Molecular design of antifungal agents

In a rational approach to the design of antifungal agents against Saccharomyces cerevisiae, a series of alkyl gallates (3,4,5-trihydroxybenzoates) were synthesized and assayed. Nonyl gallate (1) was found to be the most effective with a minimum fungicidal concentration (MFC) of 12.5 microg/mL (42 microM), followed by octyl gallate (2) with an MFC of 25 microg/mL (89 microM). These MFCs are little influenced by pH values. A time-kill curve study indicates that nonyl gallate exhibits fungicidal activity against S. cerevisiae at any growing stage. The antifungal activity of nonyl gallate is due primarily to its ability to act as a nonionic surface-active agent (surfactant). The length of the alkyl group is not a major contributor but plays a role in eliciting the activity to a large extent. As far as alkyl gallates are concerned, their antimicrobial spectra and potency depend largely on the hydrophobic portion of the molecules.     

Slow-binding inhibition of soybean lipoxygenase-1 by dodecyl gallate

Dodecyl gallate inhibited the soybean lipoxygenase-1 (EC 1.13.11.12, type-1) catalyzed peroxidation of linoleic acid with an IC50 of 0.007 microM without being oxidized. The progress curves for enzyme reactions were recorded by both spectrophotometric and polarographic methods, and the inhibition kinetics revealed competitive and slow-binding inhibition. Both the initial velocity and steady-state rate in the progress curve decreased with increasing dodecyl gallate. The kinetic parameters that described the inhibition by dodecyl gallate were evaluated by nonlinear regression fits.     

Antioxidant activity of dodecyl gallate

Dodecyl (C12) gallate exhibits both potent chain-breaking and preventive antioxidant activity. The pyrogallol moiety is responsible for both activities. Dodecyl (lauryl) gallate prevents generation of superoxide radicals by xanthine oxidase, and this activity comes from its ability to inhibit the enzyme. The inhibition kinetics analyzed by Lineweaver-Burk plots found that dodecylgallate is a noncompetitive inhibitor for the generation of superoxide anion. Dodecyl gallate also inhibits formation of uric acid. The inhibition kinetics analyzed by Lineweaver-Burk plots found that dodecyl gallate is a competitive inhibitor for this oxidation. Mitochondrial lipid peroxidation induced by Fe(III)-adenosine 5'-diphosphate/reduced nicotinamide adenine dinucleotide was inhibited by dodecyl gallate while its parent compound, gallic acid, did not show this inhibitory activity. Dodecyl gallate protected mitochondrial functions and human red blood cells against oxidative stresses, but gallic acid showed little effect. The hydrophobic dodecyl group is largely associated with the preventive antioxidative activity.     

Inhibition of radical reaction of apolipoprotein B-100 and alpha-tocopherol in human plasma by green tea catechins

(-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), (-)-epigallocatechin gallate (EGCg), and Trolox inhibited the decreases of apolipoprotein B-100 (apoB) and alpha-tocopherol in a radical reaction of human plasma initiated by Cu(2+). The concentrations of EC, EGC, ECg, EGCg, and Trolox for 50% inhibition (IC50) of apoB fragmentation were 39.1, 42.2, 14.6, 21.3, and 36.2 microM, respectively. Similar IC50 values were observed for alpha-tocopherol consumption, indicating the close relationship between apoB fragmentation and alpha-tocopherol consumption. These results demonstrate that tea catechins serve as an effective antioxidant in plasma and that the gallate group has a strong antioxidative activity.     

