Flavan-3-ols from the rhizomes of Drynaria fortunei

One monomer flavan-3-ol, 4α-carboxymethyl-(+)-catechin methyl ester, two monomer flavan-3-ol glycosides, (+)-afzelechin-3-O-β-allopyranoside, (+)-afzelechin-6-C-β-glucopyranoside, two dimer flavan-3-ols, (-)-epiafzelechin-(4β→8)-4β-carboxymethyl-(-)-epicatechin methyl ester, and -(-)-epiafzelechin-(4β→8)-4α-carboxymethyl-(-)epiafzelechin ethyl ester, and one trimer flavan-3-ol, (-)-epiafzelechin-(4β→8)-(-)-epiafzelechin-(4β→8)-4β-carboxymethyl-(-)-epiafzelechin methyl ester, together with thirteen known flavan-3-ols were isolated from the rhizomes of Drynaria fortunei (Kunze) J.Sm (Polypodiaceae). The structures were established by analysis of their HRESIMS, 1D, 2D NMR spectroscopic, and CD data. In order to obtain improved resolution, the high-resolution NMR spectra of the dimers and trimer were measured at -40 °C.     

Flavan-3-ols from the barks of Barringtonia acutangula

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Flavan-3-ols in Trichomes, Pistils and Phelloderm of Some Tree Species

Tissues from nine tree species were examined histochemicallyfor the presence of flavan-3-ols including the catechins. Itwas possible to stain these phenolics selectively with p-dimethylaminocinnamaldehyde(DMACA) and to show that they were located in trichomes, pistilsand shoot phelloderm. The staining intensity of the tissueswas categorized into four groups. The flavan-3-ols were extractedfrom three tree species and the diverse components were distinguishedusing a combination of HPLC and chemical reaction detection(CRD). Twelve flavan-3-ols were isolated from pistils of Tiliagrandifolia and 32 from leaves of Acer platanoides. The hairsof the leaves of dormant buds from Aesculus hippocastanum yielded13 components. Epicatechin and ( + )-catechin were present inall three species. Trees, trichomes, pistils, phelloderm, histochemistry, HPLC, CRD     

Membrane composition determines pardaxin's mechanism of lipid bilayer disruption

Pardaxin is a membrane-lysing peptide originally isolated from the fish Pardachirus marmoratus. The effect of the carboxy-amide of pardaxin (P1a) on bilayers of varying composition was studied using (15)N and (31)P solid-state NMR of mechanically aligned samples and differential scanning calorimetry (DSC). (15)N NMR spectroscopy of [(15)N-Leu(19)]P1a found that the orientation of the peptide's C-terminal helix depends on membrane composition. It is located on the surface of lipid bilayers composed of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and is inserted in lipid bilayers composed of 1,2-dimyristoyl-phosphatidylcholine (DMPC). The former suggests a carpet mechanism for bilayer disruption whereas the latter is consistent with a barrel-stave mechanism. The (31)P chemical shift NMR spectra showed that the peptide significantly disrupts lipid bilayers composed solely of zwitterionic lipids, particularly bilayers composed of POPC, in agreement with a carpet mechanism. P1a caused the formation of an isotropic phase in 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) lipid bilayers. This, combined with DSC data that found P1a reduced the fluid lamellar-to-inverted hexagonal phase transition temperature at very low concentrations (1:50,000), is interpreted as the formation of a cubic phase and not micellization of the membrane. Experiments exploring the effect of P1a on lipid bilayers composed of 4:1 POPC:cholesterol, 4:1 POPE:cholesterol, 3:1 POPC:1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), and 3:1 POPE:POPG were also conducted, and the presence of anionic lipids or cholesterol was found to reduce the peptide's ability to disrupt bilayers. Considered together, these data demonstrate that the mechanism of P1a is dependent on membrane composition.     

Flavan-3-ols from the leaves of Malaysian Uncaria longiflora var. pteropoda (Miq.) Ridsd

A novel flavonoid, (?)-2R,3R-3,5,4′-trihydoxyflavan-[6,7:5″,6″]-2″-pyranone, named uncariechin (1), was isolated from the methanolic extract of the leaves of Uncaria longiflora var. pteropoda (Miq.) Ridsd. along with the known (?)-epiafzelechin (2) and (?)-epicatechin (3), methyl 4-hydroxybenzoate and 4-hydroxybenzaldehyde, four pentacyclic oxindole alkaloids, isopteropodine, pteropodine, uncarine F and isopteropodic acid, previously found in the stems, and two coumarins, scopoletin and 3,4-dihydroxy-7-methoxycoumarin. Structures of the compounds were elucidated by 1D and 2D NMR, FTIR, UV, MS, and experimental as well as calculated electronic circular dichroism (ECD) data. Compounds 2 and 3 were evaluated for their neurotoxic and neuroprotective properties against differentiated SH-SY5Y neuroblastoma cell lines using the MTS assay. Compounds 2 and 3 did not show any neurotoxic effects but showed strong protective potential against hydrogen peroxide-induced neurotoxicity with maximum cell viability at a concentration of 1 μM.     

