Extractability of polyphenols of sunflower seed in various solvents

The extractability of chlorogenic acid from defatted sunflower seed flour in water and salt solutions at different pH values and also in aqueous organic solvents was determined. It increased with increase in pH and at pH 8 in water nearly 70% chlorogenic acid was removed in a single extraction, while NaCl did not increase the extraction, and, MgCl₂ and CaCl₂ increased it, especially at higher concentrations. Methanol, ethanol, isopropanol and acetone, at 20% concentration in water, caused the maximum extraction of polyphenol. These organic solvents without added water were poor solvents for the extraction of polyphenol from the flour.     

Identification of free radical species derived from caffeic acid and related polyphenols

Polyphenols are widely distributed in various fruits, vegetables and seasonings. It is well known that they have several physiological effects due to their antioxidative activities. Their activities depend on structural characteristics that favour the formation of their corresponding stable radicals. During the examination at which pH values, the polyphenol radicals are stabilized, we confirmed that polyphenol radicals were stabilized in NaHCO₃/Na₂CO₃ buffer (pH 10) rather than in physiological pH region. Then, we measured electron spin resonance (ESR) spectra at pH 10 to examine the characteristics of free radical species derived from caffeic acid (CA) with an unsaturated side chain, dihydrocaffeic acid (DCA) with a saturated side chain, chlorogenic acid (ChA) and rosmarinic acid (RA). In analyzing the radical structures, ESR simulation, determinations of macroscopic and microscopic acid dissociation constants and molecular orbital (MO) calculation were performed. In CA, the monophenolate forms were assumed to participate in the formation of free radical species, while in DCA, the diphenol form and the monophenolate forms were presumed to contribute to the formation of free radical species. On the basis of the results, we propose the possible structures of the free radical species formed from polyphenols under alkaline conditions.     

Enzymatic synthesis of a catecholic polyphenol product with excellent antioxidant activity

Abstract

Polyphenols, especially catecholic stilbene derivatives, have attracted much attention due to the huge pharmacological effects and promising health benefits. However, their chemical synthesis via regioselective ortho-hydroxylation on aromatic rings is highly challenging. In this study, 3′-hydroxypterostilbene (HPS) is taken as a model product due to its strong potential as an antitumor agent. One-step enzymatic synthesis of HPS from pterostilbene (PS) was explored, with immobilised tyrosinase as catalyst. The impact of solvent, pH, temperature, oxygen and reductant concentration on the reaction was investigated, and the conversion was optimised by employing the response surface methodology (RSM). Finally, a high yield of 77.9% was obtained in 2.7?h. This study demonstrates the first successful use of a biotechnological strategy to synthesise HPS. The antioxidant activities of both PS and HPS were evaluated by using the DPPH assay, demonstrating that HPS is more potent than PS as a radical scavenger.     

Syntheses of procyanidin B2 and B3 gallate derivatives using equimolar condensation mediated by Yb(OTf)3 and their antitumor activities

Synthesis of procyanidin B2 and B3 gallate derivatives, 3-O-gallate, 3″-O-gallate, and 3,3″-di-O-gallate, were synthesized using equimolar condensation mediated by Yb(OTf)₃. Synthesized compounds showed significant antitumor effects against human prostate PC-3 cell lines. Their activities were weaker than well-known EGCG and prodelphinidin B3.     

Antioxidant and prooxidant effects of polyphenol compounds on copper-mediated DNA damage

Inhibition of copper-mediated DNA damage has been determined for several polyphenol compounds. The 50% inhibition concentration values (IC₅₀) for most of the tested polyphenols are between 8 and 480 μM for copper-mediated DNA damage prevention. Although most tested polyphenols were antioxidants under these conditions, they generally inhibited Cu^I-mediated DNA damage less effectively than Fe^II-mediated damage, and some polyphenols also displayed prooxidant activity. Because semiquinone radicals and hydroxyl radical adducts were detected by EPR spectroscopy in solutions of polyphenols, Cu^I, and H₂O₂, it is likely that weak polyphenol-Cu^I interactions permit a redox-cycling mechanism, whereby the necessary reactants to cause DNA damage (Cu^I, H₂O₂, and reducing agents) are regenerated. The polyphenol compounds that prevent copper-mediated DNA damage likely follow a radical scavenging pathway as determined by EPR spectroscopy.     

The use of diversity profiling to characterize chemical modulators of the histone deacetylases

Target specificity and off-target liabilities are routinely monitored during the early phases of drug discovery for most kinase projects. Typically these criteria are evaluated using a profiling panel comprised of a diverse collection of in vitro kinase assays and relates compound structure to potency and selectivity. The success of these efforts has led to the design of similar panels for phosphatase, protease, and epigenetic targets. Here the implementation of an epigenetic profiling panel, comprised of eleven histone deacetylases (HDACs) and one histone acetyltransferase (HAT), was used to evaluate chemical modulators of these enzymes. HDAC inhibitors (HDACi) such as sodium butyrate and trichostatin A demonstrate diverse biological effects which have led to broad speculation about their therapeutic potential in multiple disease states. Some HDACi have demonstrated tumor suppression in vivo and recently Zolinza was the first HDACi approved by the FDA for the treatment of cutaneous T-cell lymphoma. While HDACi have demonstrated therapeutic utility, many of the first generation compounds are pan-inhibitors. Thus, use of an HDAC profiling panel will be essential in achieving isoform specificity of the next generation of inhibitors. To this end, twenty-one compounds, twelve of which are known to have activities against the HDACs, were tested to evaluate the utility of the epigenetic panel. Additionally, these compounds were tested against a larger 72 member enzyme panel comprised of kinase, phosphatase and protease activities. This effort represents the first time these compounds have been profiled with such a broad range of biochemical activities.     

