Effects of alkyl chain length of gallate on self-association and membrane binding

Alkyl gallates are anticipated for their use as anti-bacterial and anti-viral agents. Although their pharmacological activities depend on their alkyl chain length, no mechanism has yet been clarified. As described herein, we investigated the membrane binding properties of a series of alkyl gallates using fluorescence measurement to elucidate their different pharmacological activities. Membrane binding of the alkyl gallates increased concomitantly with increasing alkyl chain length, except for cetyl gallate and stearyl gallate. Dynamic light scattering revealed that alkyl gallates with a long alkyl chain are prone to self-association in the solution. Membrane binding abilities of the alkyl gallates are correlated with anti-bacterial and anti-virus activities, as described in previous reports. The partition constants of the alkyl gallates to lipid membranes depend on the membrane components and the membrane phase. Self-association and lipid binding of the alkyl gallates might be primary biophysical factors associated with their pharmacological activities.     

Luteolin and Quercetin Affect the Cholesterol Absorption Mediated by Epithelial Cholesterol Transporter Niemann–Pick C1-Like 1 in Caco-2 Cells and Rats

Niemann–Pick C1-Like 1 (NPC1L1) mediates cholesterol absorption, and ezetimibe is a potent NPC1L1 inhibitor applicable for medication of hypercholesterolemia. Epidemiological studies demonstrated that consumption of polyphenols correlates with a decreased risk for atherosclerosis due to their antioxidant effect. This activity can hardly be attributable to the antioxidant activity only, and we hypothesized that polyphenols inhibit intestinal transport of cholesterol. We elucidated the kinetic parameters of intestinal cholesterol absorption, screened several polyphenols for their ability to specifically inhibit intestinal cholesterol absorption, and determined the inhibitory effects of selected flavonoids in vitro and in vivo. The concentration-dependent uptake of cholesterol by Caco-2 cells obeyed a monophasic saturation process. This indicates the involvement of an active-passive transport, i.e., NPC1L1. Parameters of cholesterol uptake by Caco-2 cells were as follows: J _max, K _t, and K _d were 6.89±2.96 19.03±11.58 µM, and 0.11±0.02 pmol/min/mg protein, respectively. Luteolin and quercetin inhibited cholesterol absorption by Caco-2 cells and human embryonic kidney 293T cells expressing NPC1L1. When preincubated Caco-2 cells with luteolin and quercetin before the assay, cholesterol uptake significantly decreased. The inhibitory effects of these flavonoids were maintained for up to 120 min. The level of inhibition and irreversible effects were similar to that of ezetimibe. Serum cholesterol levels significantly decreased more in rats fed both cholesterol and luteolin (or quercetin), than in those observed in the cholesterol feeding group. As quercetin induced a significant decrease in the levels of NPC1L1 mRNA in Caco-2 cells, the in vivo inhibitory effect may be due to the expression of NPC1L1. These results suggest that luteolin and quercetin reduce high blood cholesterol levels by specifically inhibiting intestinal cholesterol absorption mediated by NPC1L1.     

Molecular design of multifunctional antibacterial agents against methicillin resistant Staphylococcus aureus (MRSA)

Antibacterial activity of a series of alkyl gallates (3,4,5-trihydroxybenzoates) against Gram-positive bacteria, especially methicillin resistant Staphylococcus aureus (MRSA) strains was evaluated. Gram-positive bacteria are all susceptible to alkyl gallates. Dodecyl gallate was the most effective against MRSA ATCC 33591 strain with the minimum bactericidal concentration (MBC) of 25 microg/mL (74 microM). The time-kill curve study showed that dodecyl gallate was bactericidal against this MRSA strain at any growth stage. This activity was observed even in the chloramphenicol-treated cells, but the rate of decrease of cell number was slower than that in the exponentially growing cells. The bactericidal activity of medium-chain alkyl gallates was noted in combination with their ability to disrupt the native membrane-associated function nonspecifically as surface-active agents (surfactants) and to inhibit the respiratory electron transport. Subsequently, the same series of alkyl protocatechuates (3,4-dihydroxybenzoates) were studied and the results obtained are similar to those found for alkyl gallates. The length of the alkyl chain is not a major contributor but is related to the activity.     

