Structure-dependent interactions of polyphenols with a biomimetic membrane system

Polyphenols are naturally-occurring compounds, reported to be biologically active, and through their interactions with cell membranes. Although association of the polyphenols with the bilayer has been reported, the detailed mechanism of interaction is not yet well elucidated. We report on spatio-temporal real-time membrane dynamics observed in the presence of polyphenols. Two distinct membrane dynamics, corresponding to the two classes of polyphenols used, were observed. Flavonoids (epi-gallocatechin-3-gallate, gallocatechin, theaflavin and theaflavin-3-gallate) caused lipid membrane aggregation and rigidification. As simple structural modification through opening of the aromatic C-ring into an olefin bond, present in trans-stilbenes (resveratrol and picead), completely changed the membrane properties, increasing fluidity and inducing fluctuation. There were differences in the membrane transformations within the same class of polyphenols. Structure-dependent classification of membrane dynamics may contribute to a better understanding of the physicochemical mechanism involved in the bioactivity of polyphenols. In general, an increase in the number of hydrophilic side chains (galloyl, hydroxyl, glucoside, gallate) increased the reactivity of the polyphenols. Most notable was the difference observed through a simple addition of the gallate group. Unraveling the importance of these polyphenols, at a functional group level further opens the key to tailored design of bioactive compounds as potential drug candidates.     

The relationship between the antioxidant and the antibacterial properties of galloylated catechins and the structure of phospholipid model membranes

The effects of four catechins, (+)-catechin (C), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG), on the physical properties of phospholipid model membranes and the correlation to their antioxidant and antibacterial capacities have been studied by using differential scanning calorimetry (DSC), fluorescence spectroscopy, infrared spectroscopy (IR), AAPH-induced oxidation, and leakage experiments. DSC data revealed that galloylated catechins, especially ECG, affected the physical properties of both the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) bilayers dramatically. Galloylated catechins showed higher phospholipid/water partition coefficients than their homologues and were immersed in the phospholipid palisade intercalating within the hydrocarbon chains, ECG being at the deepest position. In contrast, nongalloylated catechins presented a shallow location close to the phospholipid/water interface. ECG also exhibited the highest antioxidant capacity against lipid peroxidation, which correlated with its strong effect on DPH fluorescence anisotropy (as observed by the increase of the lipid order of fluid PC bilayers) and with the presence of highly cooperative transitions as seen by DSC. We propose that the high antioxidant capacity of some galloylated catechins such as ECG could be partially due to the formation of membrane structures showing resistance to detergent solubilization and in which the phospholipids have tightly packed acyl chains and highly hydrated phosphate groups. Significantly, PE was found to be essential to the promotion of carboxyfluorescein leakage from bacterial model membranes by galloylated catechins, indicating that their bactericidal activity, at least at the membrane level, could be due to the specific effect of these catechins on PE.     

Suppression of adipocyte differentiation by Cordyceps militaris through activation of the aryl hydrocarbon receptor

Mycelial extracts have a wide range of biological activities that modulate functions of mammalian cells. In this report, we sought to identify antiadipogenic mycelia with the use of 3T3-L1 cells and found that the extract of Cordyceps militaris exclusively suppressed differentiation of 3T3-L1 preadipocytes into mature adipocytes without affecting cell viability. This inhibitory effect was dose dependent, reversible, and associated with 1) a decrease in lipid accumulation, 2) blunted induction of adipocyte markers including adiponectin, peroxisome proliferator-activated receptor-gamma, and CCAAT/enhancer binding protein-alpha, and 3) sustained expression of a preadipocyte marker, monocyte chemoattractant protein-1. C. militaris also significantly decreased accumulation of lipid and hypertrophy in mature adipocytes and preserved their response to insulin (phosphorylation of Akt) during prolonged culture. Subsequent experiments revealed that C. militaris has the potential to activate the aryl hydrocarbon receptor (AhR). In 3T3-L1 cells, treatment with AhR agonists including benzo[a]pyrene and 3-methylcholanthrene reproduced the antiadipogenic effect of C. militaris. Furthermore, dominant-negative inhibition of AhR abrogated the suppressive effect of C. militaris on adipocyte differentiation. These results suggest that C. militaris has the potential to interfere with adipocyte differentiation through activation of AhR.     

