Effects of ultrasound on the enzymatic degradation of pectin.

Ultrasound-assisted enzymatic maceration (UAEM) has gained considerable interest in the fruit juice industry, owing to its potential to increase juice yield and content of polyphenols while simultaneously saving time and energy. In this study, the effects of UAEM (ultrasonic probe, 20 kHz, 21 W*cm-2 and 33 W*cm-2) on pectin degradation in a continuous circulation system were investigated over 60 and 90 min. Main pectinolytic enzymes activities of (polygalacturonase, pectin lyase and pectin methylesterase) of a commercial enzyme preparation were examined for individual synergistic effects with US. Pectin hydrolysis by UAEM differed significantly compared to treatment with ultrasound or enzymes alone regarding the profile of degradation products compared to treatment with ultrasound or enzymes alone. Ultrasound fragmented pectin to less branched oligomers of medium molecular weight (Mp approx. 150 kDa), which were further degraded by pectinolytic activities. The low molecular weight fraction (<30 kDa), which is known to be beneficial for juice-quality by adding nutritional value and stabilizing polyphenols, was enriched in small oligomers of homogalacturonan-derived, rhamnogalacturonan I-derived, and rhamnogalacturonan II-derived residues. Synergistic effects of ultrasound application enhanced the effective activities of polygalacturonase and pectin lyase and even prolonged their performance over 90 min, whereas the effective activity of pectin methylesterase was not affected. Final marker concentrations determined by each enzyme assay revealed a considerable higher total process output after UAEM treatment at reduced temperature (30 °C) comparable to the output after conventional batch maceration at 50 °C. The obtained results demonstrate the high potential of UAEM to produce high-quality juice by controlling pectin degradation while reducing process temperature and equally highlight the matrix and enzyme specific effects of a simultaneous US treatment.

Enhancement of Biomimetic Enzymatic Mineralization of Gellan Gum Polysaccharide Hydrogels by Plant-Derived Gallotannins.

Mineralization of hydrogel biomaterials with calcium phosphate (CaP) is considered advantageous for bone regeneration. Mineralization can be both induced by the enzyme alkaline phosphatase (ALP) and promoted by calcium-binding biomolecules, such as plant-derived polyphenols. In this study, ALP-loaded gellan gum (GG) hydrogels were enriched with gallotannins, a subclass of polyphenols. Five preparations were compared, namely three tannic acids of differing molecular weight (MW), pentagalloyl glucose (PGG), and a gallotannin-rich extract from mango kernel (Mangifera indica L.). Certain gallotannin preparations promoted mineralization to a greater degree than others. The various gallotannin preparations bound differently to ALP and influenced the size of aggregates of ALP, which may be related to ability to promote mineralization. Human osteoblast-like Saos-2 cells grew in eluate from mineralized hydrogels. Gallotannin incorporation impeded cell growth on hydrogels and did not impart antibacterial activity. In conclusion, gallotannin incorporation aided mineralization but reduced cytocompatibility.

Interactions of Anthocyanins with Pectin and Pectin Fragments in Model Solutions.

Anthocyanins determine the color and potential health-promoting properties of red fruit juices, but the juices contain remarkably less anthocyanins than the fruits, which is partly caused by the interactions of anthocyanins with the residues of cell wall polysaccharides like pectin. In this study, pectin was modified by ultrasound and enzyme treatments to residues of polysaccharides and oligosaccharides widely differing in their molecular weight. Modifications decreased viscosity and degrees of acetylation and methylation and released smooth and hairy region fragments. Native and modified pectin induced different effects on the concentrations of individual anthocyanins after short-term and long-term incubation caused by both hydrophobic and hydrophilic interactions. Results indicate that both pectin and anthocyanin structure influence these interactions. Linear polymers generated by ultrasound formed insoluble anthocyanin complexes, whereas oligosaccharides produced by enzymes formed soluble complexes with protective properties. The structure of the anthocyanin aglycone apparently influenced interactions more than the sugar moiety.

