Versatile synthesis of epicatechin series procyanidin oligomers, and their antioxidant and DNA polymerase inhibitory activity

Proanthocyanidins, known as condensed tannins or oligomeric flavonoids, exist in many edible plants and show various interesting biological activities. We have developed a simple and versatile method of synthesizing procyanidin oligomers consisting of (−)-epicatechin and (+)-catechin. This method is applicable to the synthesis of various 3-O-substituted oligomers. We report here the stereoselective and length controlled synthesis of [4-8]-condensed (−)-epicatechin series procyanidin oligomers. We described the details of the synthesis of an two tetramers, (−)-epicatechin-(−)-epicatechin-(−)-epicatechin-(−)-epicatechin and (−)-epicatechin-(−)-epicatechin-(−)-epicatechin-(+)-catechin (arecatannin A1), (−)-epicatechin pentamer and two 3,3″,3?-tri-O-galloyl trimers, (−)-epicatechin-(−)-epicatechin-(−)-epicatechin-3,3″,3?-tri-O-gallate and (−)-epicatechin-(−)-epicatechin-(+)-catechin-3,3″,3?-tri-O-gallate with the condensation method using TMSOTf as a catalyst. The ability of DPPH radical scavenging activity and DNA polymerase inhibitory activity of these oligomeric compounds were investigated.     

Method development for determination of (+)‐catechin and (−)‐epicatechin by micellar electrokinetic chromatography: Annual characterization of field grown blackberries

Berries are a rich source of antioxidants compounds, among which is the catechin group. Determination of the monomers (catechin and epicatechin) in fruits is a first step in the way to establish a relationship between polyphenols and their effects on human health. The purpose of this work is to develop a method to determine free catechins in blackberry by MEKC and to characterize levels of catechins in fresh fruits of Rubus fruticosus var. Lochness throughout the annual production period. A methanolic extract was prepared from fresh fruit. Then, it was evaporated and the residue was extracted with diethyl ether. MEKC conditions: phosphoric acid, 30 mmol/L; SDS, 40 mmol/L and triethylamine, 0.1% v/v at pH 2.3; ?15 kV of voltage; 10‐s hydrodynamic injection; 25°C temperature; and detection at 200 nm. Instrumental and interday precision were lower than 4.7 and 10% RSD, respectively. Only (?)‐epicatechin was quantified in blackberries and ranged from 120 to 620 mg/kg fresh weight, which were the lowest values in December and the highest in June. A solid–liquid extraction and an MEKC method were successfully applied to determine (?)‐epicatechins in blackberry for the first time. A strong dependence of (?)‐epicatechin on the annual average temperature was observed.     

Epicatechin,procyanidins, and phenolic microbial metabolites after cocoa intake in humans and rats

Proanthocyanidins, flavonoids exhibiting cardiovascular protection, constitute a major fraction of the flavonoid ingested in the human diet. Although they are poorly absorbed, they are metabolized by the intestinal microbiota into various phenolic acids. An analytical method, based on an optimized 96-well plate solid-phase extraction (SPE) procedure and liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) for the analysis of 19 phenolic microbial metabolites and monomeric and dimeric flavanols in urine samples, was developed and validated. Human urine samples were obtained before and after ingestion of an acute consumption of 40 g of soluble cocoa powder and rat urines before and after the prolonged administration (2 weeks) of different diets composed of natural cocoa powder. The mean recovery of analytes using the new SPE-LC-MS/MS method ranged from 87% to 109%. Accuracy ranged from 87.5% to 113.8%, and precision met acceptance criteria (<15% relative standard deviation). Procyanidin B2 has been detected and quantified for the first time in human and rat urine after cocoa consumption. Changes in human and rat urinary levels of microbial phenolic acids and flavanols were in the range of 0.001–59.43 nmol/mg creatinine and of 0.004–181.56 nmol/mg creatinine, respectively. Major advantages of the method developed include reduction of laboratory work in the sample preparation step by the use of 96-well SPE plates and the sensitive measurement of a large number of metabolites in a very short run time, which makes it ideal for use in epidemiological studies. ^a INGENIO-CONSOLIDER Program, Fun-c-food CSD2007-063 ^b CIBER 06003 Physiopathology of Obesity and Nutrition (CIBEROBN), and RETICS RD06/0045/0003, Instituto de Salud Carlos III     

