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.     

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.     

Evidence of Trolox and some gallates as synergistic protectors of erythrocytes against peroxyl radicals.

The peroxidation of human erythrocytes induced by peroxyl radical initiator and its inhibition by several gallate esters (e.g., propyl, methyl, ethyl) and Trolox (a more polar analogue of vitamin E) have been studied. The antioxidant activity was determined on erythrocytes against hemolysis generated by a thermal activator, 2,2'-azobis-(2-amidinopropane)dihydrogenchloride. It was found that propyl gallate and its two analogues were more effective than Trolox in preventing cell lysis. However, the combination of gallate esters and Trolox produced a protective effect exceeding the arithmetic sum of their individual contributions. These perceived synergisms occur at more than one level of Trolox at a given level of a gallate ester.     

Plasma membrane injury induced by nonyl gallate in Saccharomyces cerevisiae

AIMS: The aim was to investigate the antifungal actions of nonyl gallate against Saccharomyces cerevisiae ATCC 7754. METHODS AND RESULTS: The maximum potency of both the growth inhibitory and the fungicidal effect against the yeast strain was found in nonyl gallate among n-alkyl gallates tested. Nonyl gallate induced ROS generation dose-dependently in growing cells. This ester rapidly killed yeast cells even when cell division was restricted by cycloheximide. This ester inhibited glucose-induced medium acidification and promoted the efflux of intracellular potassium ions in a nongrowing condition. Moreover, nonyl gallate induced a leakage of calcein from artificially prepared liposomes to a greater extent than dodecyl gallate did. CONCLUSIONS: These results suggested nonyl gallate injured plasma membrane of S. cerevisiae, resulting in its exhibition of fungicidal effect accompanying with a leakage of intracellular materials from the cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study reveals new knowledge on the antifungal actions of nonyl gallate against S. cerevisiae. When nonyl gallate is applied as a food preservative, the level of its addition to foods may be reduced because of its potent antifungal activity compared with weak acids including sorbic acid and benzoic acid.     

Molecular characterization of the gallate dioxygenase from Pseudomonas putida KT2440. The prototype of a new subgroup of extradiol dioxygenases

In this work we have characterized the galA gene product from Pseudomonas putida KT2440, a ring-cleavage dioxygenase that acts specifically on gallate to produce 4-oxalomesaconate. The protein is a trimer composed by three identical subunits of 47.6 kDa (419 amino acids) that uses Fe2+ as the main cofactor. The gallate dioxygenase showed maximum activity at pH 7.0, and the Km and Vmax values for gallate were 144 microM and 53.2 micromol/min/mg of protein, respectively. A phylogenetic study suggests that the gallate dioxygenase from P. putida KT2440 is the prototype of a new subgroup of type II extradiol dioxygenases that share a common ancestor with protocatechuate 4,5-dioxygenases and whose two-domain architecture might have evolved from the fusion of the large and small subunits of the latter. A three-dimensional model for the N-terminal domain (residues 1-281) and C-terminal domain (residues 294-420) of the gallate dioxygenase from P. putida KT2440 was generated by comparison with the crystal structures of the large (LigB) and small (LigA) subunits of the protocatechuate 4,5-dioxygenase from Sphingomonas paucimobilis SYK-6. The expression of the galA gene was specifically induced when P. putida KT2440 cells grew in the presence of gallate. A P. putida KT2440 galA mutant strain was unable to use gallate as the sole carbon source and it did not show gallate dioxygenase activity, suggesting that the GalA protein is the only dioxygenase involved in gallate cleavage in this bacterium. This work points to the existence of a new pathway that is devoted to the catabolism of gallic acid and that remained unknown in the paradigmatic P. putida KT2440 strain.     

Initial steps in the anaerobic degradation of 3,4,5-trihydroxybenzoate by Eubacterium oxidoreducens: characterization of mutants and role of 1,2,3,5-tetrahydroxybenzene.

