Ester derivatives of gallic acid with potential toxicity toward L1210 leukemia cells

Gallic acid and gallates with the same number of hydroxyl groups and varying the length of the side carbon chain, with respective lipophilicity being defined through the ClogP values, were examined for their ability to induce apoptosis (through the DNA ladder fragmentation pattern), mitochondrial and cytoplasmic GSH depletion and NF-kappaB activation in murine lymphoblastic L1210 leukemia cells. A relationship between cytotoxic effect and a limited degree of lipophilicity was observed.     

Correlation of antimutagenic activity and suppression of CYP1A with the lipophilicity of alkyl gallates and other phenolic compounds

Alkyl gallates are widely used as food antioxidants. Methyl, ethyl, propyl, lauryl, and cetyl gallates showed antimutagenicity to activated 2-aminoanthracene (2AA)-induced SOS responses in Salmonella typhimurium TA1535/pSK1002. They also exhibited a suppressive effect on 3-methylcholanthrene (3-MC)-induced cytochrome P450 1A (CYP1A) in human hepatoma HepG2 cells, as indexed by the 7-ethoxyresorufin-O-deethylase (EROD) activity, and on CYP1A protein level. Both antimutagenicity and suppression of CYP1A appeared to be dependent on alkyl chain lengths, which suggested lipophilicity dependence. Based on those results, we investigated 26 other phenolic compounds for their lipophilicity, antimutagenicity and inhibition of EROD activity. The lipophilicity correlated well with the inhibition of EROD activity (r=0.78), and the inhibition of EROD activity correlated with the antimutagenicity of those compounds (r=0.71). The results suggest that the lipophilicity of the phenolic compounds may be an important factor in their ability to inhibit EROD activity.     

Anti-inflammatory and antioxidant activity of a medicinal tincture from Pedilanthus tithymaloides

Pedilanthus tithymaloides (L.) Poit. (Euphorbiaceae) is a low tropical American shrub with a reported wide range of healing properties such as emetic, anti-inflammatory, antibiotic, antiseptic, antihemorrhagic, antiviral, antitumoral, and abortive. In the present study, a tincture from P. tithymaloides collected in Cuba was evaluated for its in vivo anti-inflammatory activity, using the rat paw oedema assay, and for its in vitro scavenging effects on reactive oxygen species (ROS) (HO*, O2*-, HOCl, ROO* and H2O2), reactive nitrogen species (RNS) (ONOO- and *NO), and DPPH* radical. The protein, free amino acid, and phenolic contents of the tincture were also determined. Pertaining to the anti-inflammatory activity, the intraperitoneal administration of the tincture inhibited carrageenan-induced rat paw oedema, whereas in the scavenging assays the tincture showed to be effective against all the assayed ROS and RNS, specially for HO* (IC50 = 345+/-77 microg/mL), O2*- (IC50 = 143+/-7 microg/mL), HOCl (IC50 = 113+/-20 microg/mL), ONOO- (IC50 = 44+/-3 microg/mL), and *NO (IC50 = 54+/-4 microg/mL), but displayed weak activity in the DPPH* assay. The protein content of the tincture was 0.70%, and twenty free amino acids were identified and quantified. The content of total phenolics was 17.4+/-0.15 mg of gallic acid equivalents (GAE)/g dry material. These results provide scientific support for the empirical use of P. tithymaloides tincture as an anti-inflammatory medicine.     

