Yoshiaki Amakura

High-performance liquid chromatographic determination with photodiode array detection of ellagic acid in fresh and processed fruits

A high-performance liquid chromatographic (HPLC) procedure based on an isocratic elution with photodiode array detection has been developed for a simple and rapid determination of ellagic acid (EA) in fresh and processed fruits. The homogenized sample was refluxed with methanol and then the extract was refined using a solid-phase cartridge before HPLC. We analyzed EA in 40 kinds of fresh fruits and 11 kinds of processed fruits by the developed method. EA was found in several berries, fueijoa, pineapple and pomegranate. This is the first occurrence of the detection of EA in bayberry, fueijoa and pineapple.

Determination of phenolic acids in fruit juices by isocratic column liquid chromatography

A simple and rapid analytical method of five phenolic acids, gallic, chlorogenic, caffeic, ellagic and ferulic acid, which are naturally occurring bioactives, were determined in fruit juices by isocratic LC using photodiode array UV detection. The sample was pre-treated by solid-phase extraction (a combination of Sep-Pak Plus tC18 and Bond Elut PSA).

Constituents and their antioxidative effects in eucalyptus leaf extract used as a natural food additive

Abstract The components of a natural food additive, “eucalyptus leaf extract”, were isolated and identified in order to determine their structures and contents. The structures of eight major compounds, namely gallic and ellagic acids, eucalyptone and macrocarpals A–E, isolated from them were elucidated by spectroscopic methods. The antioxidative activities of these isolated compounds were estimated by several assays, and it appears that the antioxidant activity is mostly due to the gallic and ellagic acids. On the other hand, in the determined eucalyptus product, the content of 1,8-cineole, a major component of the eucalyptus oil, was lower than the isolated compounds, and its activity as an antioxidant was negligible.

Structural Features and Biological Properties of Ellagitannins in Some Plant Families of the Order Myrtales

Plant tannins, including hydrolysable and condensed varieties, are well known antioxidants in medicinal plants, foods, and edible fruits. Their diverse biological properties and potential for disease prevention have been demonstrated by various in vitro and in vivo assays. A number of ellagitannins, the largest group of hydrolysable tannins, have been isolated from dicotyledoneous angiosperms and characterized. This diverse class of tannins is sub-grouped into simple ellagitannins, C-glycosidic ellagitannins, complex tannins (condensates of C-glycosidic tannins with flavan-3-ol), and oligomers up to pentamers. This review outlines and describes the chemotaxonomic significance of structural features in various types of ellagitannins found in plants belonging to the Myrtaceae, Onagraceae, and Melastomataceae families, which are all included in the order Myrtales. Any biological activities that have been reported, including antitumor and antibacterial effects as well as enzyme inhibition, are also reviewed.

Validation of the CALUX bioassay for the screening of PCDD/Fs and dioxin-like PCBs in retail fish

The chemical-activated luciferase expression (CALUX) assay is a reporter gene assay that detects dioxin-like compounds based on their ability to activate the aryl hydrocarbon receptor (AhR) and thus expression of the reporter gene. In this paper, the CALUX assay was examined for its application in the screening of polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (dioxin-like PCBs) in retail fish. The sample extracts were cleaned up on a sulfuric acid-silica gel column followed by an activated carbon column, and the AhR activity of the separated PCDD/F and dioxin-like PCB fractions was determined using the assay. The quantitative limit for 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) was 0.98 pg ml(-1) (0.19 pg assay(-1) in the standard curve, corresponding to 0.16 pg g(-1) of CALUX-based toxic equivalency (2,3,7,8-TCDD equivalents) in the tested sample. Recovery tests in which dioxins were added to fish samples resulted in acceptable recoveries (77-117%). The CALUX assay performed well in the analysis of dioxins in fish samples and a comparative study revealed a strong correlation between the CALUX assay and high-resolution gas chromatography-high-resolution mass spectrometry analysis for the determination of PCDD/Fs (r = 0.89) and dioxin-like PCBs (r = 0.91) in retail fish (n = 22). These data revealed that the CALUX assay would be a useful screening method for PCDD/Fs and dioxin-like PCBs in retail fish.