Hemisynthesis of Anthocyanin Phase II Metabolites by Porcine Liver Enzymes.

The aim of this work was to obtain phase II metabolites of cyanidin-3- O-glucoside and its aglycone using porcine liver enzymes. For this purpose, anthocyanins extracted from blackberry concentrate and containing mostly cyanidin-3- O-glucoside were incubated with the S9, microsomal, and cytosolic fractions of porcine liver. The reactions were targeted to the direction of the respective phase II transformation by the addition of activated cofactors. LC-MS n and LC-IMS-QTOF-MS analyses showed that one methylated, three glucuronidated and three sulfated metabolites of cyanidin-3- O-glucoside were generated. The aglycone, cyanidin, was sulfated and glucuronidated by the liver enzymes. In addition, both were glucuronidated and methylated simultaneously. The detected compounds and the generated data like exact masses, mass spectra, and CCS values may serve as a basis in the search for metabolites formed in vivo. As their effects are largely unexplored, the described synthesis may contribute to a better understanding of the metabolism of anthocyanins.

A nutritive dose of pure (-)-epicatechin does not beneficially affect increased cardiometabolic risk factors in overweight-to-obese adults-a randomized, placebo-controlled, double-blind crossover study.

Background: Regular cocoa consumption has been shown to reduce blood pressure, improve lipid profiles, and increase insulin sensitivity and flow-mediated dilatation in healthy adults. It is assumed that these effects can be attributed to polyphenolic cocoa ingredients such as flavanols, especially to (-)-epicatechin. Nutritive intervention studies to prove this hypothesis are scarce. Objective: We aimed to evaluate whether regular consumption of 25 mg of pure (-)-epicatechin can affect increased cardiometabolic risk factors [blood pressure, glucose and lipid metabolism, low-density lipoprotein (LDL) oxidation] in overweight-to-obese subjects. Design: Forty-eight overweight or obese nonsmokers [body mass index (kg/m2) ≥25.0, ages 20-65 y] with clear signs of metabolic syndrome (blood pressure ≥130/85 mm Hg, glucose >5.55 mmol/L, or triglycerides >1.69 mmol/L or cholesterol >5.2 mmol/L in fasting blood) and without chronic diseases were included in a randomized, placebo-controlled, double-blind crossover study. Participants ingested daily 25 mg (-)-epicatechin (encapsulated) or placebo for 2-wk in random order (2-wk washout). After an overnight fast, blood pressure was monitored and blood samples were collected before and after both treatments. Anthropometric data were determined at each visit. Dietary intake was assessed by 3-d food records during both treatments and during run-in and washout phase. Results: Supplementation of pure (-)-epicatechin did not significantly affect blood pressure, glucose, insulin, homeostasis model assessment of insulin resistance, triglycerides, or total, LDL, or HDL cholesterol. Oxidized LDL, vitamins C and E, and ß-carotene in plasma were not modulated. Body weight, fat mass, fat distribution, and the intake of energy, nutrients, and (-)-epicatechin from food remained stable throughout the study. Conclusions: Daily intake of 25 mg of pure (-)-epicatechin for 2 wk does not reduce cardiometabolic risk factors in overweight-to-obese adults. Thus, the hypothesis that the cardioprotective effects of regular cocoa consumption are exclusively ascribed to (-)-epicatechin should be reconsidered. The study was registered at the German Clinical Trial Register as DRKS-ID: DRKS00009846.

Characterization of carotenoid profiles in goldenberry (Physalis peruviana L.) fruits at various ripening stages and in different plant tissues by HPLC-DAD-APCI-MSn.

