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We investigated the cytoprotective effect of phloroglucinol, which was isolated from Ecklonia cava (brown alga), against oxidative stress induced cell damage in Chinese hamster lung fibroblast (V79-4) cells. Phloroglucinol was found to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydrogen peroxide (H(2)O(2)), hydroxy radical, intracellular reactive oxygen species (ROS), and thus prevented lipid peroxidation. As a result, phloroglucinol reduced H(2)O(2) induced apoptotic cells formation in V79-4 cells. In addition, phloroglucinol inhibited cell damage induced by serum starvation and radiation through scavenging ROS. Phloroglucinol increased the catalase activity and its protein expression. In addition, catalase inhibitor abolished the protective effect of phloroglucinol from H(2)O(2) induced cell damage. Furthermore, phloroglucinol increased phosphorylation of extracellular signal regulated kinase (ERK). Taken together, the results suggest that phloroglucinol protects V79-4 cells against oxidative damage by enhancing the cellular catalase activity and modulating ERK signal pathway.
The nature and specificity of the Wiesner test (phloroglucinol-HCl reagent) for the aromatic aldehyde fraction contained in lignins is studied. Phloroglucinol reacted in ethanol-hydrochloric acid with coniferyl aldehyde, sinapyl aldehyde, vanillin, and syringaldehyde to yield either pink pigments (in the case of hydroxycinnamyl aldehydes) or red-brown pigments (in the case of hydroxybenzaldehydes). However, coniferyl alcohol, sinapyl alcohol, and highly condensed dehydrogenation polymers derived from these cinnamyl alcohols and aldehydes did not react with phloroglucinol in ethanol-hydrochloric acid. The differences in the reactivity of phloroglucinol with hydroxycinnamyl aldehydes and their dehydrogenation polymers may be explained by the fact that, in the latter, the unsubstituted (alpha,beta-unsaturated) cinnamaldehyde functional group, which is responsible for the dye reaction, is lost due to lateral chain cross-linking reactions involving the beta carbon. Fourier transform infrared spectroscopy and thioacidolysis analyses of phloroglucinol-positive lignifying plant cell walls belonging to the plant species Zinnia elegans L., Capsicum annuumvar. annuum, Populus albaL., and Pinus halepensisL. demonstrated the presence of 4- O-linked hydroxycinnamyl aldehyde end groups and 4- O-linked 4-hydroxy-3-methoxy-benzaldehyde (vanillin) end groups in lignins. However, given the relatively low abundance of 4- O-linked vanillin in lignifying cell walls and the low extinction coefficient of its red-brown phloroglucinol adduct, it is unlikely that vanillin contributes to a great extent to the phloroglucinol-positive stain reaction. These results suggest that the phloroglucinol-HCl pink stain of lignifying xylem cell walls actually reveals the 4- O-linked hydroxycinnamyl aldehyde structures contained in lignins. Histochemical studies showed that these aldehyde structures are assembled, as in the case of coniferyl aldehyde, during the early stages of xylem cell wall lignification.
It was found that the inhibition and enhancement by phloroglucinol of the chemiluminescence from the luminol-K3Fe(CN)6 system were dependent on the pH of luminol solution and the concentration of phloroglucinol. In Na2CO(3)-NaHCO3 buffer, phloroglucinol exhibited strong chemiluminescent enhancement at pH 9.4. On this basis, a flow injection method was developed for the determination of phloroglucinol. The method was simple, rapid, convenient and sensitive, with a detection limit of 2.0 x 10(-9) mol/L. It is effective for determining phloroglucinol in the range of 1.0 x 10(-5)-5.0 x 10(-9) mol/L. The relative standard deviation is 1.3% within one day and 3.2% between days for the determination of 5.0 x 10(-7) mol/L phloroglucinol. The method has been successfully used to determine phloroglucinol in environmental water, with satisfactory results.
An aqueous acetone extract obtained from the pericarps of Mallotus japonicus (MJE) was observed to inhibit prostaglandin (PG) E(2) production in a lipopolysaccharide (LPS)-activated murine macrophage-like cell line, RAW 264.7. Six phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against PGE(2) production. Among these phloroglucinol derivatives, isomallotochromanol showed the strongest inhibitory activity, with an IC(50) of 1.0 microM. MJE and its phloroglucinol derivatives did not effect the enzyme activity of either prostaglandin endoperoxide synthase (PGHS)-1 or PGHS-2. However, induction of PGHS-2 in LPS-activated macrophages was inhibited by MJE and its phloroglucinol derivatives, whereas the level of PGHS-1 protein was not affected. Moreover, RT-PCR analysis showed that MJE and its phloroglucinol derivatives significantly suppressed PGHS-2 mRNA expression. Therefore, the observed inhibition of PGHS-2 induction by MJE and its phloroglucinol derivatives was likely due to a suppression of PGHS-2 mRNA expression. These results suggest that MJE and its phloroglucinol derivatives have the pharmacological ability to suppress PGE(2) production by activated macrophages.