Effects of alkyl chain length of gallates on their antifungal property and potency as an environmentally benign preservative against wood-decay fungi

This study investigated effects of alkyl chain length of eight aliphatic gallates from C₁ to C₁₈ on their antifungal activity and free radical scavenging activity, which are two important indicators in developing wood preservatives. Results from the agar plate test showed that the antifungal activity against wood-rot fungi of gallates was related to alkyl chain length. It increased with increasing alkyl chain length, reaching a maximum at octyl gallate (C₈), and then decreased as chain length increased. Octyl gallate also exhibited potential antifungal activity against soft-rot Chaetomium globosum and copper-tolerant fungi Wolfiporia extensa and Poria placenta, which are difficult to combat with current copper-based wood preservatives. Octyl gallate is a potent antifungal agent with excellent antifungal activity over a broad antifungal spectrum. All of the gallates tested, regardless of their alkyl chain length, showed strong scavenging activity on the DPPH radical with EC₅₀ values around 1–5 μg ml^?1, indicating that the alkyl chain length was not directly related to this activity. Results from the soil block test showed that excellent antioxidants such as propyl gallate (C₃) and octyl gallate impart wood with good resistance against wood-decay fungi. This suggests that antioxidants have potential as environmentally benign wood preservatives.     

Anti-MRSA activity of alkyl gallates.

A series of alkyl gallates (3,4,5-trihydroxybenzoates) was found to show antibacterial activity against Gram-positive bacteria including methicillin resistant Staphylococcus aureus (MRSA) strains. For example, dodecyl (C(12)) gallate (1) exhibited bactericidal activity against MRSA ATCC 33591 strain with the minimum bactericidal concentration (MBC) of 25 microg/mL (74 microM). The time-kill curve study showed that dodecyl gallate is bactericidal against this MRSA strain. This bactericidal activity comes in part from its ability to inhibit respiratory electron transport systems. The length of the alkyl chain is not a major contributor but plays an important role in eliciting the activity.     

Non-antibiotic antibacterial activity of dodecyl gallate

Dodecyl (C(12)) gallate (3,4,5-trihydroxybenzoate) (1) was found to possess antibacterial activity specifically against Gram-positive bacteria, in addition to its potent antioxidant activity. The time-kill curve study indicates that this amphipathic gallate exhibits bactericidal activity against methicillin resistant Staphylococcus aureus (MRSA) strains. Dodecyl (lauryl) gallate inhibited oxygen consumption in whole cells and oxidation of NADH in membrane preparation. The antibacterial activity of this gallate comes in part from its ability to inhibit the membrane respiratory chain. As far as alkyl gallates are concerned, their antimicrobial spectra and potency depend in part on the hydrophobic portion of the molecule.     

Effects of alkyl chain lengths of gallates upon enzymatic wool functionalisation

The covalent grafting of alkyl gallates on wool through a laccase catalysed reaction in 80/20 (v/v, %) aqueous–ethanol mixture provided in a one-step process a multifunctional textile material with antioxidant, antibacterial and water repellent properties. Gallic acid and its alkyl esters ethyl, propyl, octyl and dodecyl gallate have been enzymatically grafted on wool fibres in order to study the effect of alkyl chain length on wool functional modification. The capacity of laccase to oxidise these phenolic compounds in an aqueous–organic medium has been verified by electrochemical techniques. The increase of CH₂, CH₃ groups in the FTIR spectra, together with the XPS analysis of the enzymatically modified fabrics confirmed the covalent grafting of ester gallates on wool. The result obtained in this work for antibacterial, water repellent as well as antioxidant properties show that the length of the alkyl chain of gallates molecule play an important role on wool functionalisation.     

Quercetin-iron chelates are transported via glucose transporters

Flavonoids are well-known antioxidants and free radical scavengers. Their metal-binding activity suggests that they could be effective protective agents in pathological conditions caused by both extracellular and intracellular oxidative stress linked to metal overload. Quercetin is both a permeant ligand via glucose transport proteins (GLUTs) and a high-affinity inhibitor of GLUT-mediated glucose transport. Chelatable “free iron” at micromolar concentrations in body fluids is a catalyst of hydroxyl radical (OH^?) production from hydrogen peroxide. A number of flavonoids, e.g., quercetin, luteolin, chrysin, and 3,6-dihydroxyflavone, have been demonstrated to chelate intracellular iron and suppress OH^? radical production in Madin Darby canine kidney cells. The most effective chelation comes from the flavonone B ring catechol found in both quercetin and luteolin. We show here that quercetin concentrations of < 1 μM can facilitate chelatable iron shuttling via GLUT1 in either direction across the cell membrane. These siderophoric effects are inhibited by raised quercetin concentrations (> 1 μM) or GLUT inhibitors, e.g., phloretin or cytochalasin B, and iron efflux is enhanced by impermeant extracellular iron chelators, either desferrioxamine or rutin. This iron shuttling property of quercetin might be usefully harnessed in chelotherapy of iron-overload conditions.     

