Purpurogallin--a natural and effective hepatoprotector in vitro and in vivo.

Purpurogallin is a plant phenol that is sometimes added as an oxidation retardant to fats-oils or to certain fuels or lubricants. However, it was unknown if purpurogallin is cytoprotective. Here we examined this issue, both in isolated hepatocytes and in vivo. From 0.5 to 2.0 mM, purpurogallin prolongs survival of rat hepatocytes substantially against oxyradicals generated with xanthine oxidase and hypoxanthine. The protection was dose dependent and surpassed that given by such antioxidants as ascorbate, mannitol, superoxide dismutase, catalase, and Trolox, when each was examined at or near its optimal concentration in the same system. When 1.5, 3, and 6 mumol of purpurogallin in saline were infused into rats with postischemic livers shortly before reperfusion, the mean hepatic salvages were 42, 76, and 86%, respectively. Such salvage effects would rank purpurogallin highly among the hepatoprotectors known. Over the range of 31 to 500 microM, purpurogallin inhibited the rate of O2 consumption in the xanthine oxidase reaction by approximately 90%, which was 2- to several-fold higher than the inhibition elicited by allopurinol over the same concentrations. Thus, purpurogallin is an effective natural hepatoprotector that may operate partly or principally as an inhibitor of xanthine oxidase.     

Anti-inflammatory functions of purpurogallin in LPS-activated human endothelial cells

Enzymatic oxidation of commercially available pyrogallol was efficiently transformed to an oxidative product, purpurogallin. Purpurogallin plays an important role in inhibiting glutathione S-transferase, xanthine oxidase, catechol O-methyltransferase activities and is effective in the cell protection of several cell types. However, the anti-inflammatory functions of purpurogallin are not well studied. Here, we determined the effects of purpurogallin on lipopolysaccharide (LPS)-mediated proinflammatory responses. The results showed that purpurogallin inhibited LPS-mediated barrier hyper-permeability, monocyte adhesion and migration and such inhibitory effects were significantly correlated with the inhibitory functions of purpurogallin on LPS-mediated cell adhesion molecules (vascular cell adhesion molecules, intracellular cell adhesion molecule, E-selectin). Furthermore, LPS-mediated nuclear factor-κB (NF-κB) and tumor necrosis factor-α (TNF-α) releases from HUVECs were inhibited by purpurogallin. Given these results, purpurogallin showed its anti-inflammatory activities and could be a candidate as a therapeutic agent for various systemic inflammatory diseases. [BMB reports 2012; 45(3): 200-205].     

Purpurogallin is a more powerful protector of kidney cells than Trolox and allopurinol.

Phase contrast and electron microscopic experiments demonstrated that oxyradicals generated with xanthine oxidase and hypoxanthine markedly damage rat kidney mesangial and porcine tubular epithelial cells. Purpurogallin, a phenol found in oak nutgalls, prolongs survival of the xanthine oxidase exposed renal cells three- to nine-fold longer than those without purpurogallin present. At levels equimolar to purpurogallin, either Trolox or allopurinol is less effective in delaying cell necrosis. Purpurogallin scavenges not only xanthine oxidase generated oxyradicals, but also non-enzymatically produced peroxyl radicals, more actively than equimolar levels of Trolox or allopurinol. Purpurogallin inhibits xanthine oxidase with severalfold higher potency than allopurinol and its more active metabolite oxypurinol. Therefore, purpurogallin is a stronger antioxidant than Trolox and a more potent inhibitor of xanthine oxidase than allopurinol as well as oxypurinol.     

Purpurogallin protects both ventricular myocytes and aortic endothelial cells of rats against oxyradical damage.

Rat ventricular myocytes have been isolated and cultured by two separate procedures. Using phase-contrast and electron microscopies, we illustrate that (a) definitive cell damage is produced when myocytes are exposed to xanthine oxidase--hypoxanthine and (b) purpurogallin between 0.25 and 1.0 mM prolongs survival of both myocyte preparations in a dose-dependent manner. The cytoprotection produced by 1 mM purpurogallin exceeds that given by 2 mM each of ascorbate, Trolox, and mannitol, or 24,200 IU superoxide dismutase/L and (or) 92,000 IU catalase/L. Furthermore, we noted, for the first time, that purpurogallin markedly protects rat aortic endothelial cells, a key target of free radical generation and attack. In contrast, Trolox has a negligible effect here. Mechanistically, we showed that purpurogallin inhibits urate formation by xanthine oxidase more potently than allopurinol. Also, the compound diminishes formation of superoxide-reduced cytochrome c. Therefore, purpurogallin is a potent protector of ventricular myocytes and aortic endothelial cells, both of which are important cells in the cardiovascular system.     