Combined application of botanical formulations and biocontrol agents for the management of Fusarium oxysporum f. sp. cubense (Foc) causing Fusarium wilt in banana

Plant products along with biocontrol agents were tested against Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense (Foc). Of the 22 plant species tested, the leaf extract of Datura metel (10%) showed complete inhibition of the mycelial growth of Foc. Two botanical fungicides, Wanis 20 EC and Damet 50 EC along with selected PGPR strains with known biocontrol activity, Pseudomonas fluorescens 1, Pf1 and Bacillus subtilis, TRC 54 were tested individually and in combination for the management of Fusarium wilt under greenhouse and field conditions. Combined application of botanical formulation and biocontrol agents (Wanis 20 EC + Pf1 + TRC 54) reduced the wilt incidence significantly under greenhouse (64%) and field conditions (75%). Reduction in disease incidence was positively correlated with the induction of defense-related enzymes peroxidase (PO) and polyphenol oxidase (PPO). Three antifungal compounds (two glycosides and one ester) in D. metel were separated and identified using TLC, RP-HPLC (Reverse Phase-High Pressure Liquid Chromatography) and mass spectrometry. In this study it is clear that combined application of botanical formulations and biocontrol agents can be very effective in the management of Fusarium wilt of banana.     

Rosmarinic acid and its esters inhibit membrane cholesterol domain formation through an antioxidant mechanism based,in nonlinear fashion,on alkyl chain length

BackgroundUnder conditions of oxidative stress, cholesterol aggregates into discrete membrane bilayer domains that precipitate the formation of extracellular crystals, a feature of advanced atheroma in cardiovascular disease. Therapeutic interventions using membrane-directed antioxidants, such as polyphenolic esters, may reduce cholesterol domains and crystal formation. In this study, the effects of rosmarinic acid (RC0) and rosmarinic esters, with alkyl chain lengths ranging from 4 to 16?carbons (RC4-RC16), on membrane lipid oxidation and cholesterol domain formation were investigated.MethodsModel membranes were prepared with 1,2-dilinoleoyl-sn-glycero-3-phosphocholine and cholesterol at different cholesterol-to-phospholipid mole ratios (0.3:1, 0.9:1, and 1.2:1), in the absence or presence of each molecule and exposed to 72 h of oxidation. Changes in lipid hydroperoxide (LOOH) and cholesterol domain formation were measured using iodometric and small angle x-ray diffraction approaches, respectively.ResultsRosmarinic acid and its esters had differential effects on LOOH formation based on alkyl chain length. RC8 exhibited the greatest antioxidant effect, reducing LOOH levels by 82%, and inhibited cholesterol domain formation. By contrast, RC0 and RC16 failed to inhibit either LOOH formation or cholesterol domain formation.ConclusionThese data indicate that the membrane antioxidant and cholesterol domain inhibition activities of rosmarinic acid esters are dependent, nonlinearly, on alkyl chain length. The mechanism for this effect is attributed to the influence of alkyl chain length on the optimal depth of the polyphenols into the lipid bilayer for trapping free radicals.General significanceThese findings provide insight into novel atheroprotective benefits of polyphenol esters that are dependent on their membrane location.     

Expression and biochemical analysis of codon-optimized polyphenol oxidase from Camellia sinensis (L.) O. Kuntze in E. coli

Polyphenol oxidases (PPOs) are copper-containing industrially important enzymes that catalyze the synthesis of many commercially important products by using polyphenols as substrate. Camellia sinensis polyphenol oxidase (CsPPO) is interesting because it oxidizes epicatechins to yield theaflavins and thearubigins. The present study aimed to optimize the expression of CsPPO in Escherichia coli. Because CsPPO had a large number of E. coli rare codons, it yielded a poor quantity of protein in E. coli Rosetta™ 2 cells, which have additional tRNAs for E. coli rare codons. Thus, synthetically constructed codon-optimized CsPPO was cloned into pET-47b(+) vector and expressed in a bacterial host. Ectopic expression led to the formation of inclusion bodies. However, extensive standardization of buffers and methods of refolding such as dialysis, on-column refolding, and rapid dilution yielded active PPO from solubilized inclusion bodies with copper content of 0.880 ± 0.095 atom/molecule of protein.Experimental data produced maximum PPO activity in a rapid dilution buffer containing 0.5 M L-arginine. Refolded CsPPO had an optimum pH of 5.0 and K_m values of 3.10, 0.479, and 0.314 mM, and a V_max of 163.9, 82.64, and 142.8 U/mg of protein for catechol, catechin, and epicatechin, respectively.     

Immobilization of polyphenol oxidase on chitosan–SiO<Subscript>2</Subscript> gel for removal of aqueous phenol

A partially purified potato polyphenol oxidase (PPO) was immobilized in a cross-linked chitosan–SiO₂ gel and used to treat phenol solutions. Under optimized conditions (formaldehyde 20 mg/ml, PPO 4 mg/ml and pH 7.0), the activity of immobilized PPO was 1370 U/g and its K _m value for catechol was 12 mm at 25°C. The highest activity of immobilized enzyme was at pH 7.4. Immobilization stabilized the enzyme with 73 and 58% retention of activity after 10 and 20 days, respectively, at 30°C whereas most of the free enzyme was inactive after 7 days. The efficiency of removing phenol (10 mg phenol/l) by the immobilized PPO was 86%, and about 60% removal efficiency was retained after five recycles. The immobilized PPO may thus be a useful for removing phenolic compounds from industrial waste-waters.