Melatonin- and Ferulic Acid-Based HDAC6 Selective Inhibitors Exhibit Pronounced Immunomodulatory Effects In Vitro and Neuroprotective Effects in a Pharmacological Alzheimer's Disease Mouse Model.

The structures of melatonin and ferulic acid were merged into tertiary amide-based histone deacetylase 6 (HDAC6) inhibitors to develop multi-target-directed inhibitors for neurodegenerative diseases to incorporate antioxidant effects without losing affinity and selectivity at HDAC6. Structure-activity relationships led to compound 10b as a hybrid molecule showing pronounced and selective inhibition of HDAC6 (IC50 = 30.7 nM, > 25-fold selectivity over other subtypes). This compound shows comparable DPPH radical scavenging ability to ferulic acid, comparable ORAC value to melatonin and comparable Cu2+ chelating ability to EDTA. It also lacks neurotoxicity on HT-22 cells, exhibits a pronounced immunomodulatory effect, and is active in vivo showing significantly higher efficacy in an AD mouse model to prevent both Aß25-35-induced spatial working and long-term memory dysfunction at lower dose (0.3 mg/kg) compared to positive control HDAC6 inhibitor ACY1215 and an equimolar mixture of the three entities ACY1215, melatonin and ferulic acid, suggesting potentially disease-modifying properties.

Accessing p-Hydroxycinnamic Acids: Chemical Synthesis, Biomass Recovery, or Engineered Microbial Production?

p-Hydroxycinnamic acids (i. e., p-coumaric, ferulic, sinapic, and caffeic acids) are phenolic compounds involved in the biosynthesis pathway of lignin. These naturally occurring molecules not only exhibit numerous attractive properties, such as antioxidant, anti-UV, and anticancer activities, but they also have been used as building blocks for the synthesis of tailored monomers and functional additives for the food/feed, cosmetic, and plastics sectors. Despite their numerous high value-added applications, the sourcing of p-hydroxycinnamic acids is not ensured at the industrial scale except for ferulic acid, and their production cost remains too high for commodity applications. These compounds can be either chemically synthesized or extracted from lignocellulosic biomass, and recently their production through bioconversion emerged. Herein the different strategies described in the literature to produce these valuable molecules are discussed.

Ferulic Acid Esters and Withanolides: In Search of Withania somnifera GABAA Receptor Modulators.

Nine compounds, including two undescribed withanolides, withasomniferolides A and B (1 and 2), three known withanolides (3-5), a ferulic acid dimeric ester (6), and an inseparable mixture of three long alkyl chain ferulic acid esters (7-9), were isolated from a GABAA receptor positive activator methanol extract of the roots of Withania somnifera. The structures of the isolated compounds were elucidated based on NMR, MS, and ECD data analysis. In order to bioassay the single ferulic acid derivatives, compounds 6-9 were also synthesized. The most active compound, docosanyl ferulate (9), was able to enhance the GABAA receptor inhibitory postsynaptic currents with an IC50 value of 7.9 µM. These results, by showing an ability to modulate the GABAA receptor function, cast fresh light on the biological activities of the secondary metabolites of W. somnifera roots.

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

Background: 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. Results: 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. Conclusions: Type C feruloyl esterase from A. ochraceus is a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system

Synthesis of novel octyl sinapate to enhance antioxidant capacity of rapeseed-linseed oil mixture.

BACKGROUND: Lipophilisation allows the formation of new functionalised antioxidants having beneficial properties compared to natural hydrophilic phenolic acids. Therefore, this work focused on the synthesis of lipophilic antioxidants, such as a new octyl sinapate, octyl caffeate and octyl ferulate using the modified Fischer esterification of selected hydroxycinnamic acids with 1-octanol. RESULTS: The lipophilic octyl sinapate was obtained for the first time with satisfactory yield (83%) after purification by column chromatography. The identity of the synthesised phenolipids was confirmed by chromatographic and spectroscopic analyses. Antioxidant capacity of phenolipids was determined by DPPH (IC50 = 35.87-52.24 µg mL-1 ) and ABTS (IC50 = 39.45-48.72 µg mL-1 ) methods and compared with IC50 values (7.37-35.30 µg mL-1 and 7.55-41.67 µg mL-1 , respectively) for well known antioxidants. The antioxidant capacity of rapeseed-linseed oil enriched with the purified esters was about two to 30 times higher in comparison with a non-supplemented oil. CONCLUSION: The novel octyl sinapate as well as octyl caffeate and octyl ferulate have antioxidant properties and lipophilic character, therefore they may be added to vegetable oils as potential antioxidants for tackling oxidative processes. © 2017 Society of Chemical Industry.