A rumen isolate, Coprococcus, sp. Pe(1)5, was found to carry phloroglucinol reductase, which catalyzed the initial step in the breakdown of phloroglucinol. The organism uses phloroglucinol as the sole source of carbon and energy when grown in the absence of oxygen. Induced levels of enzyme were detected in cells grown either on phloroglucinol or on other carbon sources in the presence of limiting quantities of phloroglucinol. Although the organism is a strict anaerobe, the enzyme from anaerobically grown cells was insensitive to air. The partially purified enzyme required reduced nicotinamide adenine dinucleotide phosphate as an electron donor and was specific for phloroglucinol. However, partial enzyme activity (14 to 17%) was also detected in the presence of 2-methyl-1,4-naphthoquinone but not in the presence of several other phenolic compounds. The enzyme exhibited a higher affinity for phloroglucinol than for reduced nicotinamide adenine dinucleotide phosphate, with K(m) values of 3.0 x 10 M and 29.0 x 10 M, respectively. The optimum pH for maximal enzyme activity was 7.4, and the molecular weight of the native protein was about 130,000, as determined by the Sephadex gel filtration technique.
Flavan-3-ol phloroglucinol adducts were synthesised through acid catalysed degradation of a procyanidins-rich grape seed extract in the presence of phloroglucinol. The reaction mixture (3.3 g) was fractionated without further sample preparation using the all-liquid chromatographic technique of high-speed counter-current chromatography (HSCCC). Selected solvent systems were hexane-ethyl acetate-methanol-water (0.1:5:0.1:5, v/v/v/v) and (1.5:10:1.5:10, v/v/v/v). The fractions obtained were found to contain almost pure compounds, in some cases final purification was achieved by preparative HPLC. The so-obtained pure standards of (+)catechin-(4alpha-->2)-phloroglucinol, (-)epicatechin-(4beta-->2)-phloroglucinol, (+)catechine, (-)epicatechin-3-O-galloyl-(4beta-->2)-phloroglucinol, (-)epicatechin, and (-)epicatechin gallate are required for quantification of acid-catalysed phloroglucinol degradation products of procyanidins.
Substantial concentrations of phloroglucinol were synthesized by Pseudomonas fluorescens Pf-5 expressing the plasmid-localized phlACBDE gene cluster responsible for biosynthesis of 2,4-diacetylphloroglucinol. Expression in Escherichia coli of a single gene in this cluster, P. fluorescens Pf-5 phlD, led to extracellular accumulation of phloroglucinol. Purification of PhlD to homogeneity afforded an enzyme that catalyzed the conversion of malonyl-CoA into phloroglucinol with Km = 5.6 muM and kcat = 10 min-1. Acetylase and deacetylase activities were observed with the catalyzed interconversions of phloroglucinol, 2-acetylphloroglucinol, and 2,4-diacetylphloroglucinol when phlACB was expressed in E. coli. Beyond the mechanistic implications attendant with the identification of an enzyme that catalyzes the conversion of malonyl-CoA into phloroglucinol, PhlD provides the basis for environmentally benign syntheses of phloroglucinol and resorcinol from glucose.
The pathway for the anaerobic catabolism of gallic acid by Eubacterium oxidoreducans was studied by using both in vivo and cell-free systems. Cells grown with gallate and crotonate, but with no formate or H2, excreted pyrogallol and phloroglucinol into the medium. Gallate was decarboxylated by crude cell extracts, with pyrogallol as the only detectable product. Whole cells converted pyrogallol to phloroglucinol. A phloroglucinol reductase catalyzed the conversion of phloroglucinol to dihydrophloroglucinol when NADPH was used as the source of electrons. Both formate dehydrogenase (EC 1.2.1.43) and hydrogenase (EC 1.18.99.1) were present in cell extracts of gallate-formate-grown cells. These two enzymes were both NADP linked. Since either H2 or formate is required for cell growth with gallate or phloroglucinol, these results suggest that the oxidation of the reduced substrate may be indirectly linked to the reduction of phloroglucinol. A dihydrophloroglucinol hydrolase was present, which hydrolyzed dihydrophloroglucinol to 3-hydroxy-5-oxohexanoate. This six-carbon ring cleavage product then presumably can be broken down by a series of reactions similar to beta-oxidation. These reactions cleaved the six-carbon acid to 3-hydroxybutyryl-coenzyme A yielding acetate and butyrate as end products. A number of key enzymes involved in beta-oxidation and substrate-level phosphorylation were demonstrated in cell extracts.
