaction temperature, time, H2 pressure, and catalyst dosage were examined in view of activity and selectivity. The loss of Zn from the catalyst is discussed as the primary cause of deactivation, and the catalytic activity and selectivity can be well preserved in at least six runs by thermal calcination. The high selectivity to 1 and 2 leads to their easy separation and purification from lignin oily product to provide sustainable monomers for the preparation of functional polyether esters and polyesters.

English

Hyoscyamus muticus is an endangered desert plant spread in the Arabian Peninsula and the Middle East deserts. The methanol extract of the aerial parts of H. muticus grown in the arid zones in the Northern regions of Saudi Arabia were subjected to primary phytochemical analysis which revealed the presence of phenolic compounds, flavonoids, tannins and sterols, the gas chromatography-mass spectrometry (GC-MS) analysis of the methanolic extract exhibited different types of phenolic compounds, including ferulic acid, 4-hydroxy-cinamic acid-ester, methyl salicylate and methyl ferulate. The accumulation of phenolic compounds supports the antioxidative properties of the plant against oxidative stress. The antioxidant testing showed that the methanolic extract of H. muticus has a noticeable antioxidant activity with an IC50 of 8.1±0.65 mg/ml and an EC50 of 12.74±1.12 mg/ml. The antimicrobial investigation on 11 microbial strains revealed that the methanolic extract of H. muticus areal parts showed average or weak antibacterial activity against gram-positive bacteria, weak antibacterial activity against gram-negative bacteria and no antifungal activity. Moreover, the investigations exhibited the presence of an antifeedant potential on the methanol extract of H. muticus on the 4th instar larvae of Spodoptera littoralis. Key words: Hyoscyamus muticus, phytochemical analysis, antioxidant capacity, antimicrobial activity, antifeedant assay Spodoptera littoralis.

Ferulic Acid, Methyl Ferulate, and Ferulic Acid Glucopyranosyl Ester Isolated from Cultured Cells of Phytolacca americana

Production of secondary metabolites in cultured plant cells of Phytolacca americana was investigated. Three compounds, i.e., ferulic acid, methyl ferulate, and ferulic acid glucopyranosyl ester, were isolated from cultured P. americana cells.

Phthalides and Other Metabolites from Roots of Ligusticum wallichii

A new phthalide that was identified using NMR and mass spectral data as (±)-3-methoxysedanenolide (1) and the nine known compounds sedanenolide (2), Z-ligustilide (3), Z-butylidenephthalide (4), methyl ferulate (5), trans-ferulic (6) and trans-isoferulic acids (7), falcarindiol (8), β-sitosterol (9), and daucosterol (10) were isolated from the MeOH extract of roots of Ligusticum wallichii. Compounds 7 and 8 exhibited high inhibitory activity against NO formation in RAW264.7 cells.

A type D ferulic acid esterase from Streptomyces werraensis affects the volume of wheat dough pastries.

A type D ferulic acid esterase (FAE) was identified in the culture supernatant of Streptomyces werraensis, purified, sequenced, and heterologously produced in E. coli BL21(DE3)Star by co-expressing chaperones groES-groEL (69UL-1). The unique enzyme with a mass of about 48kDa showed no similarity to other FAEs, and only moderate homology (78.5%) to a Streptomycete β-xylosidase. The purified reSwFAED exhibited a temperature optimum of 40°C, a pH optimum in the range from pH seven to eight and a clear preference for bulky natural substrates, such as 5-O-trans-feruloyl-L-arabinofuranose (FA) and β-D-xylopyranosyl-(1→2)-5-O-trans-feruloyl-L-arabinofuranose (FAX), compared to the synthetic standard substrate methyl ferulate. Treatment of wheat dough with as little as 0.03U or 0.3Ukg-1 reSwFAED activity resulted in a significant increase of the bun volume (8.0 or 9.7%, resp.) after baking when combined with polysaccharide-degrading enzymes from Aspergillus. For the first time, the long-standing, but rarely proven positive effect of a FAE in baking was confirmed.