Effects of phenol compounds, glutathione analogues and a diuretic drug on glutathione S-transferase, glutathione reductase and glutathione peroxidase from canine erythrocytes.

1. Phenol compounds (ellagic acid, quercetin and purpurogallin), glutathione analogues (S-hexylglutathione and S-octylglutathione) and a diuretic drug (ethacrynic acid) were compared for their inhibitory effects on glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GSH-Px) in the canine erythrocytes. 2. All these compounds inhibited GST activity; quercetin was found to be the most potent inhibitor. 3. Ellagic acid, purpurogallin, quercetin and ethacrynic acid inhibited GR activity; S-hexylglutathione and S-octylglutathione had no effect on GR and GSH-Px activities. 4. Quercetin and purpurogallin inhibited GST non-competitively toward glutathione, whereas ellagic acid showed a competitive inhibition. Ellagic acid and purpurogallin inhibited GR non-competitively toward oxidized glutathione.     

Design, synthesis, and biological evaluation of a series of simple and novel potential antimalarial compounds.

A series of compounds bearing an endocyclic -N-O- moiety with potential antimalarial activity based on simple derivatives of the tropolone purpurogallin was prepared by means of a hetero Diels-Alder reaction using nitrosobenzene as a dienophile. The rationale behind the design of these compounds is presented, together with the synthetic route to derivatives bearing aromatic and aliphatic esters of the C4'-position hydroxyl group of the purpurogallin framework, as well as biological data obtained from in vitro assays against Plasmodium falciparum and Trypanosoma cruzi. Several of the new compounds have activities in the 3-9 microM range, and provide leads for the development of a novel class of antiparasitic drugs with improved biological and pharmacological properties.     

New enhancers for the chemiluminescent peroxidase catalysed chemiluminescent oxidation of pyrogallol and purpurogallin

The effects of various boronate compounds, 4-biphenylboronic acid, 4-bromobenzeneboronic acid, trans-4-(3-propionic acid)phenylboronic acid and 4-iodophenylboronic acid, on the horseradish peroxidase (HRP) catalysed chemiluminescent oxidation of pyrogallol and purpurogallin by peroxide were investigated. trans-4-(3-Propionic acid)phenylboronic acid produced a 13.7-fold enhancement in the peak light emission from the chemiluminescent HRP catalysed pyrogallol reaction (detection limit for HRP < 1.25 fmol). At low enhancer concentration a single peak of light emission was observed and as the enhancer concentration increased the time to peak light emission became progressively longer. The chemiluminescence showed two peaks at higher concentrations (> 54.3 μmol/L) and the individual peak times depended upon the concentration of the enhancer. All of the boronates enhanced peak light emission in the chemiluminescent HRP catalysed purpurogallin reaction. 4-Biphenylboronic acid was the most effective and it enhanced peak light emission 314-fold. The practical detection limit for HRP (Type VIA) using this enhancer was 4.18 pmol (peak emission at 20 minutes). This compound also enhanced peak light emission 232-fold from a chemiluminescent HRP-purpurogallin reaction in which molecular oxygen replaced peroxide as the oxidant.     

Coordination of aluminium with purpurogallin and theaflavin digallate

Polyphenols are antioxidants, which are known to influence bioavailability of metals in the body. The theaflavins of black tea are important members of this family, which have been sparsely investigated. The complexation of aluminium with purpurogallin (2,3,4,6-tetrahydroxy-5H-benzocyclohepten-5-one) has been investigated using nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography-mass spectroscopy (LC-MS) and Fourier transform infrared (FT-IR) spectroscopy. 1H NMR was used to determine the coordination site of the aluminium ion and LC-MS to determine the stoichiometry and molecular weight of the major complex formed in solution. FT-IR spectral comparisons were used to corroborate the proposed chelating moiety. The complexation of aluminium with the high-molecular-weight, tea polyphenol theaflavin digallate was also investigated using 1H NMR and heteronuclear multiple quantum coherence experiments. Structures of the major aluminium purpurogallin and aluminium theaflavin digallate complexes are proposed.     