Chemical mutagenesis and antibiotic enrichment techniques were used to isolate five mutant strains of the obligate anaerobe Eubacterium oxidoreducens that were unable to grow on 3,4,5-trihydroxybenzoate (gallate). Two strains could not transform gallate and showed no detectable gallate decarboxylase activity. Two other strains transformed gallate to pyrogallol and dihydrophloroglucinol but lacked the hydrolase activity responsible for ring cleavage. A fifth strain accumulated pyrogallol, although it contained adequate levels of the enzymes proposed for the complete transformation of gallate to the ring cleavage product. The conversion of pyrogallol to phloroglucinol by cell extract of the wild-type strain was dependent on the addition of 1,2,3,5-tetrahydroxybenzene or dimethyl sulfoxide. This activity was induced by growth on gallate, while the other enzymes involved in the initial reactions of gallate catabolism were constitutively expressed during growth on crotonate. The results confirm the initial steps in the pathway previously proposed for the metabolism of gallate by E. oxidoreducens, except for the conversion of pyrogallol to phloroglucinol.     

In vitro characterization of a plastid terminal oxidase (PTOX).

The plastid terminal oxidase (PTOX) encoded by the Arabidopsis IMMUTANS gene was expressed in Escherichia coli cells and its quinone/oxygen oxidoreductase activity monitored in isolated bacterial membranes using NADH as an electron donor. Specificity for plastoquinone was observed. Neither ubiquinone, duroquinone, phylloquinone nor benzoquinone could substitute for plastoquinone in this assay. However, duroquinol (fully reduced chemically) was an accepted substrate. Iron is also required and cannot be substituted by Cu(2+), Zn(2+) or Mn(2+). This plastoquinol oxidase activity is independent of temperature over the 15-40 degrees C range but increases with pH (from 5.5 to 9.0). Unlike higher plant mitochondrial alternative oxidases, to which PTOX shows sequence similarity (but also differences, especially in a putative quinone binding site and in cysteine conservation), PTOX activity does not appear to be regulated by pyruvate or any other tested sugar, nor by AMP. Its activity decreases, however, with increasing salt (NaCl or KCl) concentration. Various quinone analogues were tested for their inhibitory activity on PTOX. Pyrogallol analogues were found to be inhibitors, especially octyl gallate (I50 = 0.4 microM ) that appears far more potent than propyl gallate or gallic acid. Thus, octyl gallate is a useful inhibitor for future in vivo or in organello studies aimed at studying the roles of PTOX in chlororespiration and as a cofactor for carotenoid biosynthesis.     

Antioxidants from steamed used tea leaves and their reaction behavior

The most efficient steaming conditions below 200 degrees C for extracting antioxidants from used tea leaves and their reaction behavior during the steaming treatment were investigated. The antioxidative activity of the steamed extracts increased with increasing steaming temperature, and the yield of the ethyl acetate extract fraction from each steamed extract showing the greatest antioxidative activity also increased. Caffeine, (-)-catechin, (-)-epicatechin, (-)-gallocatechin, (-)-epigallocatechin, (-)-catechin gallate, (-)-epicatechin gallate, (-)-gallocatechin gallate, (-)-epigallocatechin gallate and gallic acid were identified from the ethyl acetate extract fraction. Quantitative analyses demonstrated that the catechins with a 2,3-cis configuration decreased with increasing steaming temperature, whereas the corresponding epimers at the C-2 position increased. Each pair of epimers showed similar antioxidative activity to each other, indicating that the epimerization reaction did not contribute to the improved antioxidative activity. It is concluded from these results that the improvement in antioxidative activity at higher steaming temperatures was due to the increased yield of catechins and other antioxidants.     