Feruloyl esterase from Alternaria tenuissima that hydrolyses lignocellulosic material to release hydroxycinnamic acids

An extracellular feruloyl esterase from the culture filtrates of the isolated fungus Alternaria tenuissima was successfully purified to apparent homogeneity by anion-exchange and size-exclusion chromatography. Peptide fragments of purified enzyme (designated as AltFAE; molecular weight of 30.3 kDa determined by SDS-PAGE) were identified by mass spectrometry using a NanoLC-ESI-MS/MS system. Michaelis-Menten constants (KM) and catalytic efficiencies (kcat/KM) were determined for typical substrates of feruloyl esterase, and the lowest KM of 50.6 μM (i.e., the highest affinity) and the highest kcat/KM (3.1 × 105 s—1 M–1) were observed for methyl p-coumarate and methyl ferulate, respectively. Not least, AltFAE catalyzed conversion of lignocellulosic material (e.g. wood meal) to release hydroxycinnamic products, i.e. ferulic- and p-coumaric acids.

Effects of Different Lipophilized Ferulate Esters in Fish Oil-Enriched Milk: Partitioning, Interaction, Protein, and Lipid Oxidation.

Antioxidant effects of ferulic acid and lipophilized ferulate esters were investigated in fish oil-enriched milk. Methyl ferulate (C1) and ethyl ferulate (C2) more efficiently prevented lipid oxidation than dodecyl ferulate (C12) did, followed by ferulic acid (C0). The combination of C1 or C2 with C12 could have a "synergistic" effect indicated by peroxide value, hexanal, and 1-penten-3-ol analysis results. These antioxidants also showed protein oxidation inhibition effects. The most effective antioxidants (C1 and C2) had the highest concentration in the precipitate phase but the lowest concentration in the aqueous phase, which was the opposite of the partitioning of C0. C12 had the highest concentration in the oil and emulsion phase. In particular, the interaction between ferulates esterified with short and medium alkyl chain lengths could lead to their "synergistic" effects in fish oil-enriched milk, which could be caused by the change in their partitioning or localization at the interface.

Antioxidant Activity of Phenolic and Alkaloid Fractions Accumulated in Artemisia Judaica and Artemisia Herba Alba

Genus Artemisia are desert plants spread in the Egypt and Saudi Arabia. The methanol extract of the aerial parts of Artemisia herba alba and Artemisia judaica grown in the arid zones in the Northern regions of Saudi Arabia and North Sinai, in Egypt were subjected to the primary phytochemical analysis which, showed the presence of phenolic compounds, flavonoids, tannins and sterols. The GC-MS analysis of the methanolic extract exhibited numerous compounds, 11 compounds were identified as phenolic compounds, ferulic acid, ursodeoxycholic acid and derivatives of cinnamic acid (ethyl cinnamate). The highest phenolics content in A.herba alba grown in Saudi Arabia was observed for methyl cinnamate, (8.2 %) and the lowest methyl ferulate (0.02 %) for A. judica grown in Egypt. The quantization analysis of GC-MS revealed high amount of phenolic acid in the plant grow in Saudi Arabia than the plant collected from Egypt. The radical scavenging activities of two plant were studied by 1,1-diphenyl -2-pycrylhydrazyl (DPPH), EC (50) values were determined for A.herba alba and A.judaica (0.122, and 0.56 mg/ml) growing in Saudi Arabia and A.herba alba and A. judaica (1.7 and 0. 67 mg/ml ) growing in Egypt. Total alkaloid content was analysed and some alkaloid compounds was identified by GC-MS, viz. dasycarpidan-1-methanol, acetate, pseudosolasodine diacetate and morphinan4,5epoxy3,6-diol. Certain compounds were rich in Saudi plant but not detected in plants grown in Egypt, which reflect the role of environment adaptation of plant by accumulation of only certain compounds.