Permeabilized cells and cell extracts of Pelobacter acidigallici catalyzed the conversion of pyrogallol (1,2,3-trihydroxybenzene) to phloroglucinol (1,3,5-trihydroxybenzene) in the presence of 1,2,3,5-tetrahydroxybenzene. Pyrogallol consumption by resting cells stopped after lysis by French press or mild detergent (cetyltrimethylammonium bromide [CTAB]) treatment. Addition of 1,2,3,5-tetrahydroxybenzene to the assay mixture restored pyrogallol consumption and led to stoichiometric phloroglucinol accumulation. The stoichiometry of pyrogallol conversion to phloroglucinol was independent of the amount of tetrahydroxybenzene added. The tetrahydroxybenzene concentration limited the velocity of the transhydroxylation reaction, which reached a maximum at 1.5 mM tetrahydroxybenzene (1 U/mg of protein). Transhydroxylation was shown to be reversible. The equilibrium constant of the reaction was determined, and the free-energy change (delta G degree') of phloroglucinol formation from pyrogallol was calculated to be -15.5 kJ/mol. Permeabilized cells and cell extracts also catalyzed the transfer of hydroxyl moieties between other hydroxylated benzenes. Tetrahydroxybenzene and hydroxyhydroquinone participated as hydroxyl donors and as hydroxyl acceptors in the reaction, whereas pyrogallol, resorcinol, and phloroglucinol were hydroxylated by both donors. A novel mechanism deduced from these data involves intermolecular transfer of the hydroxyl moiety from the cosubstrate (1,2,3,5-tetrahydroxybenzene) to the substrate (pyrogallol), thus forming the product (phloroglucinol) and regenerating the cosubstrate.
Biodegradation of phloroglucinol (1,3,5-trihydroxybenzene) and other polyphenolic compounds by microbes may occur by aerobic and anaerobic metabolic pathways. Aerobic microbes may initiate the mineralization of phloroglucinol or other polyphenolics by either a reductive pathway, epoxide formation, or a specific hydroxylating mechanism. Cleavage of the various intermediates of phloroglucinol and polyphenolic degradation may occur by intradiol and extradiol mechanisms. The reductive pathway in contrast to other mechanisms utilized by aerobic microbes, seems both cumbersome and energy wasteful. The degradation of lignin and its associated phenolics follows an enzymatic combustion process which resembles a nonspecific enzyme-catalyzed burning. Anaerobic mineralization of phloroglucinol and its associated polyphenolics by several microbes seems to favour the reductive formation of a dihydrophloroglucinol (1,3-dioxo-5-hydroxycyclohexane), which is cleaved by a specific hydrolase. Mineralization of numerous other polyphenolic compounds by anaerobes seems to utilize phloroglucinol as a central metabolite.
An aqueous acetone extract of the pericarps of Mallotus japonicus (MJE) inhibited nitric oxide (NO) production by a murine macrophage-like cell line, RAW 264.7, which was activated by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Seven phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against NO production. Among these phloroglucinol derivatives, isomallotochromanol exhibited strong inhibitory activity toward NO production, exhibiting an IC(50) of 10.7 microM. MJE and the phloroglucinol derivatives significantly reduced both the induction of inducible nitric oxide synthase (iNOS) protein and iNOS mRNA expression. NO production by macrophages preactivated with LPS and IFN-gamma for 16 h was also inhibited by MJE and the phloroglucinol derivatives. Furthermore, MJE and the derivatives directly affected the conversion of L-[(14)C]arginine to L-[(14)C]citrulline by the cell extract. These results suggest that MJE and the phloroglucinol derivatives have the pharmacological ability to suppress NO production by activated macrophages. They inhibited NO production by two mechanisms: reduction of iNOS protein induction and inhibition of enzyme activity.
A sensitive and selective liquid chromatographic method coupled with mass spectrometry (LC-MS) was developed for the quantification of phloroglucinol in human plasma. Resorcinol was used as internal standard, with plasma samples extracted using ethyl acetate. A centrifuged upper layer was then evaporated and reconstituted with mobile phase. The reconstituted samples were injected into a C(18) XTerra MS column (2.1 x 100 mm) with 3.5-microm particle size. The analytical column lasted for at least 500 injections. The mobile phase was 15% acetonitrile (pH 3.0), with flow-rate at 200 microl/min. The mass spectrometer was operated in negative ion mode with selective ion monitoring (SIM). Phloroglucinol was detected without severe interferences from plasma matrix when used negative ion mode. Phloroglucinol produced a parent molecule ([M-H](-)) at m/z 125 in negative ion mode. Detection of phloroglucinol in human plasma was accurate and precise, with quantification limit at 5 ng/ml. This method has been successfully applied to a study of phloroglucinol in human specimens.