Gallic acid, a metabolite of the antioxidant propyl gallate, inhibits gap junctional intercellular communication via phosphorylation of connexin 43 and extracellular-signal-regulated kinase1/2 in rat liver epithelial cells

Propyl gallate and its metabolite, gallic acid, are widely used as antioxidants in the food industry, but they have been shown to exhibit liver toxicity and enhance carcinogenesis. In the present study, we investigated the possible undesirable effects of propyl gallate and gallic acid on gap junctional intercellular communication (GJIC), inhibition of which is closely linked to carcinogenesis. Gallic acid and propyl gallate exhibited dose-dependent free-radical-scavenging activities as determined by 1,1-diphenyl-2-picrylhydrazyl- or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)-radical-scavenging assays, and the free-radical-scavenging activity of gallic acid was stronger than that of propyl gallate. However, using WB-F344 rat liver epithelial cells, gallic acid inhibited GJIC in a dose-dependent manner, while propyl gallate had no significant effect compared with untreated controls. The gallic-acid-induced inhibition of GJIC was reversible, with a recovery of nearly 65% after 120 min. Gallic acid induced the phosphorylation of connexin 43 (Cx43) and phosphorylation of extracellular-signal-regulated kinase1/2 (ERK1/2). The gallic-acid-induced inhibition of GJIC was attenuated by treatment with mitogen-activated protein kinase kinase inhibitors (U0126 and PD098059). U0126 blocked the gallic-acid-induced phosphorylation of Cx43 and ERK1/2, indicating that the gallic-acid-induced inhibition of GJIC is mediated by phosphorylation of Cx43 via activation of ERK1/2. In addition, gallic-acid-induced inhibition of GJIC was protected by ascorbic acid and quercetin, which might represent a simple example of the different effects of natural antioxidants in carcinogenesis.     

Interference of antioxidants E/O of some free radical "scavengers" with the activity of glucose-6-phosphatase after administration of carbon tetrachloride]

G-6-Pase activity was investigated in the microsomal fraction from rat liver in the presence of carbon tetrachloride and/or propyl gallate (PG), reduced glutathione (GSH) and superoxide dismutase. Results obtained "in vitro" demonstrated that CCl4 induced a 60% inhibition of the microsomal enzyme activity. Moreover, a marked inhibition of G-6-Pase activity was found also when propyl gallate and reduced glutathione were added, at different concentrations, to incubation mixture. In addition, these drugs were unable to interfere with the dangerous effect exerted on the enzymatic activity by the haloalkane. Additional experiments carried out "in vivo" with propyl gallate produced evidence that intraperitoneal administration of the antioxidant was followed by a significant inhibition of G-6-Pase activity, while the damaging action of CCl4 was unaffected. Some possible explanations of these results are reported.     

Lauryl Gallate Inhibits the Activity of Protein Tyrosine Kinase c-Src Purified from Human Platelets

The inhibitory effect of gallic acid (3,4,5-trihydroxybenzoic acid), and its ester derivatives methyl, propyl, octyl and lauryl has been tested on the tyrosine kinase activity of affinity purified c-Src from human platelets, using the artificial substrate Poly (Glu.Na, Tyr) 4:1. When tested as inhibitor of the autophosphorylation of the enzyme and the phosphorylation of the protein tyrosine phosphatase SHP-1 by c-Src, lauryl gallate was found to be a more potent inhibitor than other widely used protein tyrosine kinase (PTK) inhibitors such as genistein and herbimycin A. However, lauryl gallate did not inhibit the activity of the serine threonine kinases protein kinase A (PKA) and casein kinase II (CKII) from rat brain.     

Lauryl gallate inhibits the activity of protein tyrosine kinase c-Src purified from human platelets

The inhibitory effect of gallic acid (3,4,5-trihydroxybenzoic acid), and its ester derivatives methyl, propyl, octyl and lauryl has been tested on the tyrosine kinase activity of affinity purified c-Src from human platelets, using the artificial substrate Poly (Glu,Na,Tyr) 4:1. When tested as inhibitor of the autophosphorylation of the enzyme and the phosphorylation of the protein tyrosine phosphatase SHP-1 by c-Src, lauryl gallate was found to be a more potent inhibitor than other widely used protein tyrosine kinase (PTK) inhibitors such as genistein and herbimycin A. However, lauryl gallate did not inhibit the activity of the serine threonine kinases protein kinase A (PKA) and casein kinase II (CKII) from rat brain.     