Functional targeting of the TGF-βR1 kinase domain and downstream signaling: A role for the galloyl moiety of green tea-derived catechins in ES-2 ovarian clear cell carcinoma

The galloyl moiety is a specific structural feature which dictates, in part, the chemopreventive properties of diet-derived catechins. In ovarian cancer cells, galloylated catechins were recently demonstrated to target the transforming growth factor (TGF)-β-mediated control of the epithelial-mesenchymal transition process. The specific impact of the galloyl moiety on such signaling, however, remains poorly understood. Here, we questioned whether the sole galloyl moiety interacted with TGF-β-receptors to alter signal transduction and chemotactic migratory response in an ES-2 serous carcinoma-derived ovarian cancer cell model. In line with the LogP and LogS values of the tested molecules, we found that TGF-β-induced Smad-3 phosphorylation and cell migration were optimally inhibited, provided that the lateral aliphatic chain of the galloyl moiety reached 8–10 carbons. Functional inhibition of the TGF-β receptor (TGF-βR1) kinase activity was supported by surface plasmon resonance assays showing direct physical interaction between TGF-βR1 and the galloyl moiety. In silico molecular docking analysis predicted a model where galloylated catechins may bind TGF-βR1 within its adenosine triphosphate binding cleft in a site analogous to that of Galunisertib, a selective adenosine triphosphate-mimetic competitive inhibitor of TGF-βR1. In conclusion, our data suggest that the galloyl moiety of the diet-derived catechins provides specificity of action to galloylated catechins by positioning them within the kinase domain of the TGF-βR1 in order to antagonize TGF-β-mediated signaling that is required for ovarian cancer cell invasion and metastasis.     

Evaluation of the anti-oxidative effect (in vitro) of tea polyphenols

Forty-three polyphenols from tea leaves were evaluated for their anti-oxidative effect against lipid peroxidation by the ferric thiocyanate method in vitro. Among these, 1,4,6-tri-O-galloyl-beta-D-glucose (hydrolyzable tannin) showed the highest anti-oxidative activity against lipid peroxidation, even stronger than that of 3-tert.-butyl-4-hydroxyanisole (BHA). The assay demonstrates that tea polyphenols, except for desgalloylated dimeric proanthocyanidins that possess a catechin structure in the upper unit and desgalloylated flavan-3-ols, and excepting theaflavin 3,3'-di-O-gallate, had more anti-oxidative activity than that of alpha-tocopherol. The chemical structure-activity relationship shows that the anti-oxidative action advanced with the condensation of two molecules of flavan-3-ols as well as with 3-O-acylation in the flavan skeleton such as that by galloyl, (3'-O-methyl)-galloyl, and p-coumaroyl groups.     

Optimisation of plant sterols incorporation in human keratinocyte plasma membrane and modulation of membrane fluidity.

The in vitro effects of plant sterols were investigated with regard to their uptake and membrane lipid fluidity in human keratinocytes. Among the different media tested to transport sterols (liposomes, micelles and organic solvents), the best results in terms of incorporation and viability were obtained by the use of the organic solvents dimethylsulfoxide and ethanol. After 48 h incubation exogenous sterol can account for about 30% of the total cell sterol content. The total sterol amount in plasma membranes increased 2-fold after incubation with cholesterol, whereas it was not altered when phytosterols were incorporated. The incorporation of cholesterol, sitosterol and stigmasterol led to an increase in the percent of unsaturated fatty acid C18:1 in the plasma membrane. The effect of this uptake on membrane fluidity was studied by means of fluorescence polarisation using DPH and TMA-DPH as fluorescent probes. Whereas cholesterol and sitosterol had no significant effect on the DPH fluorescence anisotropy (rs), the presence of stigmasterol induced a 12% decrease of rs reflecting an increase in membrane fluidity. We can conclude from this study that in the presence of sitosterol, the mean fluidity of the membrane is regulated whereas stigmasterol triggers a looseness of molecular packing of phospholipids acyl chains, in accordance with previous results obtained on purely lipid model membranes.     