Uptake of phenolic compounds from plant foods in human intestinal Caco-2 cells

In continuation of our studies on the bioaccessibility of phenolic compounds from food grains as influenced by domestic processing, we examined the uptake of phenolics from native/sprouted finger millet (Eleucine coracana) and green gram (Vigna radiata) and native/heat-processed onion (Allium cepa) in human Caco-2 cells. Absorption of pure phenolic compounds, as well as the uptake of phenolic compounds from finger millet, green gram, and onion, was investigated in Caco-2 monolayer model. Transport of individual phenolic compounds from apical compartment to the basolateral compartment across Caco-2 monolayer was also investigated. Sprouting enhanced the uptake of syringic acid from both these grains. Open-pan boiling reduced the uptake of quercetin from the onion. Among pure phenolic compounds, syringic acid was maximally absorbed, while the flavonoid isovitexin was least absorbed. Apparent permeability coefficient P_(app) of phenolic compounds from their standard solutions was 2.02 × 10^?6 cm/s to 8.94 × 10^?6 cm/s. Sprouting of grains enhanced the uptake of syringic acid by the Caco-2 cells. Open-pan boiling drastically reduced the uptake of quercetin from the onion. The permeability of phenolic acids across Caco-2 monolayer was higher than those of flavonoids.     

The antimutagenic activity of the major flavonoids of rooibos (Aspalathus linearis): some dose-response effects on mutagen activation-flavonoid interactions

The antimutagenic properties of the most prevalent flavonoids in rooibos (Aspalathus linearis) were compared in the Salmonella typhimurium mutagenicity assay using tester strains TA98 and TA100 with, respectively, 2-acetamido-fluorene (2-AAF) and aflatoxin B(1) (AFB(1)) as mutagens in the presence of metabolic activation. The flavonoids included the dihydrochalcones aspalathin and nothofagin and their flavone analogues, orientin and isoorientin, and vitexin and isovitexin, respectively, as well as luteolin, chrysoeriol, (+)-catechin, quercetin, isoquercitrin, hyperoside and rutin. Flavonoid-mutagen interactions ranged from antimutagenic, comutagenic and promutagenic to mutagenic, while dose-response effects were mutagen-specific and ranged from typical to atypical including biphasic and threshold effects. Aspalathin and nothofagin and their structural flavonoid analogues displayed moderate antimutagenic properties while luteolin and to some extent, chrysoeriol, showed activities comparable to those of the green tea flavonoid (-) epigallocatechin gallate (EGCG). Apart from their mutagenic and promutagenic properties, quercetin and isoquercitrin exhibited concentration-dependent comutagenic and/or antimutagenic effects against 2-AAF- and AFB(1)-induced mutagenesis. Different structural parameters known to affect the antimutagenic properties of flavonoids include their hydrophilic or lipophilic nature due to the extent of hydroxylation and O-methylation, glycosylation on the A and B rings, the C4-keto group and the C2-C3 double bond. The C ring does not appear to be a prerequisite when comparing for the antimutagenic activity of the dihydrochalcones when compared of the dihydrochalcones with the structural flavone analogues.     

Expression and localization of organic cation/carnitine transporter OCTN2 in Caco-2 cells

l-Carnitine is derived both from dietary sources and biosynthesis. Dietary carnitine is absorbed in the small intestine and then distributed to other organs. Previous studies using Caco-2 cells demonstrated that the transport of l-carnitine in the intestine involves a carrier-mediated system. The purpose of this study was to determine whether the uptake of l-carnitine in Caco-2 cells is mediated by the recently identified organic cation/carnitine transporter (OCTN2). Kinetics of l-[(3)H]carnitine uptake were investigated with or without specific inhibitors. l-Carnitine uptake in mature cells was sodium dependent and linear with time. K(m) and V(max) values for saturable uptake were 14.07 +/- 1.70 micro M and 26.3 +/- 0.80 pmol. mg protein(-1). 6 min(-1), respectively. l-carnitine uptake was inhibited (P < 0.05-0.01) by valproate and other organic cations. Anti-OCTN2 antibodies recognized a protein in the brush-border membrane (BBM) of Caco-2 cells with an apparent molecular mass of 60 kDa. The OCTN2 expression was confirmed by double immunostaining. Our results demonstrate that l-carnitine uptake in differentiated Caco-2 cells is primarily mediated by OCTN2, located on the BBM.     