Inhibition of angiotensin converting enzyme (ACE) activity by flavan-3-ols and procyanidins

It was determined that flavan-3-ols and procyanidins have an inhibitory effect on angiotensin I converting enzyme (ACE) activity, and the effect was dependent on the number of epicatechin units forming the procyanidin. The inhibition by flavan-3-ols and procyanidins was competitive with the two substrates assayed: N-hippuryl-L-histidyl-L-leucine (HHL) and N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG). Tetramer and hexamer fractions were the more potent inhibitors, showing Ki of 5.6 and 4.7 microM, respectively. As ACE is a membrane protein, the interaction of flavanols and procyanidins with the enzyme could be related to the number of hydroxyl groups on the procyanidins, which determine their capacity to be adsorbed on the membrane surface.     

Mechanism of lipid bilayer disruption by the human antimicrobial peptide,LL-37

LL-37 is an amphipathic, alpha-helical, antimicrobial peptide. (15)N chemical shift and (15)N dipolar-shift spectroscopy of site-specifically labeled LL-37 in oriented lipid bilayers indicate that the amphipathic helix is oriented parallel to the surface of the bilayer. This surface orientation is maintained in both anionic and zwitterionic bilayers and at different temperatures and peptide concentrations, ruling out a barrel-stave mechanism for bilayer disruption by LL-37. In contrast, electrostatic factors, the type of lipid, and the presence of cholesterol do affect the extent to which LL-37 perturbs the lipids in the bilayer as observed with (31)P NMR. The (31)P spectra also show that micelles or other small, rapidly tumbling membrane fragments are not formed in the presence of LL-37, excluding a detergent-like mechanism. LL-37 does increase the lamellar to inverted hexagonal phase transition temperature of both PE model lipid systems and Escherichia coli lipids, demonstrating that it induces positive curvature strain in these environments. These results support a toroidal pore mechanism of lipid bilayer disruption by LL-37.     

Ultrasonic studies of lipid bilayer. Phase transition in synthetic phosphatidylcholine liposomes

The ultrasonic velocity at 3 MHz and the density in the nonsonicated and sonicated liposomes of dipalmitoylphosphatidylcholine have been measured in the temperature range from 0 degrees C to 55 degrees C. The results indicate that nonsonicated multilamellar vesicles undergo a weak first order transition which is analogous to the nematic-isotropic transition of liquid crystals. A sharp change in the ultrasonic velocity associated with the first order transition disappears when the multilamellar vesicles are sonicated. The bulk modulus of the lipid bilayer calculated from the ultrasonic velocity and the density of sonicated liposomes has a value of 3.0 X 10(10) dyne/cm2 at 20 degrees C, reaches a minimum value of 2.1 X 10(10) dyne/cm2 at its transition temperature and increases slightly to 2.2 X 10(10) dyne/cm2 at 50 degrees C.     

Effect of membrane composition on lipid oxidation in liposomes

To study the effect of membrane composition on the oxidation of liposomes, different systems were prepared by adding one component at time to phosphatidylcholine (Epikuron 200). In particular, the effect of cholesterol and its ester, cholesterol stearate, on membrane structure and oxidation was studied. A first screening of the structure and net charge of the different preparation was made by means of z-potential and size measurements. Then the liposomes were oxidized by using a hydrophilic radical initiator, the (2,2-azobis(2-amidinopropane) hydrochloride, AAPH, which thermally decomposes to give a constant radical flux in water. The oxidation of liposomes, monitored by following the absorbance of the primary products of oxidation at 234 nm, was shown to be dependent on the composition of the liposomal bilayer and so on its biophysical properties. In addition, size and z-potential measurements gathered in the time course of the peroxidation reaction, revealed that the oxidation induced a modification of the superficial characteristics of the membrane bilayer so as to change its charge at the shear plane (z-potential). This behaviour was shared by all liposomal preparations independent of the composition. The change in sizes of the different liposomal preparation, instead, followed different trends, being more stable both in control samples and in oxidized ones when cholesterol was present. From the analysis of the results, it can be concluded that cholesterol affects the oxidation induced by hydrophilic radical initiator of model membranes by changing the biophysical properties of the phospholipid bilayer. The rigidity induced by cholesterol at temperatures above the T_m makes the membrane more resistant to radical attack from an external aqueous phase and this in turn delays the start of the reaction. The decrease of z-potential of the liposomal particles induced by the oxidation process can be an important clue to understand the mechanisms involved in the etiology of important diseases.     