Carotenoid profiles of goldenberry (Physalis peruviana L.) fruits differing in ripening states and in different fruit fractions (peel, pulp, and calyx of ripe fruits) were investigated by HPLC-DAD-APCI-MSn. Out of the 53 carotenoids detected, 42 were tentatively identified. The carotenoid profile of unripe fruits is dominated by (all-E)-lutein (51%), whereas in ripe fruits, (all-E)-ß-carotene (55%) and several carotenoid fatty acid esters, especially lutein esters esterified with myristic and palmitic acid as monoesters or diesters, were found. In overripe fruits, carotenoid conversion products and a higher proportion of carotenoid monoesters to diesters compared to ripe fruits were observed. Overripe fruits showed a significant decrease in total carotenoids of about 31% due to degradation. The observed conversion and degradation processes included epoxidation, isomerization, and deesterification. The peel of ripe goldenberries showed a 2.8 times higher total carotenoid content of 332.00 µg/g dw compared to the pulp.

Characterization of phenolic and other polar compounds in peel and flesh of pink guava (Psidium guajava L. cv. 'Criolla') by ultra-high performance liquid chromatography with diode array and mass spectrometric detection.

Pink guava (Psidium guajava L.) is a highly consumed fruit in tropical countries. Despite of interesting research on health effects of this fruit, investigations into the profile of secondary plant metabolites are scarce. In this study, the phenolic compounds in the peel and flesh of pink guava were characterized by ultra-high performance liquid chromatography with diode array and mass spectrometric detection. Sixty phenolic compounds were characterized by MS2 and classified as ellagitannins, flavones, flavonols, flavanols, proanthocyanidins, dihydrochalcones, and anthocyanidins, and non-flavonoids such as phenolic acid derivatives, stilbenes, acetophenones, and benzophenones. Forty-two polyphenols are reported for the first time in both peel and flesh, and twenty-four compounds were detected for the first time in P. guajava, e.g., phlorizin, nothofagin, astringin, chrysin-C-glucoside, valoneic acid bilactone, cinnamoyl-glucoside, and two dimethoxycinnamoyl-hexosides.

Profiling of iridoid glycosides in Vaccinium species by UHPLC-MS.

The iridoid profile of four Vaccinium species was investigated using UHPLC-MS to obtain further information about this group of species for phytochemical characterization. Fruits of bog bilberry (Vaccinium uliginosum L.) showed 14 different iridoid glycosides with a total amount of 20mg/kg fresh weight (FW), whereas bilberry (Vaccinium myrtillus L.) contained 11 iridoid glycosides and a total amount of 127mg/kg FW. Highbush blueberry (Vaccinium corymbosum L.) and lowbush blueberry (Vaccinium angustifolium L.) contained none of the investigated iridoid glycosides. Among the different iridoids, the isomers scandoside and deacetylasperulosidic acid as well as a dihydro derivative thereof were described for the first time in the Ericaceae family. The p-coumaroyl isomers of scandoside, deacetylasperulosidic acid and dihydromonotropein are reported for the first time in V. myrtillus and V. uliginosum. Monotropein and its p-coumaroyl isomers were found for the first time in V. uliginosum. The comparison of iridoid profiles in bilberry fruit and juice samples revealed constant proportions throughout the juice processing. Quantification and profile determination of iridoids may be used for species differentiation and thus for authentication purposes.

Differentiation of Brazilian Peppertree (Schinus terebinthifolius Raddi) and Peruvian Peppertree (Schinus molle L.) Fruits by UHPLC-UV-MS Analysis of Their Anthocyanin and Biflavonoid Profiles.

The objectives of this work were to determine the phenolic profile of Schinus terebinthifolius and Schinus molle fruits and to develop a reliable method for the differentiation of these two similar spices both known as pink pepper. Anthocyanins, biflavonoids and gallotannins, some of which are reported for the first time in these species, were identified by UHPLC-UV/vis-MS/MS. Consideration of the relative and absolute amounts of phenolics as well as indicator compounds from 18 samples revealed that the relative amounts of anthocyanins and biflavonoids are the most trustworthy parameters. Principal component analysis and cluster analysis (CA) allowed a grouping of the samples according to their species, showing that the anthocyanins are most important for the identification of species. As a result, authentication of the two Schinus species can be accomplished by UHPLC analysis of the relative amounts of anthocyanins combined with CA.