Antioxidants modulate molecular mobility, oxygen permeability, and microstructure in zein films

The effect of octyl gallate and propyl gallate on the molecular mobility, oxygen permeability, and microstructure of zein/glycerol films was studied. Films were cast from 70% ethanol/water containing 20% (w/w) glycerol and different amounts of the antioxidants propyl gallate or octyl gallate. The oxygen permeability and local mobility of these films were measured using phosphorescence from the dispersed triplet probe erythrosin B. Although both antioxidants increased the local mobility of the zein matrix to about the same extent, octyl gallate and to a lesser extent propyl gallate dramatically increased the permeability of the film to oxygen. Atomic force microscopy imaging indicated that propyl gallate induced aggregation of zein complexes, which could lead to a more condensed film. These results indicate that the addition of specific functional ingredients, such as antioxidants, to edible films may significantly affect the physical properties and structure and, thus, functional properties in ways that influence their eventual use.     

Lipid oxidation in emulsions as affected by charge status of antioxidants and emulsion droplets

The influence of charge status of both lipid emulsion droplets and phenolic antioxidants on lipid oxidation rates was evaluated using anionic sodium dodecyl sulfate (SDS) and nonionic polyoxyethylene 10 lauryl ether (Brij)-stabilized emulsion droplets and the structurally similar phenolic antioxidants gallamide, methyl gallate, and gallic acid. In nonionic, Brij-stabilized salmon oil emulsions at pH 7.0, gallyol derivatives (5 and 500 microM) inhibited lipid oxidation with methyl gallate > gallamide > gallic acid. In the Brij-stabilized salmon oil emulsions at pH 3.0, low concentrations of the galloyl derivatives were prooxidative or ineffective while high concentrations were antioxidative. In SDS-stabilized salmon oil emulsions, oxidation rates were faster and the galloyl derivatives were less effective compared to the Brij-stabilized emulsions. Differences in antioxidant activity were related to differences in the ability of the galloyl derivatives to partition into emulsion droplets and to increase the prooxidant activity of iron at low pH.     

Oxymyoglobin oxidation and membrane lipid peroxidation initiated by iron redox cycle: prevention of oxidation by enzymic and nonenzymic antioxidants.

The red color of muscle is principally due to the presence of oxymyoglobin. Oxidation of heme iron from the ferrous to the ferric state produces a brownish color, which consumers find undesirable. The aim of this study was to use enzymic and nonenzymic antioxidants to simulate in situ muscle antioxidation reactions in order to understand better the mechanism by which the iron redox cycle catalyzes membrane lipid peroxidation and oxymyoglobin oxidation. The inclusion of superoxide dismutase (SOD) in the model system decreased oxymyoglobin oxidation by 10% without affecting lipid peroxidation. Addition of catalase decreased oxymyoglobin oxidation by approximately 40% but not lipid peroxidation. Increasing the ceruloplasmin concentration inhibited lipid peroxidation but increased oxymyoglobin oxidation, which was inhibited by SOD and catalase. Conalbumin (50 microM), a specific iron chelator, inhibited peroxidation and oxymyoglobin oxidation by almost 50%. The addition of the antioxidant catechin (500 microM) decreased lipid peroxidation by 90% but oxymyoglobin oxidation by only 50%. Feeding turkeys with vitamin E at several levels significantly increased the alpha-tocopherol level of membranes, thus preventing oxymyoglobin and lipid oxidation. In conclusion, oxymyoglobin stability in the model system was affected by two pathways: (a) oxygen active species, such as O(2)*(-), H(2)O(2), HO*, and ferryl, generated during autoxidation of myoglobin and oxidation of ferrous ions and ascorbic acid; and (b) lipid radicals, such as ROO*, RO*, and hydroperoxides, generated during lipid peroxidation. Maximum inhibition could be achieved only by introducing inhibitors of both pathways into the system.     

Competitive inhibition of mushroom tyrosinase by 4-substituted benzaldehydes

A kinetic study of the inhibition of mushroom tyrosinase by 4-substituted benzaldehydes showed that these compounds behave as classical competitive inhibitors, inhibiting the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) by mushroom tyrosinase (o-diphenolase activity). The kinetic parameter (K(I)) characterizing this inhibition was evaluated for all of the seven compounds assayed. Cuminaldehyde showed the most potent inhibitory activity (K(I) = 9 microM). It also inhibited the oxidation of L-tyrosine by mushroom tyrosinase (o-monophenolase activity) in a competitive manner. The corresponding kinetic parameter for this inhibition was evaluated (K(I) = 0.12 mM).     