The structure of melittin in lipid bilayer membranes

0.15 M inorganic phosphate dramatically increased the α-helix content of melittin in aqueous solution.When melittin interacted with egg yolk phosphatidylcholine liposomes in the absence of inorganic phosphate, it was converted to an α-helix rich form, as postulated by Dawson et al. (Dawson, C.R., Drake, A.F. Helliwell, J. and Hider, R.C. (1978) Biochim. Biophys. Acta 510, 75–86).     

Incorporation of [C]Phenylalanine into Flavan-3-ols and Procyanidins in Cell Suspension Cultures of Douglas Fir

L-[¹⁴C]Phenylalanine, fed to cell suspension cultures of Douglas fir, (Pseudotsuga menziesii Franco) was incorporated simultaneously, but at different rates, into (+)-catechin, (−)-epicatechin, and procyanidins of increasing molecular weight. Asymmetric labeling of dimers and polymers was demonstrated, with more label appearing in the upper than in the lower or terminal unit. In addition, the total pool of free monomers was 10 to 30 times more highly labeled than was this lower, terminal unit of dimers and higher oligomers. Since the dimer, epicatechin-catechin, contained more label than catechin-catechin, it is concluded that the carbocation with the 2,3-cis stereochemistry of (−)-epicatechin was formed more rapidly than was that of the 2,3-trans type of (+)-catechin.     

Effect of acyl chain structure and bilayer phase state on binding and penetration of a supported lipid bilayer by HPA3

The effect of acyl chain structure and bilayer phase state on binding and penetration by the peptide HPA3 was studied using dual polarisation interferometry. This peptide is an analogue of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1) which has been shown to have antimicrobial and cell-penetrating properties. The binding of HPA3 to zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1-palmitolyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and negatively charged membranes composed of DMPC and 1,2-dimyristoyl-sn-glycero-3-(phosphor-rac-(1-glycerol)) (DMPG) or POPC and 1-palmitolyl-2-oleyl-sn-glycero-3-(phosphor-rac-(1-glycerol)) (POPG) was determined using dual polarisation interferometry (DPI). Mass and birefringence were measured in real time, enabling the creation of birefringence–mass plots for detailed analysis of the changes in lipid bilayer order during the peptide-binding process. HPA3 bound to all four lipids and the binding progressed as a single phase for the saturated gel phase bilayers DMPC and DMPC–DMPG. However, the binding process involved two or more phases, with penetration of the unsaturated fluid phase POPC and POPC–POPG bilayers. Structural changes in the saturated bilayer were partially reversible whereas binding to the unsaturated bilayer resulted in irreversible changes in membrane structure. These results demonstrate that more disordered unsaturated bilayers are more susceptible to further disorganisation and have a lower capacity to recover from peptide-induced structural changes than saturated ordered bilayers. In addition, this study further establishes DPI as powerful tool for analysis of multiphase peptide-insertion processes associated with complex structural changes in the liquid-crystalline membrane.     

Using fluorescence to locate intercalants within the lipid bilayer of liposomes, bioliposomes and erythrocyte ghosts

In previous work, we have shown the utility of the “NMR technique” in locating intercalants within the lipid bilayer. We describe herein the development of a more sensitive and complementary “fluorescence technique” for this purpose and its application to liposomes, bioliposomes and erythrocyte ghosts. This technique is based on the observation in selected compounds of an excellent correlation between the emission wavelength (λ_em) and Dimroth–Reichardt E_T(30) polarity parameter for the solvent in which the fluorescence emission spectrum was obtained.     

Effect of the nature of the 3beta-substitution in manoyl oxides on the thermotropic behavior of DPPC lipid bilayer and on DPPC liposomes