Structure-activity relationships in the hydrophobic interactions of polyphenols with cellulose and collagen

Polyphenol interactions with both cellulose and collagen in the solid state have been studied by using chromatography on cellulose and by evaluating the hydrothermal stability of the polyphenol treated sheepskin collagen. Twenty-four polyphenolic compounds were studied, including seven glucose-based gallotannins, five polyalcohol-based gallotannins, and twelve ellagitannins. In the cellulose-polyphenols systems, the polyphenol's affinity to cellulose is positively correlated with their molecular masses, the number of galloyl groups, and their hydrophobicity (logP). The polyphenol treatment increased the hydrothermal stability of collagen samples, and such effects are also positively correlated with the molecular masses, total number of galloyl groups and the hydrophobicity of polyphenols. Ellagitannins showed much weaker interactions with both biopolymers than gallotannins having similar molecular mass, the same number of galloyl groups, and the same number of phenolic hydroxyl groups. It is concluded that, for the polyphenol interactions with both cellulose and collagen, (1) the galloyl group of polyphenols is the functional group; (2) the strength of interactions are positively correlated with molecular size, the number of galloyl groups and the hydrophobicity of polyphenols; (3) the hydrophobic interactions are of great significance; and (4) the interactions are strongly dependent on the flexibility of galloyl groups.     

Rohitukine inhibits in vitro adipogenesis arresting mitotic clonal expansion and improves dyslipidemia in vivo

We developed a common feature pharmacophore model using known antiadipogenic compounds (CFPMA). We identified rohitukine, a reported chromone anticancer alkaloid as a potential hit through in silico mapping of the in-house natural product library on CFPMA. Studies were designed to assess the antiadipogenic potential of rohitukine. Rohitukine was isolated from Dysoxylum binacteriferum Hook. to ⬧95% purity. As predicted by CFPMA, rohitukine was indeed found to be an antiadipogenic molecule. Rohitukine inhibited lipid accumulation and adipogenic differentiation in a concentration- and exposure-time-dependent manner in 3T3-L1 and C3H10T1/2 cells. Rohitukine downregulated expression of PPARγ, CCAAT/enhancer binding protein α, adipocyte protein 2 (aP2), FAS, and glucose transporter 4. It also suppressed mRNA expression of LPL, sterol-regulatory element binding protein (SREBP) 1c, FAS, and aP2, the downstream targets of PPARγ. Rohitukine arrests cells in S phase during mitotic clonal expansion. Rohitukine was bioavailable, and 25.7% of orally administered compound reached systemic circulation. We evaluated the effect of rohitukine on dyslipidemia induced by high-fat diet in the hamster model. Rohitukine increased hepatic expression of liver X receptor α and decreased expression of SREBP-2 and associated targets. Rohitukine decreased hepatic and gonadal lipid accumulation and ameliorated dyslipidemia significantly. In summary, our strategy to identify a novel antiadipogenic molecule using CFPMA successfully resulted in identification of rohitukine, which confirmed antiadipogenic activity and also exhibited in vivo antidyslipidemic activity.     

Effects of fatty acids on the growth of Caco-2 cells

Epidemiological studies suggest that polyunsaturated fatty acids may protect against colorectal neoplasia. In order to explore this observation, cell proliferation and viability, lipid composition, membrane fluidity, and lipid peroxidation were measured in Caco-2 cells after 48h incubation with various fatty acids. Saturated and monounsaturated fatty acids incorporated less well in the membranes than polyunsaturated fatty acids (PUFAs). All of the PUFAs tested had an inhibitory effect on cell proliferation/viability whereas the saturated and monounsaturated fatty acids did not. Addition of palmitic acid had no significant effect on membrane fluidity whereas unsaturated fatty acids increased membrane fluidity in a dose-dependent manner. PUFAs strongly increased tumor cell lipid peroxidation in a dose-dependent manner. Saturated and monounsaturated fatty acids increased lipid peroxidation in this cell line only at high concentration. Preincubation of Caco-2 cells with vitamin E prevented the inhibition of proliferation/viability, the elevation of the MDA concentration and the increased membrane fluidity induced by PUFAs. Our data indicate that PUFAs are potent inhibitors of the growth of colon cancer cells in vitro.     