Cytoprotective effect of hydroxytyrosyl alkyl ether derivatives after oral administration to rats in a model of glucose-oxygen deprivation in brain slices

This study was designed to determine whether the oral administration of hydroxytyrosol (HT) alkyl ether derivatives has a neuroprotective effect in rats. The animals were treated for 7 days with HT or ethyl, butyl, hexyl, octyl, and dodecyl HT ether. A method of in vitro hypoxia-reoxygenation in brain slices was used. Hexyl, octyl, and dodecyl HT derivatives reduced brain cell death (LDH efflux). Lipid peroxidation and nitrite concentrations were inhibited most by hexyl, octyl, and dodecyl derivatives. Concentrations of 3-nitrotyrosine were reduced by HT butyl, hexyl, octyl, and dodecyl ether derivatives. Interleukin-1β was significantly reduced in brain slices from rats treated with all HT ether derivatives. LDH efflux showed a linear correlation with brain concentrations of lipid peroxides, nitrites plus nitrates, and interleukin 1β. The reduction in oxidative and nitrosative stress and decreased production of pro-inflammatory interleukins may be the basis for the observed neuroprotective effects.     

Procyanidin fractions from pine (Pinus pinaster) bark: radical scavenging power in solution, antioxidant activity in emulsion, and antiproliferative effect in melanoma cells

Pine (Pinus pinaster) bark is a rich source of procyanidin oligomers. From a total polyphenolic extract, we have generated fractions of different procyanidin composition. The mixtures, devoid of gallate esters, were active as free radical scavengers against ABTS(*+), DPPH, and HNTTM. Pine bark fractions were tested for antioxidant activity in solution (hydrogen donation and electron transfer) and emulsion (inhibition of lipid peroxidation) and compared with their galloylated counterparts from grape origin. While galloylation clearly influenced the free radical scavenging efficiency in solution, it did not seem to play a determinant role in protection against lipid peroxidation in emulsion. The fractions were very mild inhibitors of cell proliferation. Because gallate esters appear to interfere with crucial cell functions, gallate free pine procyanidins may be the innocuous chemopreventative agents of choice for many applications in food and skin protection.     

Free radical scavengers and antioxidants from Lemongrass (Cymbopogon citratus (DC.) Stapf.)

Methanol, MeOH/water extracts, infusion, and decoction of Cymbopogon citratus were assessed for free radical scavenging effects measured by the bleaching of the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical, scavenging of the superoxide anion, and inhibition of the enzyme xanthine oxidase (XO) and lipid peroxidation in human erythrocytes. The extracts presented effect in the DPPH and superoxide anion assay, with values ranging between 40 and 68% and 15-32% at 33 and 50 microg/mL, respectively, inhibited lipid peroxidation in erythrocytes by 19-71% at 500 microg/mL and were inactive toward the XO at 50 microg/mL. Isoorientin, isoscoparin, swertiajaponin, isoorientin 2' '-O-rhamnoside, orientin, chlorogenic acid, and caffeic acid were isolated and identified by spectroscopic methods. Isoorientin and orientin presented similar activities toward the DPPH (IC(50): 9-10 microM) and inhibited lipid peroxidation by 70% at 100 microg/mL. Caffeic and chlorogenic acid were active superoxide anion scavengers with IC(50) values of 68.8 and 54.2 microM, respectively, and a strong effect toward DPPH. Caffeic acid inhibited lipid peroxidation by 85% at 100 microg/mL.     