A series of vanillin-reactive compounds has been isolated from extracts of the brown alga Fucus vesiculosus. The first three members of this series have been examined by mass spectrometry, nuclear magnetic resonance spectrometry, and chemical methods, and were shown to be phloroglucinol, its phenyl-linked dimer, and a timer also composed of phloroglucinol units. Evidence is presented for the presence of tetrameric, pentameric, and hexameric phloroglucinol derivatives. Polymeric vanillin-reactive compounds were isolated and separated into two fractions. Oxidative hydrolysis of the major one produced phloroglucinol and related oligomers in low yields. The classical vanillin reactivity of physodes may thus be attributed to phloroglucinol-containing derivatives, as originally proposed by Crato (Crato, E.: Ber. Dtsch. Bot, Ges. 10, 295-302 (1892)).
Twenty patients (15 women, 5 men, mean age: 46.3 +/- 11.82 years, mean weight: 70.9 +/- 8.83 kg) presenting with a 8.5 +/- 4.7 years' history of irritable bowel syndrome associated predominantly with postprandial abdominal pain were recruited into the study. They underwent, after an enema, a rectosigmoid manometry (4 channels, low compliant infusion pump and catheters) before and after a 1000 kcal standardized meal. At the end of the meal they were administered intravenously phloroglucinol (two 40 mg ampoules) or placebo (two ampoules). Both treatments were strictly similar and the order of administration was randomly assigned Motor activity at the rectum, rectosigmoid, lower sigmoid and sigmoid level was determined using a motor index calculated for each 15 min period. Three preprandial and four postprandial indices were calculated. Both groups were statistically similar for sex ratio, age and history of disease. A slight difference was noted for mean weight: the caloric instake per kg of weight was a little higher in the phloroglucinol group. Mean preprandial indices were comparable in both groups. Variance analysis showed that the increase in postprandial motor indices was statistically less pronounced in the phloroglucinol group than in the placebo group. In the placebo group a clear cut increase in motor activity was assessed, which was not observed in the phloroglucinol group. This variation of motricity, which was observed at every level, was more pronounced for the first two postprandial indices than for the last two ones. These results suggest that phloroglucinol is able to reduce rectosigmoid motor response after a test meal. This could explain its activity in abdominal pain associated with irritable bowel syndrome.
Chemiluminescence reaction of phloroglucinol with potassium permanganate in the presence of formaldehyde as an enhancer was investigated. A new chemiluminescence method for the determination of phloroglucinol with flow injection technique was developed. The linear range for phloroglucinol is 5.0 x 10(-9)-5.0 x 10(-5) mol x L(-1) with a detection limit of 3.0 x 10(-9) mol x L(-1). The relative standard deviation is 2.5% for 1.0 x 10(-6) mol x L(-1) phloroglucinol in 11 repeated measurements. The method has been used to determine the content of phloroglucinol in simulated water with satisfactory results.
Phloroglucinol reductase was purified 90-fold to homogeneity from the anaerobic rumen organism Eubacterium oxidoreducens strain G-41. The enzyme is stable in the presence of air and is found in the soluble fraction after ultracentrifugation of cell extract. Ion-exchange, hydrophobic interaction, and affinity chromatography were used to purify the enzyme. The native Mr is 78,000, and the subunit Mr is 33,000 indicating an alpha 2 homodimer. The enzyme is specific for phloroglucinol and NADPH. The Km and Vmax are 600 microM and 640 mumol min-1 mg-1 (pH 7.2) for phloroglucinol, and 6.7 microM and 550 mumol min-1 mg-1 (pH 6.8) for NADPH; the Km and Vmax for the reverse direction are 290 microM and 140 mumol min-1 mg-1 (pH 7.2) for dihydrophloroglucinol, and 27 microM and 220 mumol min-1 mg-1 (pH 7.2) for NADP. Temperature and pH optima are 40 degrees C and 7.8 in the forward direction. The pure enzyme is colorless in solution and flavins are absent. Analysis for cobalt, manganese, molybdenum, vanadium, tungsten, selenium, copper, nickel, iron, and zinc indicated that these metals are not components of the phloroglucinol reductase. Cupric chloride, n-ethylmaleimide, and p-chloromercuribenzoate are potent inhibitors of enzyme activity. The properties of phloroglucinol reductase indicate that it functions in the pathway of anaerobic degradation of trihydroxybenzenes by catalyzing reduction of the aromatic nucleus prior to ring fission.
In this paper, a new and efficient method for synthesis of phloroglucinol with an overall yield of 60% was described. As well, the phloroglucinol association on an immobilized human serum albumin (HSA) column was analyzed in biochromatography by the determination of its Langmuir distribution isotherms. The role of the magnesium cation Mg2+ on the phloroglucinol-HSA binding process was as well analyzed. The results showed that in the Mg2+ concentration range (0.7-2 mM) (including its biological concentration range, i.e. 0.75-0.90 mM), increasing the Mg2+ concentration increased the fraction of free phloroglucinol (not linked with HSA) and thus its biological effect.