Separation of alk(en)ylresorcinols from rye bran with saturated, monoenoic, dienoic, trienoic and hydroxylated monoenoic side chains using an octyl phase in ultra-high performance liquid chromatography and their differentiation by tandem mass spectrometrie.

Alkylresorcinols (ARs) occur in bran of cereals and in fruits from the Anacardiaceae family. Their separation by liquid chromatography is challenging, especially in rye (Secale cereale L.) that has a complex AR composition. An octyl phase (C8) with 1.8µm particles was used for the analysis of an acetone extract of rye bran. The ARs were detected by UV at 205 and 275nm and by MS applying selected ion monitoring (SIM) of known and hypothetical m/z values in positive and negative mode. The compounds found were subjected to product ion scans in a triple quadrupole mass spectrometer. The C8 UHPLC column has a suitable selectivity for the analysis of ARs from rye. In combination with the sub-2µm particles, baseline separation of most ARs was achieved. The MS2 spectra in positive mode show diagnostic fragments that allow identifying the ARs subclasses (saturated, monoenoic, dienoic, trienoic and hydroxylated monoenoic) unambiguously. Several minor ARs were detected for the first time: C23:3, C27:1OH, C20:0, C22:0, C24:0 and some minor alkenylresorcinol isomers. The chromatographic resolution on the C8 column is unprecedented in the field of rye ARs. Thus, isolation and quantification using non-mass-selective detectors is now possible for each AR. Since rye bran has the most complex AR composition, this method is expected to facilitate the analysis of ARs also in other samples.

Fast and comprehensive analysis of secondary metabolites in cocoa products using ultra high-performance liquid chromatography directly after pressurized liquid extraction.

Fast methods for the extraction and analysis of various secondary metabolites from cocoa products were developed and optimized regarding speed and separation efficiency. Extraction by pressurized liquid extraction is automated and the extracts are analyzed by rapid reversed-phase ultra high-performance liquid chromatography and normal-phase high-performance liquid chromatography methods. After extraction, no further sample treatment is required before chromatographic analysis. The analytes comprise monomeric and oligomeric flavanols, flavonols, methylxanthins, N-phenylpropenoyl amino acids, and phenolic acids. Polyphenols and N-phenylpropenoyl amino acids are separated in a single run of 33 min, procyanidins are analyzed by normal-phase high-performance liquid chromatography within 16 min, and methylxanthins require only 6 min total run time. A fourth method is suitable for phenolic acids, but only protocatechuic acid was found in relevant quantities. The optimized methods were validated and applied to 27 dark chocolates, one milk chocolate, two cocoa powders and two food supplements based on cocoa extract.

A fast isocratic liquid chromatography method for the quantification of xanthophylls and their stereoisomers.

A fast isocratic liquid chromatography method was developed for the simultaneous quantification of eight xanthophylls (13-Z-lutein, 13'-Z-lutein, 13-Z-zeaxanthin, all-E-lutein, all-E-zeaxanthin, all-E-canthaxanthin, all-E-ß-apo-8'-carotenoic acid ethyl ester and all-E-ß-apo-8'-carotenal) within 12 min, compared to 90 min by the conventional high-performance liquid chromatography method. The separation was achieved on a YMC C30 reversed-phase column (100 mm x 2.0 mm; 3 µm) operated at 20°C using a methanol/tert-butyl methyl ether/water solvent system at a flow rate of 0.8 mL/min. The method was successfully applied to quantify lutein and zeaxanthin stereoisomers in egg yolk, raw and cooked spinach, and a dietary supplement. The method can be used for the rapid analysis of xanthophyll isomers in different food products and for quality control purposes.

Influence of Accelerated Solvent Extraction and Ultrasound-Assisted Extraction on the Anthocyanin Profile of Different Vaccinium Species in the Context of Statistical Models for Authentication.