Inhibition of lysophospholipase D activity by unsaturated lysophosphatidic acids or seed extracts containing 1-linoleoyl and 1-oleoyl lysophosphatidic acid

Lysophospholipase D (lysoPLD), generating lipid mediator lysophosphatidic acid (LPA) from lysophosphatidyclcholine (LPC), is known to be inhibited by lysophosphatidic acids. Meanwhile, some plant lipids are known to contain lysophospholipids as minor components. Therefore, it is interesting to test whether edible seed samples, rich in phospholipids, may contain lysophospholipids, which express a strong inhibition of lysoPLD activity. First, the structural importance of fatty acyl group in LPAs was examined by determining the inhibitory effect of various LPAs on bovine lysoPLD activity. The most potent in the inhibition of lysoPLD activity was linoleoyl-LPA ( K i, 0.21 microM), followed by arachidonoyl-LPA ( K i, 0.55 microM), oleoyl-LPA ( K i, 1.2 microM), and palmitoyl-LPA ( K i, 1.4 microM), based on the fluoresecent assay. The same order of inhibitory potency among LPA analogs with different acyl chains was also found in the spectrophotometric assay. Subsequently, the extracts of 12 edible seeds were screened for the inhibition of lysoPLD activity using both spectrophotometric and fluorescent assays. Among seed extracts tested, the extract from soybean seed, sesame seed, or sunflower seed (30 mg seed weight/mL) was found to exhibit a potent inhibition (>80%) of lysoPLD activity. In further study employing ESI-MS/MS analysis, major LPA components in seed extracts were identified to be 1-linoleoyl LPA, 1-oleoyl LPA, and 1-palmitoyl LPA with 1-linoleoyl LPA being more predominant. Thus, the potent inhibition of lysoPLD activity by seed extracts might be ascribed to the presence of LPA with linoleoyl group rather than other acyl chains.     

Food components inhibiting recombinant human histidine decarboxylase activity

Histidine decarboxylase (HDC) catalyzes histamine formation from histidine. Histamine is a bioactive amine acting as a neurotransmitter as well as a chemical mediator. Phenolic food components have been tested for their ability to inhibit recombinant human HDC. Epicatechin gallate (ECG) was found to be a potent inhibitor as it inhibited HDC activity in a competitive manner with Ki = 10 muM against l-histidine. Epigallocatechin gallate (EGCG) showed time-dependent inhibition which disappeared under anaerobic conditions. It is probable that time-dependent inhibition could be due to the result of autoxidation of EGCG. The initial burst observed for EGCG suggests that EGCG itself is involved in HDC inhibition as observed for ECG. Our present results have shown that the tested food components can inhibit HDC activity. This inhibition likely affects histamine biosynthesis and possibly leads to controlling the biological action induced by histamine. Therefore, those food components exhibiting HDC inhibitory activity might be potentially useful in controlling histamine-induced biological actions.     

Anti-Salmonella activity of alkyl gallates

A series of alkyl gallates (3,4,5-trihydroxybenzoates) was synthesized and tested for their antibacterial activity against Salmonella choleraesuis. Nonyl (C(9)) and octyl (C(8)) gallates were noted to be the most effective against this food-borne bacterium, each with a minimum bactericidal concentration (MBC) of 12.5 microg/mL, followed by decyl (C(10)) gallate, with a MBC of 25 microg/mL. Dodecyl (C(12)) gallate exhibited activity against S. choleraesuis, with a MBC of 50 microg/mL. Propyl (C(3)) gallate showed no activity against S. choleraesuis up to 3200 microg/mL. The length of the alkyl group is not a major contributor but plays a role in eliciting the activity to a large extent. The same series of alkyl gallates, regardless of alkyl chain length, all showed nearly the same potent scavenging activity on the 1,1-diphenyl-2-picrylhydrazyl radical, indicating that the length of the alkyl group is not associated with the activity.     