没食子酸丙酯和没食子酸异丁酯对人红细胞膜结构与功能的影响

本文应用荧光探剂ANS(1—苯胺—8萘磺酸)、NPN(N—苯基—1—萘胺)和DPH(1.6—二苯基—1.3.5—已三烯)观察没食子酸丙醋和没食子酸异丁酯对人红细胞膜流动性和相变温度以及Na~ -K~ ATP酶活性的影响.实验结果指出该两种化合物均能:(1)降低与膜结合的荧光探剂强度但不改变探剂在水相与膜相的分配比例:(2)降低膜脂的相变温度,增加膜的流动性;(3)抑制红细胞膜Na~ -K~ ATP酶活性;(4)标记红细胞膜的DPH偏振度随化合物浓度的增加而降低,膜的流动性增加.在给定的浓度范围内,两种化合物的效应表现为明显的量效关系与构效关系.从上述结果推测该两种化合物可能是通过改变膜脂结构、膜蛋白的脂类环境而调节膜的功能,成为其治疗疾病的机理之一.     

Deodorizing effects of tea catechins on amines and ammonia

Deodorizing effects of tea catechins on amines were examined under alkaline conditions to eliminate the neutralization reaction. They showed deodorizing activity on ethylamine, but none on dimethylamine or trimethylamine. Deodorizing activity on ethylamine was found to be in the order of (-)-epigallocatechin gallate > gallic acid > (-)-epigallocatechin (EGC) > (-)-epicatechin gallate > ethyl gallate > (+)-catechin = (-)-epicatechin. Further, reaction products of EGC with methylamine, ethylamine, and ammonia were detected by HPLC, indicating that a deodorizing reaction other than neutralization occurs. From structural analysis of the reaction product with the methylamine isolated as a peracetylated derivative, the product was presumed to be methylamine substituted EGC, in which the hydroxyl group of EGC at the 4' position is replaced by the methylamino group. The same replacement reaction took place in the case of ethylamine and ammonia.     

Green tea catechins as a BACE1 (beta-secretase) inhibitor

In the course of searching for BACE1 (beta-secretase) inhibitors from natural products, the ethyl acetate soluble fraction of green tea, which was suspected to be rich in catechin content, showed potent inhibitory activity. (-)-Epigallocatechin gallate, (-)-epicatechin gallate, and (-)-gallocatechin gallate were isolated with IC(50) values of 1.6 x 10(-6), 4.5 x 10(-6), and 1.8 x 10(-6) M, respectively. Seven additional authentic catechins were tested for a fundamental structure-activity relationship. (-)-Catechin gallate, (-)-gallocatechin, and (-)-epigallocatechin significantly inhibited BACE1 activity with IC(50) values of 6.0 x 10(-6), 2.5 x 10(-6), and 2.4 x 10(-6) M, respectively. However, (+)-catechin, (-)-catechin, (+)-epicatechin, and (-)-epicatechin exhibited about ten times less inhibitory activity. The stronger activity seemed to be related to the pyrogallol moiety on C-2 and/or C-3 of catechin skeleton, while the stereochemistry of C-2 and C-3 did not have an effect on the inhibitory activity. The active catechins inhibited BACE1 activity in a non-competitive manner with a substrate in Dixon plots.     

Polyhydroxybenzoates inhibit ascorbic acid activation of mitochondrial glycerol-3-phosphate dehydrogenase: implications for glucose metabolism and insulin secretion

Glycerol-3-phosphate dehydrogenase from pig brain mitochondria was stimulated 2.2-fold by the addition of 50 microm l-ascorbic acid. Enzyme activity, dependent upon the presence of l-ascorbic acid, was inhibited by lauryl gallate, propyl gallate, protocatechuic acid ethyl ester, and salicylhydroxamic acid. Homogeneous pig brain mitochondrial glycerol-3-phosphate dehydrogenase was activated by either 150 microm L-ascorbic acid (56%) or 300 microm iron (Fe(2+) or Fe(3+) (62%)) and 2.6-fold by the addition of both L-ascorbic acid and iron. The addition of L-ascorbic acid and iron resulted in a significant increase of k(cat) from 21.1 to 64.1 s(-1), without significantly increasing the K(m) of L-glycerol-3-phosphate (10.0-14.5 mm). The activation of pure glycerol-3-phosphate dehydrogenase by either L-ascorbic acid or iron or its combination could be totally inhibited by 200 microm propyl gallate. The metabolism of [5-(3)H]glucose and the glucose-stimulated insulin secretion from rat insulinoma cells, INS-1, were effectively inhibited by 500 microm or 1 mm propyl gallate and to a lesser extent by 5 mm aminooxyacetate, a potent malate-aspartate shuttle inhibitor. The combined data support the conclusion that l-ascorbic acid is a physiological activator of mitochondrial glycerol-3-phosphate dehydrogenase, that the enzyme is potently inhibited by agents that specifically inhibit certain classes of di-iron metalloenzymes, and that the enzyme is chiefly responsible for the proximal signal events in INS-1 cell glucose-stimulated insulin release.     