Polyphenolic compounds isolated from the leaves of Myrtus communis

Four hydrolyzable tannins [oenothein B (1), eugeniflorin D2 (2), and tellimagrandins I (3) and II (4)], two related polyphenolic compounds [gallic acid (5) and quinic acid 3,5-di-O-gallate (6)], and four myricetin glycosides [myricetins 3-O-β-d-xyloside (7), 3-O-β-d-galactoside (8), 3-O-β-d-galactoside 6″-O-gallate (9), and 3-O-α-l-rhamnoside (10)] were isolated from the leaves of Myrtus communis. Antioxidant activities of the isolated compounds were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay.

Marker Constituents of the Natural Antioxidant Eucalyptus Leaf Extract for the Evaluation of Food Additives

In order to establish the marker constituents of the natural antioxidant food-additive Eucalyptus leaf extract, the UV-absorbing constituents of two eucalyptus leaf extracts registered as food additives (eucalyptus A and B) were investigated. Several major peaks on the reversed-phase HPLC chromatogram of eucalyptus A were characterized as gallic acid, ellagic acid, 3-O-β-D-glucuronides of quercetin and kaempferol, and a hydrolyzable tannin dimer, oenothein B, by direct comparison with authentic specimens isolated from Eucalyptus globulus leaves. A new gallotannin was found in the E. globulus leaf extract, and its structure was found to be 1,2,3,6-tetra-O-galloyl-β-D-galactose. Two major peaks on the HPLC chromatogram of eucalyptus B were identified as gallic acid and ellagic acid, indicative of degradation products from hydrolyzable tannins in the leaves. Considering the evaluation of antioxidant activity by radical scavenging ability, a standardization of eucalyptus leaf extract, including the antioxidative polyphenol, oenothein B, is proposed.

Antioxidative Properties of Functional Polyphenols and Their Metabolites Assessed by an ORAC Assay

We compared the antioxidative activities of polyphenol metabolites with those of intact functional polyphenols by an assay of the oxygen radical absorbance capacity (ORAC). The metabolites of ellagitannin geraniin, chlorogenic acid, and (−)-epigallocatechin gallate displayed more potent antioxidative activity than their respective original compounds. Our findings suggest that these metabolites may play important roles as biological antioxidants after their consumption.

Structure of the tannin geraniin based on conventional X-ray data at 295 K and on synchrotron data at 293 and 120 K

Geraniin, C41H28O27.7H2O, is the main tannin from Geranium thunbergii Sieb. et Zucc., which is one of the most popular folk medicines and also an official antidiarrheic drug in Japan. Conventional X-ray diffractometer data at 295 K and synchrotron data at 293 and 120 K (at EMBL/DESY Hamburg with a MARRESEARCH imaging plate) were measured. The structure could not be determined with any of the currently distributed direct-methods programs, but was easily solved with DIRDIF using several conformers of the hexahydroxydiphenoyl group as input fragments into a vector-search procedure. A molecular structure was established where a cyclohexenetrione moiety attached to O4 of a central glucose unit was in a hydrated six-membered hemiacetal ring structure, as expected from the NMR study of crystalline geraniin. Owing to the high oligocyclic substitution the glucopyranosyl ring is in the unusual 1C4 conformation. Refinement of the low-temperature synchrotron data allowed identification of almost all the H atoms, even within the seven water molecules, so that a rather complex system of approximately 30 different hydrogen bonds can be studied in some detail.

Isolation and Characterization of Activators of ERK/MAPK from Citrus Plants

Extracellular signal-regulated kinases 1/2 (ERK1/2), components of the mitogen-activated protein kinase (MAPK) signaling cascade, have been recently shown to be involved in synaptic plasticity and in the development of long-term memory in the central nervous system (CNS). We therefore examined the ability of Citrus compounds to activate ERK1/2 in cultured rat cortical neurons, whose activation might have a protective effect against neurodegenerative neurological disorders. Among the samples tested, extracts prepared from the peels of Citrus grandis (Kawachi bankan) were found to have the greatest ability to activate ERK1/2. The active substances were isolated by chromatographic separation, and one of them was identified to be 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF). HMF significantly induced the phosphorylation of cAMP response element-binding protein (CREB), a downstream target of activated ERK1/2, which appears to be a critical step in the signaling cascade for the structural changes underlying the development of long-term potentiation (LTP). In addition, the administration of HMF into mice treated with NMDA receptor antagonist MK-801 restored the MK-801-induced deterioration of spatial learning performance in the Morris mater-maze task. Taken together, these results suggest that HMF is a neurotrophic agent for treating patients with memory disorders.