The Antioxidant and Membrane Activities of the Benzotropalone Pigment Purpurogallin

Biophysics - Abstract—The phenolic compound purpurogallin (PPG) is found in oak nutgalls and is a red pigment with a benzotropolone ring structure. PPG shows pronounced cytoprotective and...     

Protection by tropolones against H2O2-induced DNA damage and apoptosis in cultured Jurkat cells

Tropolones, the naturally occurring compounds responsible for the durability of heartwood of several cupressaceous trees, have been shown to possess both metal chelating and antioxidant properties. However, little is known about the ability of tropolone and its derivatives to protect cultured cells from oxidative stress-mediated damage. In this study, the effect of tropolones on hydrogen peroxide-induced DNA damage and apoptosis was investigated in cultured Jurkat cells. Tropolone, added to the cells 15 min before the addition of glucose oxidase, provided a dose dependent protection against hydrogen peroxide induced DNA damage. The IC₅₀ value observed was about 15 μM for tropolone. Similar dose dependent protection was also observed with three other tropolone derivatives such as trimethylcolchicinic acid, purpurogallin and β-thujaplicin (the IC₅₀ values were 34, 70 and 74 μM, respectively), but not with colchicine and tetramethyl purpurogallin ester. Hydrogen peroxide-induced apoptosis was also inhibited by tropolone. However, in the absence of exogenous H₂O₂ but in the presence of non-toxic concentrations of exogenous iron (100 μM Fe³⁺), tropolone dramatically increased the formation of single strand breaks at molar ratios of tropolone to iron lower than 3 to 1, while, when the ratio increased over 3, no toxicity was observed. In conclusion, the results presented in this study indicate that the protection offered by tropolone against hydrogen peroxide-induced DNA damage and apoptosis was due to formation of a redox-inactive iron complex, while its enhancement of iron-mediated DNA damage at ratios of [tropolone]/[Fe³⁺] lower than 3, was due to formation of a lipophilic iron complex which facilitates iron transport through cell membrane in a redox-active form.     

Virtual screening of National Cancer Institute database for claudin-4 inhibitors: Synthesis,biological evaluation,and molecular dynamics studies

Claudin-4 (CLDN4) is a vital member of tight-junction proteins that is often overexpressed in cancer and other malignancies. The three-dimensional structure of human CLDN4 was constructed based on homology modeling approach. A total of 265 242 molecules from the National Cancer Institute (NCI) database has been utilized as a dataset for this study. In the present work, structure-based virtual screening is performed with the NCI database using Glide. By molecular docking, 10 candidate molecules with high scoring functions, which binds to the active site of CLDN4 were identified. Subsequently, molecular dynamics simulations of membrane protein were used for optimization of the top-three lead compounds (NCI110039, NCI344682, and NCI661251) with CLDN4 in a dynamic system. The lead molecule from NCI database NCI11039 (purpurogallin carboxylic acid) was synthesized and cytotoxic properties were evaluated with A549, MCF7 cell lines. Our docking and dynamics simulations predicted that ARG31, ASN142, ASP146, and ARG158 as critically important residues involved in the CLDN4 activity. Finally, three lead candidates from the NCI database were identified as potent CLDN4 inhibitors. Cytotoxicity assays had proved that purpurogallin carboxylic acid had an inhibitory effect towards breast (MCF7) and lung (A549) cancer cell lines. Computational insights and in vitro (cytotoxicity) studies reported in this study are expected to be helpful for the development of novel anticancer agents.     