Microwave‐assisted extraction followed by CE for determination of catechin and epicatechin in green tea

In this work, for the first time, microwave‐assisted extraction (MAE) followed by CE was developed for the fast analysis of catechin and epicatechin in green tea. In the proposed method, catechin and epicatechin in green tea samples were rapidly extracted by MAE technique, and then analyzed by CE. The MAE conditions and the method's validation were studied. It is found that the extraction time of 1 min with 400 W microwave irradiation is enough to completely extract catechin and epicatechin in green tea sample, whereas the conventional ultrasonic extraction (USE) technique needs long extraction time of 60 min. The method validations were also studied in this work. The calibration curve shows good linearity in 0.01–3 mg/mL for catechin (R²=0.993), and 0.005–3 mg/mL for epicatechin (R²=0.996), respectively. The RSD values for catechin and epicatechin are 0.65 and 2.58%, respectively. This shows that the proposed method has good reproducibility. The proposed method has good recoveries, which are 118% for catechin and 120% for epicatechin. The proposed method was successfully applied to determination of the catechin and epicatechin in different green tea samples. The experiment results have demonstrated that the MAE following CE is a simple, fast and reliable method for the determination of catechin and epicatechin in green tea.     

Separation of Phenols from the Leaves of Toona Sinensis (Meliaceae) by Capillary Electrophoresis

A micellar electrokinetic capillary chromatography (MEKC) method, using UV detection, was developed for the determination of polyphenols in Toona sinensis (Meliaceae); the procedure involved precipitation of polyphenols from the leaves of T. sinensis using methanol. The structures can be established with fifteen compounds including methyl gallate, gallic acid, kaempferol, quercitin, quercitrin, rutin, kaempferol‐glucoside, catechin, epicatechin, stearic acid, palmitic acid, β‐sitosterol, stigmasterol, β‐sitosteryl‐glucoside, and stigmasteryl‐glucoside by spectroscopic analysis. However, there has been no investigation to quantitate the polyphenols that form T. sinensis. Thus, seven polyphenols of T. sinensis with UV absorbance, catechin (C), epicatechin (EC), methyl gallate (MG), rutin (R), gallic acid (G), quercitrin (Q), and kaempferol (K) were separated within 10 min with a 40 cm uncoated fused‐silica column, with the RSD < 3% (migration times), voltage at 15 kV using this method. On‐column detection was carried out at 254 nm. The detection limit of this method for all analytes ranged from 19.5 to 0.02 μM (RSD < 3.1%). The method provided a rapid and sensitive identification of polyphenols of interest in T. sinensis and is suitable for biological activity studies.     

Asymmetric total synthesis of B-ring modified (−)-epicatechin gallate analogues and their modulation of β-lactam resistance in Staphylococcus aureus

Two enantiomerically pure B-ring modified analogues of (−)-epicatechin gallate were synthesised and their modulation of β-lactam resistance using three strains of methicillin resistant Staphylococcus aureus (BB 568, EMRSA-15 and EMRSA-16) evaluated. Sub-inhibitory concentrations (12.5 and 25 mg/L) of the two analogues fully sensitised each of the three MRSA strains to oxacillin, reducing the MIC to less than 0.5 mg/L, identical to levels achieved with ECg. Lower concentrations demonstrated that the position and degree of hydroxylation of the B-ring is important for activity.     

Adsorption Isotherms of Quercetin and Catechin Compounds on Quercetin-MIP

A molecular imprinted polymer(MIP)was prepared with quercetin as the template and methacrylic acid(MAA)as the functional monomer.Acetonitrile and methanol were used as the porogen with ethylene glycol dimethacrylate(EGDMA)as the crosslinker and 2,2'-azobis(isobutyronitrile)(AIBN)as the initiator.The experimental parameters of the equilibrium isotherms were estimated via linear and nonlinear regression analyses.The linear equation as the functions of the adsorption concentration of the single compound in its solution and the competitive adsorption of the single compound in its mixed compounds solution was then expressed,and the adsorption equilibrium data were correlated to Langmuir and Freundlich isotherm models.The mixture compounds show competitive adsorption on the specific binding sites of quercetin-MIP.Furthermore,the competitive Langmuir isotherms were applied to the mixture compounds.The adsorption concentrations of quercetin,( )catechin( C),and(-)epicatechin(EC)on the quercetin molecular imprinted polymer were compared.The quercetin-imprinted polymer shows extraordinarily higher adsorption ability for quercetin than for the two catechin compounds that were also assessed.     