Preparation and properties of multifunctional sinapic acid corn bran arabinoxylan esters.

In the present study, corn bran arabinoxylan (CAX) were modified with sinapic acid (SA) by esterification to generate sinapic acid corn bran arabinoxylan esters (SA-CAX) with various substituted degrees. The structure of SA-CAX was characterized by FT-IR, NMR and UV spectroscopy. And the antioxidant activities of SA-CAX were evaluated by scavenging the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical, emulsion lipid oxidation test and the lipid peroxidant level test. Compared with CAX, SA-CAX exhibited superior antioxidant activities in vitro, which indicated that the attachment of SA to CAX could enhance antioxidant activities of CAX. Moreover, the aqueous solution behavior of CAX and SA-CAX was investigated by light scattering, scanning electron microscopy and rheological measurement. The SA-CAX could form the aggregates even at diluted solutions. The hydrophobic association led to a higher viscosity and stronger gel behavior of the SA-CAX aqueous solution than that of CAX aqueous solution.

p-Coumaroyl Malate Derivatives of the Pandanus amaryllifolius Leaf and Their Isomerization.

A novel p-coumaroyl dimethyl malate (1) was isolated from the Pandanus amaryllifolius leaf in addition to three known analogs of p-coumaroyl dimethyl malate (2-4), and their structures were elucidated by analysis of the spectroscopic data. The p-coumaroyl malate derivatives were isolated as a mixture of E and Z isomers. To determine the cause of isomerization, the p-coumaroyl malate isolated in this study was synthesized. We concluded that the Z isomer might be an artifact generated from the E isomer through purification steps.

A newly synthesized sinapic acid derivative inhibits endothelial activation in vitro and in vivo.

Inhibition of oxidative stress and inflammation in vascular endothelial cells (ECs) may represent a new therapeutic strategy against endothelial activation. Sinapic acid (SA), a phenylpropanoid compound, is found in natural herbs and high-bran cereals and has moderate antioxidant activity. We aimed to develop new SA agents with the properties of antioxidation and blocking EC activation for possible therapy of cardiovascular disease. We designed and synthesized 10 SA derivatives according to their chemical structures. Preliminary screening of the compounds involved scavenging hydroxyl radicals and 2,2-diphenyl-1-picrylhydrazyl (DPPH(⋅)), croton oil-induced ear edema in mice, and analysis of the mRNA expression of adhesion molecules in ECs. 1-Acetyl-sinapic acyl-4-(3'-chlorine-)benzylpiperazine (SA9) had the strongest antioxidant and anti-inflammatory activities both in vitro and in vivo. Thus, the effect of SA9 was further studied. SA9 inhibited tumor necrosis factor α-induced upregulation of adhesion molecules in ECs at both mRNA and protein levels, as well as the consequent monocyte adhesion to ECs. In vivo, result of face-to-face immunostaining showed that SA9 reduced lipopolysaccharide-induced expression of intercellular adhesion molecule-1 in mouse aortic intima. To study the molecular mechanism, results from luciferase assay, nuclear translocation of NF-κB, and Western blot indicated that the mechanism of the anti-inflammatory effects of SA9 might be suppression of intracellular generation of ROS and inhibition of NF-κB activation in ECs. SA9 is a prototype of a novel class of antioxidant with anti-inflammatory effects in ECs. It may represent a new therapeutic approach for preventing endothelial activation in cardiovascular disorders.

Synthesis of stable isotope-labeled precursors for the biosyntheses of capsaicinoids, capsinoids, and capsiconinoids.