The effect of propoxycaine.HCl on the physical properties of neuronal membranes

Fluorescent probe techniques were used to evaluate the effect of propoxycaine.HCl on the physical properties (transbilayer asymmetric lateral and rotational mobilities, annular lipid fluidity and protein distribution) of synaptosomal plasma membrane vesicles (SPMVs) isolated from bovine cerebral cortex. An experimental procedure was used based on selective quenching of both 1,3-di(1-pyrenyl)propane (Py-3-Py) and 1,6-diphenyl-1,3,5-hexatriene (DPH) by trinitrophenyl groups, and radiationless energy transfer (RET) from the tryptophans of membrane proteins to Py-3-Py. Propoxycaine.HCl increased the bulk lateral and rotational mobilities, and annular lipid fluidity in SPMVs lipid bilayers, and had a greater fluidizing effect on the inner monolayer than that of the outer monolayer. The magnitude of increasing effect on annular lipid fluidity in SPMVs lipid bilayer induced by propoxycaine.HCl was significantly far greater than magnitude of increasing effect of the drug on the lateral and rotational mobilities of SPMVs lipid bilayer. It also caused membrane proteins to cluster. These effects of propoxycaine.HCl on neuronal membranes may be responsible for some, though not all, of the local anesthetic actions of propoxycaine.HCl.     

The effect of bupivacaine·HCl on the physical properties of neuronal membranes

Fluorescent probe techniques were used to evaluate the effect of bupivacaine.HCl on the physical properties (transbilayer asymmetric lateral and rotational mobilities, annular lipid fluidity and protein distribution) of synaptosomal plasma membrane vesicles (SPMVs) isolated from bovine cerebral cortex. An experimental procedure was used based on selective quenching of both 1,3-di(1-pyrenyl)propane (Py-3-Py) and 1,6-diphenyl-1,3,5-hexatriene (DPH) by trinitrophenyl groups, and radiationless energy transfer (RET) from the tryptophans of membrane proteins to Py-3-Py. Bupivacaine.HCl increased the bulk lateral and rotational mobilities, and annular lipid fluidity in SPMVs lipid bilayers, and had a greater fluidizing effect on the inner monolayer than that of the outer monolayer. The magnitude of increasing effect on annular lipid fluidity in SPMVs lipid bilayer induced by bupivacaine.HCl was significantly far greater than magnitude of increasing effect of the drug on the lateral and rotational mobilities of bulk SPMVs lipid bilayer. It also caused membrane proteins to cluster. These effects of bupivacaine.HCl on neuronal membranes may be responsible for some, though not all, of the local anesthetic actions of bupivacaine.HCl.     

Quercetin and epigallocatechin gallate effects on the cell membranes biophysical properties correlate with their antioxidant potential

Quercetin and epigallocatechin gallate are two of the most abundant polyphenols in dietary plants, including apples, onions, red wine and green tea. The bioactivity of polyphenols is linked to their ability to interact with cell membranes without being internalized. The aim of the present study was to assess the short-time effect of these polyphenols on membrane anisotropy and transmembrane potential of U937 monocytes and Jurkat T lymphoblasts. Results showed that quercetin and epigallocatechin gallate induced, after 20 minutes cell exposure, a dose-dependent increase of membrane anisotropy and polarization. Anisotropy increase was correlated with the reduction of lipid peroxidation. Our results could indicate that the antioxidant capacity of the tested polyphenols is due to their stabilizing effect on the cell membranes, thus contributing to cell protection in various pathologies and as adjuvant therapy in highly toxic treatment regimens.     