A review on structure–affinity relationship of dietary flavonoids with serum albumins

Flavonoids are a class of plant secondary metabolites and among thousands of flavonoids few are considered as dietary flavonoids. Serum albumin (SA), the most abundant protein in plasma, functions as the most important carrier of vital drugs, including dietary flavonoids. The binding affinity of dietary flavonoids to SA is demonstrated to be governed by structure–affinity relationship (SAR) and its bioavailability. The present review summarizes the interactions of flavonoids categorized as flavanol, flavonol, flavone, isoflavone, flavanones, and anthocyanidins with SAs (bovine serum albumin and human serum albumin) in light of SAR. The key findings are: (1) the position and degree of hydroxylation highly influence the affinity of flavonoids to SAs, (2) glycosylation decreases and substitution of methoxy group increases the affinity of flavonoids for SAs, (3) catechin gallates have higher binding affinity to SAs than catechins and gallocatechins, (4) inorganic metal ions modulate the binding affinity of flavonoids to SAs, and (5) hydrophobic interaction plays a major role in the interactions of all flavonoids with SAs.     

Effect of three flavonoids, 5,7,3',4'-tetrahydroxy-3-methoxy flavone, luteolin, and quercetin, on the stimulus-induced superoxide generation and tyrosyl phosphorylation of proteins in human neutrophil

The effect of three flavonoids, 5,7,3',4'-tetrahydoxy-3-methoxy flavone (THMF), luteolin, and quercetin, on the stimulus-induced superoxide generation and tyrosyl phosphorylation of proteins in human neutrophils were investigated. When the cells were preincubated with these flavonoids, the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was significantly suppressed, showing a dependence on amounts of the flavonoid. The suppressing effect of the flavonoid was THMF > luteolin > quercetin. These flavonoids also suppressed the superoxide generation induced by phorbol 12-myristate 13-acetate. In this case also, THMF was more effective than luteolin and quercetin. On the other hand, the superoxide generation induced by arachidonic acid was markedly suppressed by quercetin. The suppressing effect was quercetin > THMF > luteolin. THMF, luteolin, and quercetin significantly suppressed tyrosyl phosphorylation of 80.1-, 58.0-, and 45.0-kDa proteins in fMLP-treated human neutrophils. The suppression depended on the concentration of the flavonoids, and the inhibition of tyrosyl phosphorylation was in parallel to that of the fMLP-induced superoxide generation, respectively. While luteolin and quercetin showed a weak hemolytic activity at 2.5 mM, THMF showed almost no hemolytic activity even at 5 mM, suggesting an advantage of THMF for its clinical use.     

Antifungal activity of alkyl gallates against plant pathogenic fungi

The antifungal activity of alkyl gallates against plant pathogenic fungi was evaluated. All of the fungi tested in this study were susceptible to some alkyl gallates, and the effect of linear alkyl gallates against plant pathogenic fungi was similar to the previously reported effects against Gram-negative and Gram-positive bacteria. We found that branched alkyl gallates showed stronger activity than did linear alkyl gallates with similar log P values. In addition, the antifungal activity of alkyl gallates was correlated with gallate-induced inhibition of the activity of mitochondrial complex II. The antifungal activity of alkyl gallates likely originates, at least in part, from their ability to inhibit the membrane respiratory chain.     

Protective effects of flavonoids on the cytotoxicity of linoleic acid hydroperoxide toward rat pheochromocytoma PC12 cells

The protective effects of nine flavonoids, including apigenin, eriodictyol, 3-hydroxyflavone, kaempherol, luteolin, quercetin, rutin, and taxifolin (Table 1), on the cytotoxicity of linoleic acid hydroperoxide (LOOH) toward rat pheochromocytoma PC12 cells were examined. The cytotoxicity was assessed by the trypan blue exclusion test and so-called MTT assay. When cells were preincubated with each flavonoid prior to LOOH exposure, quercetin, 3-hydroxyflavone, or luteolin decreased LOOH cytotoxicity toward undifferentiated cells, while only luteolin decreased efficiently LOOH cytotoxicity toward differentiated cells. On the other hand, when cells were coincubated with each flavonoid and LOOH, kaempherol, eriodictyol, quercetin, 3-hydroxyflavone, luteolin, or taxifolin decreased LOOH cytotoxicity toward undifferentiated and differentiated cells. On both preincubation prior to LOOH exposure and coincubation with LOOH, luteolin acted as the most efficiently protective agent against LOOH cytotoxicity. Further, these flavonoids showed protective effects on coincubation rather than preincubation. Flow cytometry using the fluorescence probe 2',7'-dichlorofluorescin diacetate revealed that LOOH increases the intracellular level of reactive oxygen species in undifferentiated cells in a dose-dependent manner, and that desferrioxamine mesylate suppresses the LOOH-induced increase in the level. These flavonoids suppress the LOOH-induced increase. Further, the protective effect of flavonoids on LOOH cytotoxicity correlates with the suppression of the LOOH-induced increase. These results suggest that such flavonoids are beneficial for neuronal cells under oxidative stress.     