Stable isotope-labeled precursors were synthesized for an analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to elucidate the biosynthetic flow of capsaicinoids, capsinoids, and capsiconinoids. [1'-(13)C][5-(2)H]-Vanillin was prepared by the condensation of guaiacol with [(13)C]-chloroform and a D(2)O treatment. Labeled vanillylamine, vanillyl alcohol, ferulic acid, and coniferyl alcohol were prepared from the labeled vanillin. The labeled vanillylamine was converted to labeled capsaicinoid in a crude enzyme solution extracted from pungent Capsicum fruits.

[Preparation and anti-oxidant activity of cinnamic acid derivatives-g-CTS].

OBJECTIVE: To prepare cinnamic acid derivatives-g-CTS and to study its antioxidation activity. METHOD: The ability of catching oxygen of the products and raw material were determined through two methods, Marklund method and trace pyrogallic acid method, with autoxidation reaction of pyrogallol as the oxygen anion source. RESULT: The antioxidation activities of all products were better than the raw material. CONCLUSION: Cinnamic acid derivatives-g-CTS is suitable as the O2-* -capture agent.

[Synthesis of ferulic acid derivatives and their inhibitory effect on platelet aggregation].

Ferulic acid, an useful compound of Chinese traditional medicine, was used as leading compound. Six ferulic acid derivatives were designed and synthesized based on bioisosterism. Their structures were characterized by IR, 1H NMR, 13C NMR and mass spectra. In vivo experiment showed that ferulic acid derivatives had good inhibitory effects on adenosine diphosphate (ADP) induced platelet aggregation, which were significantly higher than that of Ozagrel.

N1,N5,N10-Tris(4-hydroxycinnamoyl)spermidines from Microdesmis keayana roots.

Three new N1,N5,N10-tris(4-hydroxycinnamoyl)spermidines were isolated from a methanolic root extract of Microdesmis keayana. They were identified as N5,N10-di(p-coumaroyl)-N1-feruloylspermidine,N5-(p-coumaroyl)-N1,N10-diferuloylspermidine, and N1,N5,N10-triferuloylspermidine, and were named keayanidines A, B, and C (1-3), respectively. Their structures were established by spectral techniques(electrospray mass spectrometry, one- and two-dimensional NMR). A 4',4'',4'''-trimethylated derivative was prepared by methylation of keayanidine C, and the same compound was synthesized fromspermidine and 3,4-dimethoxycinnamic acid to confirm the spectral attributions of the NMR data of the natural compounds. Radical-scavenging properties of all compounds were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical spectrophotometric assay.

Novel chemo-enzymatic oligomers of cinnamic acids as direct and indirect inhibitors of coagulation proteinases.

Thrombin and factor Xa, two important procoagulant enzymes, have been prime targets for regulation of clotting through the direct and indirect mechanism of inhibition. Our efforts on exploiting the indirect mechanism led us to study a carboxylic acid-based scaffold, which displayed major acceleration in the inhibition of these enzymes [J. Med. Chem.2005, 48, 1269, 5360]. This work advances the study to chemo-enzymatically prepared oligomers of 4-hydroxycinnamic acids, DHPs, which display interesting anticoagulant properties. Oligomers, ranging in size from tetramers to pentadecamers, were prepared through peroxidase-catalyzed oxidative coupling of caffeic, ferulic, and sinapic acids, and sulfated using triethylamine-sulfur trioxide complex. Chromatographic, spectroscopic, and elemental studies suggest that the DHPs are heterogeneous, polydisperse preparations composed of inter-monomer linkages similar to those found in natural lignins. Measurement of activated thromboplastin and prothrombin time indicates that both the sulfated and unsulfated derivatives of the DHPs display anticoagulant activity, which is dramatically higher than that of the reference polyacrylic acids. More interestingly, this activity approaches that of low-molecular-weight heparin with the sulfated derivative showing approximately 2- to 3-fold greater potency than the unsulfated parent. Studies on the inhibition of factor Xa and thrombin indicate that the oligomers exert their anticoagulant effect through both direct and indirect inhibition mechanisms. This dual inhibition property of 4-hydroxycinnamic acid-based DHP oligomers is the first example in inhibitors of coagulation. This work puts forward a novel, non-heparin structure, which may be exploited for the design of potent, dual action inhibitors of coagulation through combinatorial virtual screening on a library of DHP oligomers.