Interaction of natural polyphenols with α-amylase in vitro: molecular property-affinity relationship aspect

The relationship between the structural properties of natural polyphenols and their affinities for α-amylase were investigated by fluorescence titration analysis. The binding process with α-amylase was strongly influenced by the structural differences of the compounds under study. For instance, the methylation of the hydroxyl group in flavonoids increased their binding affinities for α-amylase by 2.14 to 7.76 times. The hydroxylation on rings A, B, and C of flavonoids also significantly affected their affinities for α-amylase. The glycosylation of isoflavones and flavanones reduced their affinities for α-amylase and the glycosylation of flavones and flavonols enhanced their affinities for α-amylase. Hydrogenation of the C2=C3 double bond of flavonoids decreased the binding affinities. The galloylated catechins had higher binding affinities with α-amylase than non-galloylated catechins and the pyrogallol-type catechins had higher affinities than the catechol-type catechins. The presence of the galloyl moiety is the most decisive factor. The glycosylation of resveratrol decreased its affinity for α-amylase. The esterification of gallic acid significantly reduced the affinity for α-amylase. The binding interaction between polyphenols and α-amylase was mainly caused by hydrophobic forces.     

The effect of temperature on the growth and lipid composition of the extremely halophilic coccus,Sarcina marina

Sarcina marina (NCMB 778) grew over the temperature range 20–45°C but no growth was recorded at 15°C or 50°C. At the optimum growth temperature of 34°C the doubling time was 14.5 h.The major polar lipid components, tentatively identified as the diether analogues of phosphatidyl glycerophosphate (PGP), phosphatidyl glycerol (PG), diglycosyl diglyceride (DGD) and triglycosyl diglyceride (TGD), and the major neutral lipid components, tentatively identified as squalene, dihydrosqualene, tetrahydrosqualene, vitamin MK8, geranyl geraniol and di-O-phytanyl glycerol, are identical to those found in other extremely halophilic rods and cocci.The total lipid content varied with growth conditions from 0.6 – 3.2% of the dry cell weight, polar lipids accounted for between 94.3 and 83.6% of the total lipid, the remainder being neutral lipid.In response to both the transition from exponential to stationary phase and a reduction of 14°C in growth temperature, batch cultures showed: (i) an increase in total lipid content; (ii) a decrease in PG and (iii) an increase in PGP. Specific responses to the temperature decrease were (i) increased total lipid content; (ii) no decrease in neutral lipids in stationary phase; (iii) marked reduction in PG and (iv) raised DGD. (i) and (ii) could be mechanisms for increasing membrane fluidity.In common with all other extreme halophiles investigated the alkyl side chains of S. marina polar lipids were identified as the phytanyl (3R, 7R, 11R, 15-tetramethylhexadecyl) group. Its structure did not appear to vary with temperature so that the normal mechanisms for modifying the structure of lipid alkyl side chains to modulate membrane fluidity in response to temperature changes probably does not occur in this group of microorganisms.     

Orientation of paramecium swimming in a static magnetic field: Dependence on membrane lipid fluidity

We studied the swimming orientation of the ciliated protozoan Paramecium aurelia in a static magnetic field (0.78 T). P. aurelia is a complex of species termed syngens, whose cell morphology appears similar on microscopic examination. In the magnetic field, the cells of some syngens gradually changed their swimming orientation so that they were swimming perpendicular or parallel to the magnetic field, although such sensitivity to magnetic fields differs between syngens. When the temperature of the cell suspension was raised, the magnetic sensitivity of the cells was decreased. On the other hand, when the cells were cultured beforehand at a high temperature, their magnetic sensitivity was increased. These results raise the possibility that membrane lipid fluidity, which is inversely proportional to the membrane lipid order, contributes to the magnetic orientation of syngens. In this study, measurements of membrane lipid fluidity obtained using fluorescence image analysis with the lipophilic dye, laurdan (6-lauroyl-2-dimethylaminonaphtalene), showed that the degree of membrane lipid fluidity was correlated with the differences in magnetic orientation between syngens. That is, the syngens with decreased membrane fluidity showed an increased degree of magnetic orientation. Therefore, the membrane lipid order is a key factor in the magnetic orientation of Paramecium swimming.     