Drug solubilization effect of lauroyl-L-glutamate

This article proposes a new technique for the solubilization of poorly soluble drugs using lauroyl-L-glutamate, which is one of the amino acid detergents, with additional small additives. Lauroyl-L-glutamate was highly effective in solubilizing long-chain alkyl gallates, e.g. dodecyl gallate. Furthermore, lauroyl-L-glutamate and small additives, particularly arginine, acted to increase the solubility of alkyl gallates. The synergistic effect was not observed by sodium dodecyl sulphate with arginine. The solubilizing system can be applied to other drugs because of the low toxicity of both lauroyl-L-glutamate and arginine.     

Inhibition of beta-carotene-15,15'-dioxygenase activity by dietary flavonoids

Beta-carotene-15,15'-dioxygenase is an enzyme responsible for providing vertebrates with vitamin A by catalyzing oxidative cleavage of beta-carotene at its central double bond to two molecules of retinal in intestinal cells. However, little data have been reported regarding regulation of the enzyme activity. We have evaluated the effects of antioxidants and dietary flavonoids on the beta-carotene dioxygenase activity in vitro using a pig intestinal homogenate as the enzyme source. 2,6-Di-tert-butyl-4-methylphenol (BHT), a synthetic antioxidant, strongly inhibited the activity at the level of 10(-6) M (a mixed-type inhibition), whereas butylated hydroxyanisole, nor-dihydroguaiaretic acid, n-propyl gallate, and curcumin were moderately inhibitory. Flavonoids such as luteolin, quercetin, rhamnetin, and phloretin remarkably inhibited the dioxygenase activity noncompetitively, whereas flavanones, isoflavones, catechins, and anthocyanidins were less inhibitory. The structure-activity relationship indicated that catechol structure of ring B and a planar flavone structure were essential for inhibition. The enzyme inhibition was also indicated in the cultured Caco-2 cells by the significantly reduced conversion of beta-carotene to retinol when incubated with BHT and rhamnetin at 2 microM and 5 microM, respectively. The results suggest that some dietary antioxidants derived from food sources modulate conversion of beta-carotene to vitamin A in intestinal cells.     

苦苣菜属5种植物的黄酮类成分含量分析

菊科（Compositae）苦苣菜属（Sonchus L．）的许多种类在民间作为药用植物使用,大多具有清热解毒、消肿排脓及凉血利湿等功效。苦苣菜属植物主要含有黄酮类及倍半萜类活性成分,其中苦苣菜（S．oleraceus L．）中的总黄酮对脑缺血缺氧小鼠及实验性肝损伤均有明显的保护作用。     

Characteristics of L-glutamine transport during Caco-2 cell differentiation

Glutamine is the main fuel of intestinal epithelial cells, as well as a precursor for the intense nucleotide biosynthesis which arises with the rapid turnover of enterocytes. In order to determine whether glutamine uptake may vary as a function of metabolic demand, glutamine transport across the brush-border membrane of differentiating Caco-2 cells has been investigated. The uptake of L-[(3)H]glutamine was measured between day 7 and day 21 post-seeding. Kinetic analysis with glutamine concentrations ranging from 6.25 microM to 12.8 mM revealed the involvement of high affinity Na(+)-dependent (K(t)=110 microM) and low affinity Na(+)-independent (K(t)=900 microM) transport components at day 7. Both components were partially inhibited by L-lysine in a competitive fashion, suggesting that four different systems were responsible for glutamine uptake: B(0), B(0,+), b(0,+) and L. All four systems were present during the differentiation process, with systems L and B(0) being responsible for up to 80% of glutamine uptake. Caco-2 cell differentiation was associated with a marked decrease in L-glutamine uptake, which affected both the Na(+)-dependent and the Na(+)-independent components. In contrast to glucose uptake, the development of L-glutamine uptake across the brush-border membrane of Caco-2 cells may reflect an adjustment to cell metabolic demand rather than the progressive appearance of a vectorial transport process.