Anthocyanins are frequently discussed as marker compounds for fruit product authenticity. Proper analysis including sample preparation for the determination of anthocyanin concentrations is crucial for the comparability of authenticity data. The present study determined the influence of accelerated solvent extraction (ASE) and ultrasound-assisted extraction (UAE), using two different solvent compositions on the anthocyanin profile of bilberries (Vaccinium myrtillus L.), lowbush blueberries (Vaccinium angustifolium Ait.), and American cranberries (Vaccinium macrocarpon Ait.). Besides differences in total anthocyanin concentrations in the extracts, significant deviations (p ≤ 0.05) in the individual anthocyanin concentration were observed, resulting in differing anthocyanin proportions. Linear discriminant analysis comparing the differences caused by the extraction method to the natural differences within a set of 26 bilberry and lowbush blueberry samples of different origins was conducted. It revealed that profile variations induced by the extraction methods are in a similar scale to profile variations as a result of geographic and climatic differences.

Optimization and single-laboratory validation of a method for the determination of flavonolignans in milk thistle seeds by high-performance liquid chromatography with ultraviolet detection.

Seeds of milk thistle, Silybum marianum (L.) Gaertn., are used for treatment and prevention of liver disorders and were identified as a high priority ingredient requiring a validated analytical method. An AOAC International expert panel reviewed existing methods and made recommendations concerning method optimization prior to validation. A series of extraction and separation studies were undertaken on the selected method for determining flavonolignans from milk thistle seeds and finished products to address the review panel recommendations. Once optimized, a single-laboratory validation study was conducted. The method was assessed for repeatability, accuracy, selectivity, LOD, LOQ, analyte stability, and linearity. Flavonolignan content ranged from 1.40 to 52.86% in raw materials and dry finished products and ranged from 36.16 to 1570.7 µg/mL in liquid tinctures. Repeatability for the individual flavonolignans in raw materials and finished products ranged from 1.03 to 9.88% RSDr, with HorRat values between 0.21 and 1.55. Calibration curves for all flavonolignan concentrations had correlation coefficients of >99.8%. The LODs for the flavonolignans ranged from 0.20 to 0.48 µg/mL at 288 nm. Based on the results of this single-laboratory validation, this method is suitable for the quantitation of the six major flavonolignans in milk thistle raw materials and finished products, as well as multicomponent products containing dandelion, schizandra berry, and artichoke extracts. It is recommended that this method be adopted as First Action Official Method status by AOAC International.

The role of intestinal microbiota in development of irinotecan toxicity and in toxicity reduction through dietary fibres in rats.

CPT-11 is a drug used as chemotherapy for colorectal cancer. CPT-11 causes toxic side-effects in patients. CPT-11 toxicity has been attributed to the activity of intestinal microbiota, however, intestinal microbiota may also have protective effects in CP!-11 chemotherapy. This study aimed to elucidate mechanisms through which microbiota and dietary fibres could modify host health. Rats bearing a Ward colon carcinoma were treated with a two-cycle CPT-11/5-fluorouracil therapy recapitulating clinical therapy of colorectal cancer. Animals were fed with a semi-purified diet or a semi-purified diet was supplemented with non-digestible carbohydrates (isomalto-oligosaccharides, resistant starch, fructo-oligosaccharides, or inulin) in 3 independent experiments. Changes in intestinal microbiota, bacteria translocating to mesenteric lymphnodes, cecal GUD activity, and cecal SCFA production, and the intestinal concentration of CPT-11 and its metabolites were analysed. Non-digestible carbohydrates significantly influenced feed intake, body weight and other indicators of animal health. The identification of translocating bacteria and their quantification in cecal microbiota indicated that overgrowth of the intestine by opportunistic pathogens was not a major contributor to CPT-11 toxicity. Remarkably, fecal GUD activity positively correlated to body weight and feed intake but negatively correlated to cecal SN-38 concentrations and IL1-ß. The reduction in CPT-11 toxicity by non-digestible carbohydrates did not correlate to stimulation of specific bacterial taxa. However, cecal butyrate concentrations and feed intake were highly correlated. The protective role of intestinal butyrate production was substantiated by a positive correlation of the host expression of MCT1 (monocarboxylate transporter 1) with body weight as well as a positive correlation of the abundance of bacterial butyryl-CoA gene with cecal butyrate concentrations. These correlations support the interpretation that the influence of dietary fibre on CPT-11 toxicity is partially mediated by an increased cecal production of butyrate.