Synthesis and evaluation as potential anticancer agents of novel tetracyclic indenoquinoline derivatives

We report the synthesis and evaluation as potential anticancer agents of a series of tetracyclic indenoquinolines. The compounds, which are obtained through the photoisomerization of Diels–Alder adducts formed between purpurogallin derivatives and nitrosobenzene, have in vitro antiproliferative activities in the μM to nM range against breast (MCF-7), lung epithelial (A-549), and cervical (HeLa) adenocarcinoma cells. The cytotoxicities of several of the novel tetracycles are comparable to or better than that of camptothecin. A strong correlation between the activity of the compounds and their aromaticity and planarity was observed, suggesting a mode of action similar to that of topoisomerase poisons.     

Characterization of an UV- and VIS-absorbing, purpurogallin-derived secondary pigment new to algae and highly abundant in Mesotaenium berggrenii (Zygnematophyceae, Chlorophyta), an extremophyte living on glaciers

Mesotaenium berggrenii is one of few autotrophs that thrive on bare glacier surfaces in alpine and polar regions. This extremophilic alga produces high amounts of a brownish vacuolar pigment, whose chemical constitution and ecological function is largely unknown until now. Field material was harvested to isolate and characterize this pigment. Its tannin nature was determined by photometric methods, and the structure determination was carried out by means of HPLC-MS and 1D- and 2D-NMR spectroscopy. The main constituent turned out to be purpurogallin carboxylic acid-6-O-β-d-glucopyranoside. This is the first report of such a phenolic compound in this group of algae. Because of its broad absorption capacities of harmful UV and excessive VIS radiation, this secondary metabolite seems to play an important role for the survival of this alga at exposed sites. Attributes and abundances of the purpurogallins found in M.?berggrenii strongly suggest that they are of principal ecophysiological relevance like analogous protective pigments of other extremophilic microorganisms. To prove that M.?berggrenii is a true psychrophile, photosynthesis measurements at ambient conditions were carried out. Sequencing of the 18S rRNA gene of this alpine species and of its arctic relative, the filamentous Ancylonema nordenski?ldii, underlined their distinct taxonomic position within the Zygnematophyceae.     

Studies on Phyto-peroxidase

Peroxidase c was isolated and purified from Japanese-radish roots by means of a chromatographic technique with carboxymethyl cellulose. Two or more components exhibiting the absorption spectrum of peroxidase c were separated chromatographically, and the most basic component was crystallized from ammonium sulphate solution. The Reinheit Zahl and the purpurogallin number of the crystalline preparation were found to be 3.55 and 1100 respectively. The absorption maxima were found at 420 and 540 mμ, for the oxidized form and at 425 and 560 mμ for the reduced form. The crystalline preparation contained 1.57% protohematin as the prosthetic group, and then the minimum molecular weight of peroxidase c was found to be 41500.     

Peroxidases in grass dew derived from guttation: possible role in polymerization of soil organic matter

Peroxidases are enzymes that catalyze the oxidative cross-linking and polymerization of certain organic compounds by hydrogen peroxide and other organic peroxides. This study demonstrates that peroxidases are present in dew (droplets formed as the result of guttation) collected from Bermuda grass hybrids 419 and Tifway 2 [Cynodon dactylon (L.) × Cynodon transvaalensis Davy], which are warm-season C₄ grasses, and Kentucky bluegrass (Poa pratensis L.), which is a cool-season C₃ grass. Peroxidase activity [quantified with horseradish peroxidase (HRP) (activity 152 purpurogallin units/mg) as standard] in guttational fluids collected from grasses during early morning was in the 80 to 120 µg/L range. Isoelectric focusing was used to determine isoelectric points (pI) of the isozymes present in the Bermuda grass dew following dialysis and lyophilization of the collected dew. The pI values ranged from 4.3 to 8.3 with 14 isozymes being detected using guaiacol and hydrogen peroxide as substrates. Peroxidases also were extracted from soil supporting the growth of Bermuda grass. Peroxidases in these soils were most abundant in the top 5 cm layer (activity was in the 6.8 to 16 purpurogallin units/g range). Stability and activity of these peroxidases in the presence of fulvic and humic acids were evaluated. Compared to controls with no added humic substances, peroxidase activity was inhibited by a soil fulvic acid and prolonged by a humic acid. Field measurements indicated that peroxidase activity did not greatly decrease during the winter when the grass was dormant, indicating that the peroxidases released into the soil remain active for a considerable time. Based on results in these studies and previously determined dry and wet deposition of atmospheric peroxides, we estimate that peroxidase-catalyzed reactions in areas planted in these grasses may convert about 8 g C m^-2 yr^-1 of labile soil organic compounds to more persistent oligomers and polymers.     

