1H Quantitative NMR analyses of ß-asarone and related compounds for quality control of Acorus rhizome herbal drugs in terms of the effects of their constituents on in vitro acetylcholine esterase activity.

We used quantitative nuclear magnetic resonance analyses to measure the contents of major constituents of Acorus rhizome materials used as herbal drugs. The inhibitory effects of crude n-hexane extracts and their individual constituents on in vitro acetylcholine esterase activity were evaluated. The crude extracts had unexpectedly weak inhibitory effects (46-64% inhibition at 1.0 mg/mL), despite the high content (46-64%) of ß-asarone, which independently had a potent effect (IC50 2.9 µM [0.61 µg/mL]). Further investigation revealed participation of eudesmin A, a lignan constituent, in the suppression of the inhibitory effect of ß-asarone.

Coriariin M, a trimeric hydrolysable tannin with dehydrodigalloyl and valoneoyl groups as linking units, and accompanying dimeric hydrolysable tannins from Coriaria japonica.

Three oligomeric hydrolysable tannins, coriariins K, L, and M, which were previously undescribed, together with five known hydrolysable tannins were isolated from dried leaves of Coriaria japonica. Their structures were determined based on 1D and 2D NMR spectroscopy, HR-ESI-MS, and ECD spectroscopy experiments. Among the isolated compounds, coriariin M has a unique trimer structure where both dehydrodigalloyl and valoneoyl group linkages were found between the hydrolysable tannin monomers. Dimeric hydrolysable tannins coriariins K and L, having a dehydrodigalloyl group as the linking unit, were structurally related to coriariin A, the main hydrolysable tannin of this plant species. Additionally, the complexation of the eight hydrolysable tannins isolated in this study with bovine serum albumin (BSA) to form water-soluble macromolecules was analyzed using native polyacrylamide gel electrophoresis (PAGE). A comparison of the behaviors of the oligomeric hydrolysable tannins suggested the participation of the hexahydroxydiphenoyl group and the importance of the molecular sizes of the hydrolysable tannins in the formation of macromolecules.

Structures and Antibacterial Properties of Isorugosins H-J, Oligomeric Ellagitannins from Liquidambar formosana with Characteristic Bridging Groups between Sugar Moieties.

Three new ellagitannin oligomers, isorugosins H (1), I (2), and J (3), together with 11 known hydrolyzable tannins were isolated from an aqueous acetone extract of the fresh leaves of Liquidambar formosana. Their chemical structures were elucidated based on spectroscopic data and chemical conversion into known hydrolyzable tannins. The bridging mode of the valoneoyl groups between their sugar moieties has been identified only in this plant species. Additionally, the effects of the isorugosins isolated from this species on drug-resistant bacteria were evaluated and showed that isorugosin A (4) exhibited the most potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The isorugosins also had a suppressing effect on pigment formation in Pseudomonas aeruginosa. The isorugosin-protein complexes were analyzed using size-exclusion chromatography and polyacrylamide gel electrophoresis to clarify the relationship of their antibacterial properties with their protein interaction potency as hydrolyzable tannins. The results suggested that the antibacterial properties of hydrolyzable tannins are not simply a result of their binding activity to proteins, but are due to other factors such as the accessibility of polyphenolic acyl groups to bacterial membranes.

Ellagitannins of Davidia involucrata. I. Structure of Davicratinic Acid A and Effects of Davidia Tannins on Drug-Resistant Bacteria and Human Oral Squamous Cell Carcinomas.

We isolated a new ellagitannin, davicratinic acid A (5), together with four known ellagitannins, davidiin (1), granatin A (2), pedunculagin (3), and 3-O-galloylgranatin A (4), from an aqueous acetone extract of dried Davidia involucrata leaves. The known ellagitannins were identified based on spectroscopic data. The structure of davicratinic acid A (5), a monomeric ellagitannin possessing a unique, skew-boat glucopyranose core, was established based on spectroscopic data. Additionally, we examined the effects of several tannins with good yields from this plant on drug-resistant bacteria and human oral squamous cell carcinomas, and found that davidiin (1) exhibited the most potent antibacterial and antitumor properties among the tannins examined.