Functionalized manoyl oxide derivatives have been proved over the years to evoke several biological responses. Among them, 3beta-hydroxy-manoyl oxide (1) and 3beta-acetoxy-manoyl oxide (2) have been shown to exhibit in vitro antimicrobial and cytotoxic activity, while N-imidazole-3 beta-thiocarbonyl ester of manoyl oxide (3) was found to exhibit potent cytotoxic effect. Their partitioning into phospholipid bilayers may lead to membrane structure modifications that are crucial in liposome development as they may influence their maintenance and integrity. DSC was used to study the modifications induced in DPPC bilayers by incorporating increasing concentrations of the three manoyl oxide derivatives. All derivatives were found to strongly affect the bilayer structural organization in terms of a decrease of the cooperativity, the fluidization and partially destabilization of the gel phase and the induction of a lateral phase separation in clustering domains. Derivatives 1 and 3 were incorporated into DPPC liposomes and their physicochemical stability was monitored at 4 degrees C. The stability of liposomes was strongly influenced by the presence of 1 and 3 at any molar ratio studied. DPPC/1 liposomes were found to retain its stability for 48 h at low concentration of 10% mol, while at higher concentrations up to 30% mol they collapsed into aggregated material. In all cases DPPC/3 liposomes were found unstable and sticky aggregated structures precipitated from the bulk suspension.     

Surface modified liposomes by mannosylated conjugates anchored via the adamantyl moiety in the lipid bilayer

The aim of the present study was to encapsulate mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides, namely [(2R)-N-(adamant-1-yl)-3-(α,β-d-mannopyranosyloxy)-2-methylpropanamide and (2R)-N-[3-(α-d-mannopyranosyloxy)-2-methylpropanoyl]-d,l-(adamant-2-yl)glycyl-l-alanyl-d-isoglutamine] in liposomes. The characterization of liposomes, size and surface morphology was performed using dynamic light scattering (DLS) and atomic force microscopy (AFM). The results have revealed that the encapsulation of examined compounds changes the size and surface of liposomes. After the concanavalin A (ConA) was added to the liposome preparation, increase in liposome size and their aggregation has been observed. The enlargement of liposomes was ascribed to the specific binding of the ConA to the mannose present on the surface of the prepared liposomes. Thus, it has been shown that the adamantyl moiety from mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides can be used as an anchor in the lipid bilayer for carbohydrate moiety exposed on the liposome surface.     

Transport of Ca2+ by diltiazem across the lipid bilayer in model liposomes.

The calcium channel antagonist diltiazem was examined for its ability to translocate Ca2+ from an aqueous medium to the nonpolar lipid milieu. We monitored the spectral changes caused by the drug-mediated cation transport at 37 degrees C in unilamellar vesicles made of dimyristoyl phosphatidylcholine (DMPC) and containing the calcium-sensitive dye arsenazo III trapped inside. Vesicle leakage or membrane fusion caused by diltiazem was assessed by the use of vesicles containing fluorescent indicators. These effects were, however, found to be insignificant compared with ion transport. The transport was negligible at temperatures below the liquid crystalline to gel transition temperature of DMPC indicating a carrier mechanism of ion transport. A quantitative analysis of the transport kinetics indicated that a 1:2 Ca(2+)-drug complex is formed inside the lipid. The calcium ionophoretic ability of diltiazem, combined with other related data, suggests a possible role for Ca2+ in the conformation of the drug in the lipid membrane milieu and in its interaction with the calcium channel.     

Experimental verification of lipid bilayer structure through multi-scale modeling

Great progress has been made in applying coarse-grain molecular dynamics (CGMD) simulations to the investigation of membrane biophysics. In order to validate the accuracy of CGMD simulations of membranes, atomistic scale detail is necessary for direct comparison to structural experiments. Here, we present our strategy for verifying CGMD lipid bilayer simulations. Through reverse coarse graining and subsequent calculation of the bilayer electron density profile, we are able to compare the simulations to our experimental low angle X-ray scattering (LAXS) data. In order to determine the best match to the experimental data, atomistic simulations are run at a range of areas (in the NP_NAT ensemble), starting from distinct configurations extracted from the CGMD simulation (run in the NPT ensemble). We demonstrate the effectiveness of this procedure with two small, single-component bilayers, and suggest that the greater utility of our algorithm will be for CGMD simulations of more complex structures.     

The effect of methemoglobin and ferricytochrome c on the lipid bilayer structure

The effect of ferricytochrome c and methemoglobin on the model phospholipid membrane structure has been investigated using fluorescent probes: 1-anilinonaphthalene-8-sulphonate, 4-dimethylaminochalcone and 3-methoxybenzanthrone. Ferricytochrome c and methemoglobin is found to cause the disordering of the phospholipid bilayer surface region.     

The structure of melittin in lipid bilayer membranes.

0.15 M inorganic phosphate dramatically increased the alpha-helix content of melittin in aqueous solution. When melittin interacted with egg yolk phosphatidylcholine liposomes in the absence of inorganic phosphate, it was converted to an alpha-helix rich form, as postulated by Dawson et al. (Dawson, C.R., Drake, A.F. Helliwell, J. and Hider, R.C. (1978) Biochim. Biophys. Acta 510, 75--86).