Methyl gallate and its 3-glucoside promote rooting in bean cuttings

Methyl gallate stimulated adventitious root formation in cuttings of bean (Phaseolus vulgaris L.). This polyphenol was quickly metabolized into 3-glucosyl methyl gallate to such an extent that 4 h after application no methyl gallate was detected. The isolated glucoside when supplied exogenously at 0.5 mM also enhanced rooting; the effect was 2-fold greater than that of methyl gallate. The glucoside persisted in the cuttings for 72 h after treatment. Because methyl gallate is rapidly transformed to a stable glucoside, we suggest that the root stimulation effect could be ascribed to its glucoside.     

Antioxidants from Steamed Used Tea Leaves and Their Reaction Behavior

The most efficient steaming conditions below 200 °C for extracting antioxidants from used tea leaves and their reaction behavior during the steaming treatment were investigated. The antioxidative activity of the steamed extracts increased with increasing steaming temperature, and the yield of the ethyl acetate extract fraction from each steamed extract showing the greatest antioxidative activity also increased. Caffeine, (?)-catechin, (?)-epicatechin, (?)-gallocatechin, (?)-epigallocatechin, (?)-catechin gallate, (?)-epicatechin gallate, (?)-gallocatechin gallate, (?)-epigallocatechin gallate and gallic acid were identified from the ethyl acetate extract fraction. Quantitative analyses demonstrated that the catechins with a 2,3-cis configuration decreased with increasing steaming temperature, whereas the corresponding epimers at the C-2 position increased. Each pair of epimers showed similar antioxidative activity to each other, indicating that the epimerization reaction did not contribute to the improved antioxidative activity. It is concluded from these results that the improvement in antioxidative activity at higher steaming temperatures was due to the increased yield of catechins and other antioxidants.     

New apoptosis cascade mediated by lysosomal enzyme and its protection by epigallo-catechin gallate

We found a novel procaspase-3 activating cascade mediated by lysosomal enzyme. The activating enzyme of procaspase-3, named lysoapoptase having the molecular weight of 78 kDa was determined to be a lactoferrin located in the lysosome. Recombinant lactoferrin accelerated the processing of procaspase-3 to form active caspase-3 in vitro. d-Galactosamine is a well-known inducer of hepatocyte apoptosis. The caspase-3 which plays a common central role in the final step of various apoptosis cascades, was dramatically increased in the cytoplasm by the d-galactosamine administration in vivo. When d-galactosamine was administrated as a death signal in vivo, the lysosomal lactoferrin was released into the cytoplasm and procaspase-3 located in the cytoplasm was processed to form active caspase-3. The cotreatment of epigallo-catechin gallate resulted in the complete protection of the hepatocyte apoptosis suppressing the increases of caspase-3 in the cytoplasm. The caspase-3 activity was also inhibited directly by the epigallo-catechin gallate. Therefore, all apoptosis cascades mediated by caspase-3 should be suppressed by epigallo-catechin gallate. The caspase-3 activity was not only directly inhibited by epigallo-catechin gallate in vitro, but the release of lactoferrin from the lysosomes into the cytoplasm was also suppressed by epigallo-catechin gallate treatment in vivo. Therefore, the apoptosis induction was suppressed at the two successive steps by cotreatment of epigallo-catechin gallate in vivo.