Purification of Phenylalkanoids and monoterpene glycosides from Rhodiola rosea L. roots by high-speed counter-current chromatography.

INTRODUCTION: Rhodiola rosea L. is a medicinal herb used for its adaptogenic properties. The main active components are the phenylpropanoids collectively referred to as rosavins. OBJECTIVES: To develop an isolation method for phytochemicals present in Rhodiola rosea roots using high-speed counter-current chromatography (HSCCC). METHODOLOGY: The roots of Rhodiola rosea were extracted with methanol and fractionated using liquid-liquid partition and polyamide column clean-up. The purified fraction (100 mg) was subjected to semi-preparative HSCCC using the two-phase solvent system ethyl acetate:butanol:water (3:2:5). The head-to-tail elution mode was employed with a flow rate of 1.5 mL/min and a rotary speed of 1000 rpm. RESULTS: The separation yielded six main fractions with four components more than 90% pure. The sixth fraction was further purified using semi-preparative HPLC with a Synergi-hydro RP C18 -column to obtain rosin and geranyl 1-O-α-l-arabinopyranosyl(1 → 6)-ß-d-glucopyranoside. The main components isolated were rosavin (3.4 mg, 97% purity), salidroside (0.5 mg, 90% purity), benzyl-O-ß-d-glucopyranoside (1.2 mg, 85% purity), rosarin (1.3 mg, 99% purity), rosiridin (1.8 mg, 92% purity), rosin (1.2 mg, 95% purity) and geranyl 1-O-α-l-arabinopyranosyl(1 → 6)-ß-d-glucopyranoside (6.5 mg, 97% purity). The identity and purity of these components were confirmed using ultrafast liquid chromatography-diode-array detector-MS/MS analysis, ¹H- and ¹³C-NMR spectroscopy. CONCLUSION: High-speed counter-current chromatography was successful in the isolation of several phytochemicals present in Rhodiola rosea roots, including two components that are not commercially available.

Analysis of alkylamides in Echinacea plant materials and dietary supplements by ultrafast liquid chromatography with diode array and mass spectrometric detection.

Alkylamides are a class of compounds present in plants of the genus Echinacea (Asteraceae), which have been shown to have high bioavailability and immunomodulatory effects. Fast analysis to identify these components in a variety of products is essential to profile products used in clinical trials and for quality control of these products. A method based on ultrafast liquid chromatography (UFLC) coupled with diode array detection and electrospray ionization mass spectrometry was developed for the analysis of alkylamides from the roots of Echinacea angustifolia (DC.) Hell., Echinacea purpurea (L.) Moench, and commercial dietary supplements. A total of 24 alkylamides were identified by LC-MS. The analysis time for these components is 15 min. Compared to the alkylamide profiles determined in the Echinacea root materials, the commercial products showed a more complex profile due to the blending of root and aerial parts of E. purpurea. This versatile method allows for the identification of alkylamides in a variety of Echinacea products and presents the most extensive characterization of alkylamides in E. angustifolia roots so far.

Inhibitory spectra and modes of antimicrobial action of gallotannins from mango kernels (Mangifera indica L.).