Identification of 4-hydroxy-2-nonenal-histidine adducts that serve as ligands for human lectin-like oxidized LDL receptor-1.

LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) is an endothelial scavenger receptor that is important for the uptake of OxLDL (oxidized low-density lipoprotein) and contributes to the pathogenesis of atherosclerosis. However, the precise structural motifs of OxLDL that are recognized by LOX-1 are unknown. In the present study, we have identified products of lipid peroxidation of OxLDL that serve as ligands for LOX-1. We used CHO (Chinese-hamster ovary) cells that stably express LOX-1 to evaluate the ability of BSA modified by lipid peroxidation to compete with AcLDL (acetylated low-density lipoprotein). We found that HNE (4-hydroxy-2-nonenal)-modified proteins most potently inhibited the uptake of AcLDL. On the basis of the findings that HNE-modified BSA and oxidation of LDL resulted in the formation of HNE-histidine Michael adducts, we examined whether the HNE-histidine adducts could serve as ligands for LOX-1. The authentic HNE-histidine adduct inhibited the uptake of AcLDL in a dose-dependent manner. Furthermore, we found the interaction of LOX-1 with the HNE-histidine adduct to have a dissociation constant of 1.22×10(-8) M using a surface plasmon resonance assay. Finally, we showed that the HNE-histidine adduct stimulated the formation of reactive oxygen species and activated extracellular-signal-regulated kinase 1/2 and NF-κB (nuclear factor κB) in HAECs (human aortic endothelial cells); these signals initiate endothelial dysfunction and lead to atherosclerosis. The present study provides intriguing insights into the molecular details of LOX-1 recognition of OxLDL.

4-Hydroperoxy-2-nonenal is not just an intermediate but a reactive molecule that covalently modifies proteins to generate unique intramolecular oxidation products.

α,ß-Unsaturated aldehydes generated during lipid peroxidation, such as 4-oxoalkenals and 4-hydroxyalkenals, can give rise to protein degeneration in a variety of pathological states. Although the covalent modification of proteins by these end products has been well studied, the reactivity of unstable intermediates possessing a hydroperoxy group, such as 4-hydroperoxy-2-nonenal (HPNE), with protein has received little attention. We have now established a unique protein modification in which the 4-hydroperoxy group of HPNE is involved in the formation of structurally unusual lysine adducts. In addition, we showed that one of the HPNE-specific lysine adducts constitutes the epitope of a monoclonal antibody raised against the HPNE-modified protein. Upon incubation with bovine serum albumin, HPNE preferentially reacted with the lysine residues. By employing N(α)-benzoylglycyl-lysine, we detected two major products containing one HPNE molecule per peptide. Based on the chemical and spectroscopic evidence, the products were identified to be the N(α)-benzoylglycyl derivatives of N(ε)-4-hydroxynonanoic acid-lysine and N(ε)-4-hydroxy-(2Z)-nonenoyllysine, both of which are suggested to be formed through mechanisms in which the initial HPNE-lysine adducts undergo Baeyer-Villiger-like reactions proceeding through an intramolecular oxidation catalyzed by the hydroperoxy group. On the other hand, using an HPNE-modified protein as the immunogen, we raised a monoclonal antibody against the HPNE-modified protein and identified one of the HPNE-specific lysine adducts, N(ε)-4-hydroxynonanoic acid-lysine, as an intrinsic epitope of the monoclonal antibody. Furthermore, we demonstrated that the HPNE-specific epitopes were produced not only in the oxidized low density lipoprotein in vitro but also in the atherosclerotic lesions. These results indicated that HPNE is not just an intermediate but also a reactive molecule that could covalently modify proteins in biological systems.