Some Effects of Light on Excised Wheat Roots with Special Reference to Peroxide Metabolism

When excised wheat roots are exposed to blue light, catalase activity changes in a way to he expected as a result of photodestruction with first order kinetics. Wheat root catalase in vivo is less light sensitive than animal catalase in vitro, possibly due to internal screening. Illumination of the roots with red light causes some increase in catalasc activity. Peroxidase activity is much less affected by light. No relation has been found between catalase destruction and chlorophyll formation. The ability of roots to oxidize pyrogallol to purpurogallin (by other workers interpreted as peroxide production) is decreased by light, especially blue light. On the contrary, one peroxide producing enzyme, glycolic acid oxidase, was detected only in roots grown in blue light. The total flavin content, or the fraction present as FAD, is not affected by light. The ascorbic acid content is low, but slightly increased by blue light.     

Antiplatelet and antithrombotic activities of purpurogallin in vitro and in vivo

Enzymatic oxidation of pyrogallol was efficiently transformed to an oxidative product, purpurogallin (PPG). Here, the anticoagulant activities of PPG were examined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), and the activities of thrombin and activated factor X (FXa). And, the effects of PPG on expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were evaluated in tumor necrosis factor (TNF)-α activated human umbilical vein endothelial cells (HUVECs). Treatment with PPG resulted in prolonged aPTT and PT and inhibition of the activities of thrombin and FXa, as well as inhibited production of thrombin and FXa in HUVECs. In addition, PPG inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation. PPG also elicited anticoagulant effects in mice. In addition, treatment with PPG resulted in significant reduction of the PAI-1 to t-PA ratio. Collectively, PPG possesses antithrombotic activities and offers a basis for development of a novel anticoagulant. [BMB Reports 2014; 47(7): 376-381]     

Benzotropolone inhibitors of estradiol methylation: kinetics and in silico modeling studies

Natural and synthetic benzotropolone compounds were assessed in vitro for their ability to inhibit hydroxyestradiol methylation by catechol-O-methyltransferase (COMT). The compounds were also modeled in silico with a homology model of human COMT. Purpurogallin (1), purpurogallin carboxylic acid (2), and theaflavin-3,3'-digallate (6) were the most potent inhibitors of 2-hydroxy and 4-hydroxyestradiol methylation (IC(50) 0.22-0.50microM). Compounds 1 and 6 decreased the V(max) and increased the K(m) of COMT, indicating a mixed-type inhibition. Compounds 1 and 2 bound to COMT by inserting the six-membered ring of the benzotropolone into the active site. Decreased acidity of the hydroxyl groups on this ring or increased bulkiness reduced potency. Compound 6 bound by inserting the galloyl ester into the active site, which allowed the compound to overcome increased bulkiness and resulted in restored potency. Further studies are needed to determine the impact in vivo of COMT inhibition by these compounds.     

Kinetics and inhibition studies of catechol O-methyltransferase from the yeast Candida tropicalis.

The Kms for esculetin and S-adenosyl-L-methionine for catechol O-methyltransferase from the yeast Candida tropicalis were 6.2 and 40 microM, respectively. S-Adenosyl-L-homocysteine was a very potent competitive inhibitor with respect to S-adenosyl-L-methionine, with a Ki of 6.9 microM. Of the catechol-related inhibitors, purpurogallin, with a Ki of 0.07 microM, showed the greatest inhibitory effect. Sulfhydryl group-blocking reagents, such as thiol-oxidizing 2-iodosobenzoic acid and mercaptide-forming p-chloromercuribenzoic acid, provided evidence for sulfhydryl groups in the active site of the enzyme. Yeast catechol O-methyltransferase is a metal-dependent enzyme and requires Mg2+ for full activity. Zn2+ and Mn2+ but not Ca2+ were able to substitute for Mg2+. Mn2+ showed optimal enzyme activation at concentrations 50- to 100-fold lower than those of Mg2+.