Synthesis and cytotoxic activity of 4-O-β-D-galactopyranosyl derivatives of phenolic acids esters

The glycosylation of naturally occurring phenolic acids has a significant impact on their solubility, stability and physiochemical properties. D-Galactose residue was found to form a part of glycoconjugates in several tissues and involved in a variety of physiological process. To the best of our knowledge, we have noticed a little information about the glycosylation of the phenolic acids with galactose residue. In this work, we describe the glycosylation of methyl vanillate and methyl ferulate with peracetylated-β-D-galactopyranose in the presence of BF3·OEt2. The coupling reaction yielded efficiently and selectively only the acetylated β-D-galactopyranosides 3 and 6. Removal of the acetyl groups using sodium methoxide afforded the corresponding β-D-galactopyranosides 4 and 7 in good yields. Anticancer activity in vitro was evaluated against two human cancer cell lines (MCF-7 breast cancer cell lines and PC-3 prostate cancer cell lines). β-D-galactopyranosides 4 and 7 demonstrated improved cytotoxic activity compared to the parental esters.

Melanogenesis-Inhibitory and Cytotoxic Activities of Limonoids, Alkaloids, and Phenolic Compounds from Phellodendron amurense Bark.

Four limonoids, 1-4, five alkaloids, 5-9, and four phenolic compounds, 10-13, were isolated from a MeOH extract of the bark of Phellodendron amurense (Rutaceae). Among these, compound 13 was new, and its structure was established as rel-(1R,2R,3R)-5-hydroxy-3-(4-hydroxy-3-methoxyphenyl)-6-methoxy-1-(methoxycarbonylmethyl)indane-2-carboxylic acid methyl ester (γ-di(methyl ferulate)) based on the spectrometric analysis. Upon evaluation of compounds 1-13 against the melanogenesis in the B16 melanoma cells induced with α-melanocyte-stimulating hormone (α-MSH), four compounds, limonin (1), noroxyhydrastinine (6), haplopine (7), and 4-methoxy-1-methylquinolin-2(1H)-one (8), exhibited potent melanogenesis-inhibitory activities with almost no toxicity to the cells. Western blot analysis revealed that compound 6 inhibited melanogenesis, at least in part, by inhibiting the expression of protein levels of tyrosinase, TRP-1, and TRP-2 in α-MSH-stimulated B16 melanoma cells. In addition, when compounds 1-13 were evaluated for their cytotoxic activities against leukemia (HL60), lung (A549), duodenum (AZ521), and breast (SK-BR-3) cancer cell lines, five compounds, berberine (5), 8, canthin-6-one (9), α-di-(methyl ferulate) (12), and 13, exhibited cytotoxicities against one or more cancer cell lines with IC50 values in the range of 2.6-90.0μm. In particular, compound 5 exhibited strong cytotoxicity against AZ521 (IC50 2.6μm) which was superior to that of the reference cisplatin (IC50 9.5μm).

Characterization of Feruloyl Esterases Produced by the Four Lactobacillus Species: L. amylovorus, L. acidophilus, L. farciminis and L. fermentum, Isolated from Ensiled Corn Stover.

Lactic acid bacteria (LAB) play important roles in silage fermentation, which depends on the production of sufficient organic acids to inhibit the growth of undesirable microorganisms. However, LAB are not able to degrade cellulose and hemicellulose. Bacteria and fibrolytic enzymes are usually used as inoculants to improve the silage quality and digestibility. In the present study, we isolated four Lactobacillus strains (L. amylovorus CGMCC 11056, L. acidophilus CCTCC AB2010208, L. farciminis CCTCC AB2016237 and L. fermentum CCTCC AB2010204) with feruloyl esterase (FAE) activities from ensiled corn stover (CS) by a plate screening assay. The genes encoding FAEs were cloned and hetero-expressed in Escherichia coli. The optimal temperature and pH of these purified enzymes ranged from 45 to 50°C and from 7.0 to 8.0, respectively. They could hydrolyze hydroxycinnamoyl esters in a substrate-specific manner when methyl ferulate, methyl caffeate, methyl ρ-coumarate and methyl sinapinate were used as substrates. Moreover, these four FAEs were able to hydrolyze CS to release hydroxycinnamic acids. Furthermore, these strains could degrade hydroxycinnamic esters, and L. amylovorus CGMCC 11056 was the most efficient strain among these four isolates. These results provided a new target for the development of inoculants to improve silage quality and digestibility.