Selective determination of catechin, epicatechin and ascorbic acid in human urine using chiral capillary electrophoresis

Chiral CE was successfully applied to the separation and quantification of catechin, epicatechin and ascorbic acid in some commercial drinks and human urine. Analysis involved the separation of analytes in less than 5.0 min at 240 nm with an untreated fused-silica capillary under hydrodynamic injection mode. The running buffer consisted of 50 mM borate buffer with 3 mM beta-CD at pH 8.35. Detection limits for catechin, epicatechin and ascorbic acid were 0.028, 0.011 and 0.004 microg/mL, respectively. Linearity was investigated by selecting the ranges of calibration according to the amount of analytes in urine giving correlation coefficient percent (% r(2)) ranging between 99.4 and 99.6 at 99% confidence level. The maximum urinary excretion of catechin and epicatechin were noted at 2.0 and 4.0 h of the administrated dose. Unchanged catechin, epicatechin and ascorbic acid amounted to about 1.500, 8.696 and 0.003% of the administered dose in the 48.0 h urine collection. The proposed method achieved 99.2% completeness (n = 20) in urine media.     

New methods for spectrometric peak purity analysis in chromatography

Simple methods for peak purity analysis in chromatography are proposed for accurately assaying the chromatographic or electrophoretic process by spectroscopy. With these methods, the purity at each point of a chromatographic peak containing an unknown type and number of components can be detected and visualized. The ‘absorbance (A) diagram' is constructed by plotting the absorbance values of different wavelengths against each other. When only straight lines are obtained for different wavelength combinations, only one component was detected in the corresponding peak. Bent curves in the A diagrams mean that at least two components were registered. Straight lines in the absorbance difference quotient (ADQ) diagrams indicate that two components occur in the corresponding peak. Bent curves signify that at least three components were registered. The new methods are demonstrated using the HPLC technique for the separation of chlorogenic acid and epicatechin. Integral A and ADQ diagrams and the corresponding mean diagrams complete the new concept.     

Targeted metabolic profiling of phenolics in urine and plasma after regular consumption of cocoa by liquid chromatography–tandem mass spectrometry

The biological properties of cocoa (Theobroma cacao L.) polyphenols are strictly dependent on their bioavailability. A long-term cocoa feeding trial was performed with subjects at high risk for cardiovascular disease. Subjects (n = 42) received two sachets of 20 g of cocoa powder/day with 250 mL of skimmed milk each, or only 500 mL/day of skimmed milk, both for two 4-week periods. The phenolic metabolic profile including phase II conjugated metabolites and phenolic acids derived from the intestinal microbiota was determined by LC-MS/MS in both 24-h urine and fasting plasma. The analysis of 24-h urine revealed significant increases of phase II metabolites, including glucuronides and sulfate conjugates of (−)-epicatechin, O-methyl-epicatechin, 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone and 5-(3′-methoxy-4′-hydroxyphenyl)-γ-valerolactone, after regular cocoa intake. In the case of plasma, only glucuronide conjugates of dihydroxyphenylvalerolactones increased. Regular consumption of cocoa also resulted in a significant increase in the urinary excretion of colonic microbial-derived phenolic metabolites, including vanillic, 3,4-dihydroxyphenylacetic and 3-hydroxyphenylacetic acids, and particularly 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone, whereas only the two latter metabolites showed a significant increase in fasting plasma. The results found herein indicate that 5-(3′,4′-dihydroxyphenyl)-γ-valerolactone and hydroxyphenylacetic acids could be good biomarkers of the regular consumption of cocoa and therefore, of flavanol-rich foods.