This study investigated the antimicrobial activities and modes of action of penta-, hexa-, hepta-, octa-, nona-, and deca-O-galloylglucose (gallotannins) isolated from mango kernels. The MICs and minimum bactericidal concentrations (MBCs) against food-borne bacteria and fungi were determined using a critical dilution assay. Gram-positive bacteria were generally more susceptible to gallotannins than were Gram-negative bacteria. The MICs of gallotannins against Bacillus subtilis, Bacillus cereus, Clostridium botulinum, Campylobacter jejuni, Listeria monocytogenes, and Staphylococcus aureus were 0.2 g liter(-1) or less; enterotoxigenic Escherichia coli and Salmonella enterica were inhibited by 0.5 to 1 g liter(-1), and lactic acid bacteria were resistant. The use of lipopolysaccharide mutants of S. enterica indicated that the outer membrane confers resistance toward gallotannins. Supplementation of LB medium with iron eliminated the inhibitory activity of gallotannins against Staphylococcus aureus, and siderophore-deficient mutants of S. enterica were less resistant toward gallotannins than was the wild-type strain. Hepta-O-galloylglucose sensitized Lactobacillus plantarum TMW1.460 to hop extract, indicating inactivation of hop resistance mechanisms, e.g., the multidrug resistance (MDR) transporter HorA. Carbohydrate metabolism of Lactococcus lactis MG1363, a conditionally respiring organism, was influenced by hepta-O-galloylglucose when grown under aerobic conditions and in the presence of heme but not under anaerobic conditions, indicating that gallotannins influence the respiratory chain. In conclusion, the inhibitory activities of gallotannins are attributable to their strong affinity for iron and likely additionally relate to the inactivation of membrane-bound proteins.

Purification of alkylamides from Echinacea angustifolia (DC.) Hell. Roots by high-speed countercurrent chromatography.

High-speed countercurrent chromatography (HSCCC) was used for the separation of alkylamides from the roots of Echinacea angustifolia (DC.) Hell. For this purpose, the alkylamides were extracted with hexane and subjected to semipreparative HSCCC using a two-phase solvent system consisting of n-hexane, ethyl acetate, methanol, and water (4:1:2:1). The lower aqueous phase was used as the mobile phase at a flow rate of 3 mL/min and a rotary speed of 1000 rpm. This procedure led to the isolation of four pure alkylamides, that is, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide (38.9 mg, 97% purity), dodeca-2E,4E,8Z-trienoic acid isobutylamide (4.4 mg, 92% purity), dodeca-2E,4E-dienoic acid isobutylamide (3.2 mg, 99% purity), and dodeca-2E,4E-dienoic acid 2-methylbutylamide (0.3 mg, 92% purity). The identity and purity of the isolated alkylamides were confirmed by LC-ESI-MS and (1)H NMR and (13)C NMR data. To the best of the authors' knowledge, this is the first report of dodeca-2E,4E-dienoic acid 2-methylbutylamide in E. angustifolia roots.

Fractionation of Gallotannins from mango (Mangifera indica L.) kernels by high-speed counter-current chromatography and determination of their antibacterial activity.

High-speed counter-current chromatography was applied to the separation of gallotannins from mango (Mangifera indica L.) kernels. The kernels were defatted and subsequently extracted with aqueous acetone [80% (v/v)]. The crude extract was purified by being partitioned against ethyl acetate. A hexane/ethyl acetate/methanol/water solvent system [0.5:5:1:5 (v/v/v/v)] was used in the head-to-tail mode to elute tannins according to their degree of galloylation (tetra-O-galloylglucose to deca-O-galloylglucose). The compounds were characterized using liquid chromatography and mass spectrometry in the negative ionization mode. Purities ranged from 72% (tetra-O-galloylglucose) to 100% (octa-O-galloylglucose). The iron binding capacity of gallotannins was dependent on the number of galloyl groups in the molecule, with a larger capacity at lower degrees of galloylation. The minimum inhibitory concentration against Bacillus subtilis did not change among the different gallotannins tested and was in the range of 0.05-0.1 g/L in Luria-Bertani broth but up to 20 times higher in media containing more iron and divalent cations.