Hydroxytyrosol prevents oxidative deterioration in foodstuffs rich in fish lipids

Hydroxytyrosol, a natural phenolic compound obtained from olive oil byproduct, was characterized as an antioxidant in three different foodstuffs rich in fish lipids: (a) bulk cod liver oil (40% of omega-3 PUFAs), (b) cod liver oil-in-water emulsions (4% of omega-3 PUFAs), and (c) frozen minced horse mackerel ( Trachurus trachurus) muscle. Hydroxytyrosol was evaluated at different concentration levels (10, 50, and 100 ppm), and its antioxidant capacity was compared against that of a synthetic phenolic, propyl gallate. Results proved the efficiency of hydroxytyrosol to inhibit the formation of lipid oxidation products in all tested food systems, although two different optimal antioxidant concentrations were observed. In bulk oil and oil-in-water emulsions, a higher oxidative stability was achieved by increasing the concentration of hydroxytyrosol, whereas an intermediate concentration (50 ppm) showed more efficiency, delaying lipid oxidation in frozen minced fish muscle. The endogenous depletion of alpha-tocopherol and omega-3 polyunsaturated fatty acids (omega-3 PUFAs) was also inhibited by supplementing hydroxytyrosol in minced muscle; however, the consumption of the endogenous total glutathione was not efficiently reduced by either hydroxytyrosol or propyl gallate. A concentration of 50 ppm of hydroxytyrosol was best to maintain a longer initial level of alpha-tocopherol (approximately 300 microg/g of fat), whereas both 50 and 100 ppm of hydroxytyrosol were able to preserve completely omega-3 PUFAs. Hydroxytyrosol and propyl gallate showed comparable antioxidant activities in emulsions and frozen fish muscle, and propyl gallate exhibited better antioxidant efficiency in bulk fish oil.     

Pachypodol from Croton ciliatoglanduliferus Ort. as water-splitting enzyme inhibitor on thylakoids

A bioactivity-guided chemical study of aerial parts of Croton ciliatoglanduliferus Ort. led to the isolation for the first time of the flavonoids retusin (5-hydroxy-3,7,3',4'-tetramethoxyflavone) (1) and pachypodol (5,4'-dihydroxy-3,7,3'-trimethoxyflavone) (2) from the n-hexane extract. Compounds 1 and 2 were separated by preparative thin-layer chromatography. Compound 2 was the most active compound on ATP synthesis inhibition. The I50 value was 51 microM. Pachypodol behaves as a Hill reaction inhibitor. It inhibited the uncoupled electron flow on photosystem II partial reaction from water to dichlorophenol indophenol (DCPIP) and from water to sodium silicomolybdate. However, the uncoupled partial reaction from diphenylcarbazide to DCPIP and the uncoupled photosystem I from DCPIPred to MV were not inhibited by 2. These results were corroborated by fluorescence decay data. Therefore, pachypodol inhibits the water-splitting enzyme activity. Compound 1 with a 4'-methoxy group was a weak inhibitor, indicating that the 4' free -OH group is important for strong inhibition.     

Mechanisms involved in the antiplatelet activity of rutin, a glycoside of the flavonol quercetin, in human platelets

The aim of this study was to systematically examine the inhibitory mechanisms of rutin, a well-known flavonoid in platelet aggregation. In this study, rutin concentration-dependently (250 and 290 microM) inhibited platelet aggregation in human platelets stimulated by agonists (i.e., collagen). Rutin (250 and 290 microM) did not significantly interfere with the binding of FITC-triflavin to the glycoprotein IIb/IIIa complex in human platelets. Rutin (250 and 290 microM) markedly inhibited intracellular Ca(2+) mobilization and thromboxane A(2) formation in human platelets stimulated by collagen. Rapid phosphorylation of a platelet protein of M(r) 47000 (P47), a marker of protein kinase C activation, was triggered by collagen (1 microg/mL). This phosphorylation was markedly inhibited by rutin (250 and 290 microM). On the other hand, rutin (250 and 290 microM) did not significantly increase the formations of cyclic AMP and nitric oxide/cyclic GMP in platelets. In conclusion, these results indicate that the antiplatelet activity of rutin may involve the following pathways: rutin inhibited the activation of phospholipase C, followed by inhibition of protein kinase C activity and thromboxane A(2) formation, thereby leading to inhibition of the phosphorylation of P47 and intracellular Ca(2+) mobilization, finally resulting in inhibition of platelet aggregation.     