Biosynthesis of podophyllotoxin in Linum album cell cultures.

Cell cultures of Linum album Kotschy ex Boiss. (Linaceae) showing high accumulation of the lignan podophyllotoxin (PTOX) were established. Enzymological studies revealed highest activities of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, 4-hydroxycinnamate:CoA ligase and cinnamoyl-CoA:NADP oxidoreductase immediately prior to PTOX accumulation. To investigate PTOX biosynthesis, feeding experiments were performed with [2-(13)C]3',4'-dimethoxycinnamic acid, [2-(13)C]3',4'-methylenedioxycinnamic acid (MDCA), [2-(13)C]3',4',5'-trimethoxycinnamic acid, [2-(13)C]sinapic acid, [2-(13)C]- and [2,3-(13)C(2)]ferulic acid. Analysis of the metabolites by HPLC coupled to tandem mass spectrometry revealed incorporation of label from ferulic acid into PTOX and deoxypodophyllotoxin (DOP). In addition, MDCA was also unambiguously incorporated intact into PTOX. These observations suggest that in L. album both ferulic acid and methylenedioxy-substituted cinnamic acid can be incorporated into lignans. Furthermore, it appears that, in this species, the hydroxylation of DOP is a rate-limiting point in the pathway leading to PTOX. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/wo.1007/s00425-002-0834-1.

Synthesis and characterization of N-coumaroyltyramine as a potent phytochemical which arrests human transformed cells via inhibiting protein tyrosine kinases.

Numerous phytochemicals are believed to have beneficial effects on human health. N-Coumaroyltyramine accumulates in plants in response to wounding and pathogen attack. Due to the scarcity of N-coumaroyltyramine, its biological activities have not been studied in human cells. In this study, N-coumaroyltyramine was chemically synthesized and then purified by an HPLC with a UV-visible absorbance detector. Retention times of major peaks were 14.3 and 20.7 min, and the peak at 20.7 min was confirmed by LC-MS as N-coumaroyltyramine with a mass/charge (m/z) unit of 284.1. The synthesis procedure was relatively easy and had an acceptable yield (approximately 55%). The compound exhibited a new activity, suppression of growth of human tumor cells such as U937 and Jurkat cells. In addition, the suppressed growth of the cells was strongly associated with an increased percentage of cells in the S phase of the cell cycle progression. Furthermore, N-coumaroyltyramine was able to inhibit the protein tyrosine kinases including epidermal growth factor receptor (EGFR). This is the first report of the growth suppressing activity of N-coumaroyltyramine and its arrest of cells at the S phase of the cell cycle, possibly by inhibition of protein tyrosine kinases.

Synthesis and anti-human immunodeficiency virus type 1 integrase activity of hydroxybenzoic and hydroxycinnamic acid flavon-3-yl esters.

A series of new hydroxybenzoic and hydroxycinnamic acid flavon-3-yl esters were synthesized in order to obtain compounds targeting the human immunodeficiency virus (HIV) type 1 integrase (IN). The esters were tested for anti-IN and anti-reverse transcriptase (RT) activity in enzyme assays and for anti-HIV-1, anti-proliferative and anti-topoisomerase activity in cell-based assays. In enzyme assays, the two gallic acid flavon-3-yl esters showed a notable IN inhibition (IC50 values were 8.3 and 9.1 microM, respectively), while the two caffeic acid flavon-3-yl esters exhibited a modest activity (IC50 75 and 60 microM, respectively). Replacement of hydroxyl groups resulted in loss of potency. Caffeic acid 3',4'-dichloroflavon-3-yl ester also inhibited the RT activity whereas it was not active on human topoisomerases. It therefore represents an interesting example of a compound specifically targeting more than one step of the virus replication cycle.