Alterations in Membrane Lipid Dynamics of Leukemic Cells Undergoing Growth Arrest and Differentiation: Dependency on the Inducing Agent

The effect of various differentiation inducers on membrane cell dynamics was studied using HL-60 and K562 leukemic cell lines. Membrane lipid dynamics was measured by the steady-state fluorescence polarization (P) method utilizing either 1,6-diphenyl-1,3,5-hexatriene (DPH) or the trimethyl ammonium derivative of DPH (TMA-DPH), which ascertains anchorage of the label to the membrane–water–lipid interface. Decrease in membrane microfluidity was observed in HL-60 cells undergoing differentiation into macrophages by 1,25-dihydroxyvitamin D₃and by K562 cells induced to differentiate by DMSO. Sodium butyrate caused an increase in membrane fluidity in K562 cells undergoing differentiation into erythroid-like cells while in HL-60 cells a dual effect was observed. At 0.4 mM concentration, in which the cells were induced to differentiate along the monocyte pathway, a decrease in membrane fluidity was observed, while at 1 mM concentration an increase in membrane fluidity occurred. Interferon-γ (IFN-γ) induced an increase in membrane fluidity in both cell lines. Using HL-60 cells fluorescently labeled by TMA-DPH, similar results indicating fluidization of the membrane following IFN-γ treatment were obtained. Advanced fluorescence lifetime measurements, evaluated either by phase modulation spectrofluorometry or by single photon correlation fluorometry confirmed that the decrease in fluorescence polarization by IFN-γ resulted from membrane fluidization and not from elongation of the probe's excited state lifetime. It is suggested that the inducer mode of action, and not the differentiation route, determine the outcome of changes in membrane microviscosity.     

RFP134对UMR106细胞膜脂流动性的影响(英文)

癌细胞具有与正常细胞不同的膜脂流动性,导致细胞对生长因子和癌基因产物反应敏感;引起细胞增殖失控。本实验室从植物中发现一种二萜类活性物质──RFP134,在细胞周期和信号传递等多方面表现出有抑制癌细胞增殖,促进细胞分化的作用。本文以大鼠成骨肉瘤细胞（UMR106）和正常大鼠成骨细胞为模型,研究其对癌细胞膜脂流动性的影响。细胞系UMR106由美国麻省总医院内分泌室赠送。成骨细胞由本实验室分离培养。以不同浓度（20、40、60、80、100μM／L）的RFP134,在同一时间处理细胞,或以最适浓度（50μM／L）在不同时间作用于细胞。DPH为荧光标记物,测得的荧光偏振值和微粘度值为膜膜流动性指标。结果显示,无论在恒定的时间、以不同浓度的RFP134作用于UMR106细胞（Fig.1B）,或以恒定的浓度、在不同时间处理UMR106细胞（Fig．1D）,结果均表现为显著降低膜脂流动性。前者,RFP134作用于细胞时,细胞荧光偏振值与微粘度值逐步升高,其变化呈量效关系;而后者,呈时效关系。但在最适浓度与最佳作用时间,荧光偏振值和微粘度值达饱和状态。在同样条件下,RFP134对正常成骨细胞的膜脂流动性影响极小。即：荧光偏振值和微粘度值均在正常范围内保持恒定（Fig．1A;Fig．1C）。RFP134降低癌细胞的膜脂流动性     

Effects of long-chain cis-unsaturated fatty acids and their alcohol analogs on aggregation of bovine platelets and their relation with membrane fluidity change

The effects of long-chain cis-unsaturated fatty acids with different alkyl chain lengths and different numbers of double bonds on aggregation of bovine platelets and membrane fluidity were investigated. All the cis-unsaturated fatty acids tested inhibited aggregation and at the same time increased membrane fluidity in accordance with their inhibitory effects. The saturated fatty acids and trans-unsaturated fatty acid tested for comparison had much lower or no effects on aggregation and membrane fluidity. The inhibitory effects of mono cis-unsaturated fatty acids increased with increase of their alkyl chain length. cis-Unsaturated fatty acids with two or more double bonds had more inhibitory effects than mono-unsaturated fatty acids. The position of the double bonds had less influence than the number of double bonds. We also examined the effects of cis-unsaturated fatty acids on membrane fluidity with diphenylhexatriene and anthroyloxy derivatives of fatty acids as probes and observed increased fluidity to be considerable in the membrane. The alcohol analogs of cis-unsaturated fatty acids also inhibited aggregation and increased membrane perturbation. These results suggest that the inhibition of platelet aggregation by cis-unsaturated compounds is due to perturbation of the lipid layer.