Allelopathic potential and an allelopathic substance in mango leaves

The present study was aimed to determine the allelopathic potential of mango (Mangifera indica L.) leaves and to identify allelopathic substances in the leaves. The aqueous methanol extracts of mango leaves inhibited seedling growth of garden cress, radish, rapeseed, foxtail fescue and crabgrass, and the inhibitory effects increased with the increasing extract concentration, suggesting that mango leaves may contain allelopathic substances. The extract was then purified by several chromatographic runs through a bioassay-directed fractionation, and an growth inhibitory substance was isolated and identified by spectral data as methyl-3,4,5-trihydroxybenzoate (methyl gallate). Methyl gallate at the concentration of 10 mM inhibited 98.6% and 99.8% of root and shoot growth of garden cress relative to the control, respectively, and 94.4% and 49.3% of those of foxtail fescue, respectively. The concentrations of methyl gallate required for 50% growth inhibition (I₅₀) on garden cress roots and shoots were 3.9 and 3.3 mM, and those on foxtail fescue roots and shoots were 1.5 and 9.5 mM, respectively. It is the first report of an allelopathic substance in mango leaves and allelopathic activity of methyl gallate. Therefore, the mango leaves may have potential as a soil additive material for the weed management options.     

Polyphenol-induced inhibition of the response of na(+)/glucose cotransporter expressed in Xenopus oocytes

To study the effects of polyphenols on the Na(+)/glucose cotransporter (SGLT1) response, SGLT1 was expressed in Xenopus oocytes by injecting cRNA synthesized from the cloned cDNA of the small intestine cotransporter of rats, and the electrical response elicited by glucose or galactose was measured by a voltage clamping method. Most phenol derivatives had no effect on the response. However, the polyphenols (+)-catechin, (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg), which are components of green tea, caused an inhibition of the response, which was almost independent of glucose concentration. The inhibition constants were estimated to be 2.3 mM for (+)-catechin and 0.45 mM for both ECg and EGCg, assuming the noncompetitive inhibition mechanism. Saponin prepared from tea seeds also inhibited the response significantly. Tannic acid and aqueous extracts of teas induced nonspecific electrical responses in both cRNA-injected and noninjected oocytes at lower concentrations than those that caused an inhibition of the SGLT1 response when their dose-dependent effects were examined. These results are possibly helpful in the development of a dietary supplement for diabetic patients.     

Immunomodulatory activity of a new family of antioxidants obtained from grape polyphenols

We examined the potential antioxidant activity and the immunopharmacological activity of new epicatechin conjugates obtained by depolymerization of grape polymeric flavanols in the presence of cysteamine or cysteine and with or without gallate. The compounds studied were (-)-epicatechin (1), cysteinyl-epicatechin (2), cysteamine-epicatechin (3), (-)-epicatechin gallate (4), cysteinyl-epicatechin gallate (5), and cysteamine-epicatechin gallate (6) When incubated with an erythrocyte suspension, flavanols protected the erythrocyte membrane from hemolysis induced by 2,2'-azobis(2-amidinopropane) dihydrochloride, an azo free-radical initiator. All the epicatechin derivatives tested were more efficient as antioxidant than epicatechin. The most potent antioxidant was compound 6. The compounds were tested for their capacity to modulate IL-1beta and IL-6, which are the main cytokine factors influencing the acute phase of the inflammatory response. (-)-Epicatechin and its related compounds inhibited the production of IL-1beta and IL-6 in whole blood incubated in the presence of Escherichia coli lipopolysaccharide. The most efficient inhibitor of cytokine formation was compound 3.