Lipid peroxidation modification of protein generates Nepsilon-(4-oxononanoyl)lysine as a pro-inflammatory ligand.

4-Oxo-2(E)-nonenal (ONE), a peroxidation product of ω-6 polyunsaturated fatty acids, covalently reacts with lysine residues to generate a 4-ketoamide-type ONE-lysine adduct, N(ε)-(4-oxononanoyl)lysine (ONL). Using an ONL-coupled protein as the immunogen, we raised the monoclonal antibody (mAb) 9K3 directed to the ONL and conclusively demonstrated that the ONL was produced during the oxidative modification of a low density lipoprotein (LDL) in vitro. In addition, we observed that the ONL was present in atherosclerotic lesions, in which an intense immunoreactivity was mainly localized in the vascular endothelial cells and macrophage- and vascular smooth muscle cell-derived foam cells. Using liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for quantification of the ONL and confirmed that the ONL was indeed formed during the lipid peroxidation-mediated modification of protein in vitro and in vivo. To evaluate the biological implications for ONL formation, we examined the recognition of ONL by the scavenger receptor lectin-like oxidized LDL receptor-1 (LOX-1). Using CHO cells stably expressing LOX-1, we evaluated the ability of ONL to compete with the acetylated LDL and found that both the ONE-modified and ONL-coupled proteins inhibited the binding and uptake of the modified LDL. In addition, we demonstrated that the ONL-coupled protein was incorporated into differentiated THP-1 cells via LOX-1. Finally, we examined the effect of ONL on the expression of the inflammation-associated gene in THP-1 and observed that the ONL-coupled proteins significantly induced the expression of atherogenesis-related genes, such as the monocyte chemoattractant protein-1 and tumor necrosis factor-α, in a LOX-1-dependent manner. Thus, ONL was identified to be a potential endogenous ligand for LOX-1.

Identification of advanced reaction products originating from the initial 4-oxo-2-nonenal-cysteine Michael adducts.

4-Oxo-2-nonenal (ONE), an aldehyde originating from the peroxidation of omega6 polyunsaturated fatty acids, preferentially reacts with the cysteine residues of protein. Despite the fact that there has been significant recent interest in the protein reactivity and biological activity of ONE, the structural basis of the ONE-cysteine adducts remain to be established. In the present study, to gain a structural insight into the sulfhydryl modification by ONE, we characterized reaction products that originated from the initial ONE-cysteine Michael adducts. N-Acetyl-L-cysteine (10 mM) was incubated with an equimolar concentration of ONE in 0.1 M phosphate buffer (pH 7.4) at 37 degrees C. Within 1 h of incubation, the reaction of N-acetyl-L-cysteine with ONE resulted in the formation of two (C-2 and C-3) Michael addition products possessing a carbonyl functionality. Subsequent incubation of the reaction mixture resulted in their disappearance and concomitant formation of advanced reaction products, including a minor product III and major products IVa, IVb, and V. Product III was identified to be a thiomorpholine derivative, 4-acetyl-5-hydroxyl-6-(2-oxoheptyl)thiomorpholine-3-carboxylic acid, which might have originated from the C-2 Michael addition product. The major products were identified to be the novel 2-cyclopentenone derivatives, that is, 2-(acetylamino)-3-[(3-butyl-4-oxocyclopent-2-en-1-yl)sulfanyl]propionic acid (IVa and its isomer IVb) and 2-(acetylamino)-3-[(4-butyl-5-oxocyclopent-3-en-1-yl)sulfanyl]propionic acid (V), which might be generated through the base-catalyzed cyclization of the C-2 and C-3 Michael addition products, respectively. The furan derivative, which has been reported as the end product of the Michael adducts, was found to be formed only under acidic conditions. Thus, this study identified the novel ONE-cysteine adducts, including the most prominent 2-cyclopentenone derivatives, that originated from the initial Michael adducts.