Cytotoxic activity of orsellinates

The series of 2,4-dihydroxy-6-methylbenzoates 2-10 (methyl to hexyl orsellinates) prepared by alcoholysis of lecanoric acid (1)--a natural product from the lichen Parmotrema tinctorum (Nyl.) Hale - was submitted to the brine shrimp lethality test (BST), which was also performed for 2,4-dihydroxy-6-methylbenzoic acid (11) (orsellinic acid) and the derivative ethyl-2-hydroxy-4-methoxy-6-methylbenzoate (12) (4-methoxy-ethyl orsellinate), in order to detect new substances with probable antineoplasic activity. Results showed that chain elongation--increase in lipophilicity (log P)--causes a rise in the cytotoxic activity of orsellinates. Hexyl orsellinate (7) showed the highest cytotoxic activity (LC50 = 31 microM). A correlation between lipophilicity (log P) and cytotoxic activity (log 1/LC50) is presented. Compounds with ramified chains--iso-propyl, sec-butyl and tert-butyl orsellinates (8-10)--were less active than those with the correspondent linear chain. The activities presented by 4-methoxy-ethyl orsellinate (12) and ethyl orsellinate (3) suggest that the hydroxy group at the C-4 position causes effect in the cytotoxic activity of orsellinates against Artemia salina.     

Effect of acyl donor chain length on isoquercitrin acylation and biological activities of corresponding esters

The enzymatic acylation of isoquercitrin with fatty acid esters of various carbon chain lengths was carried out in 2-methyl-2-butanol using Novozym 435^®. The conversion yield and the initial rate decreased from 66% to 38% and from 17.7 to 10.1 μmol/h respectively, as the carbon chain of the acyl donor increased from C4 to C18. Isoquercitrin acylated derivatives showed higher xanthine oxidase inhibition activities than isoquercitrin. This property increased with the lipophilicity of the derivative esters. The scavenging activity of isoquercitrin esters against ABTS and DPPH radicals decreased with the acyl chain length. Conversely, for esters from C6 to C18, a linear growing relationship can be established between the chain length and the superoxide radical scavenging activity. Furthermore, an improved antiproliferative effect on Caco2 cancer cells was induced by addition of isoquercitrin esters comparing with isoquercitrin.     

Structure-activity relationship analysis of antioxidant ability and neuroprotective effect of gallic acid derivatives

Gallic acid and its derivatives are a group of naturally occurring polyphenol antioxidants which have recently been shown to have potential healthy effects. In order to understand the relationship between the structures of gallic acid derivatives, their antioxidant activities, and neuroprotective effects, we examined their free radical scavenging effects in liposome and anti-apoptotic activities in human SH-SY5Y cell induced by 6-hydrodopamine autooxidation. It was found that these polyphenol antioxidants exhibited different hydrophobicity and could cross through the liposome membrane to react with 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical in a time and dose-dependent manner. At the same time, the structure-antioxidant activity relationship of gallic acid derivatives on scavenging DPPH free radical in the liposome was also analyzed based on theoretical investigations. Analysis of cell apoptosis, intracellular GSH levels, production of ROS and the influx of Ca(2+) indicated that the protective effects of gallic acid derivatives in cell systems under oxidative stress depend on both their antioxidant capacities and hydrophobicity. However, the neuroprotective effects of gallic acid derivatives seem to depend more on their molecular polarities rather than antioxidant activities in the human SH-SY5Y cell line. In conclusion, these results reveal that compounds with high antioxidant activity and appropriate hydrophobicity are generally more effective in preventing the injury of oxidative stress in neurodegenerative diseases.     

Tea catechol oxidase: Isolation,purification and kinetic characterization

Cathechol oxidase extracted from tea leaves was purified over 200-fold, using isoelectric focusing. The purified catechol oxidase was free of peroxidase and flavanol gallate esterase activities. Further, this enzyme was shown to have optimum activity near pH 5·7 and a K_m of 2·3 × 10^?3 M (at 25°) for (?)-epigallocatechin gallate. The purified enzyme was found to be capable of epimerizing tea flavanols at their C-2 position whether oxidation of the flavanol occurs (aerobic conditions) or not (anaerobic conditions). When oxygen is present, gallic acid is formed as a result of oxidation of either (?)-epigallocatechin gallate or (?)-epicatechin gallate. Formation of gallic acid is a side reaction of the oxidation of the flavanol gallates and is named oxidative degallation; no esterase per se is involved in this reaction.     

Activation of rat liver microsomal glutathione S-transferase by gallic acid

The effect of phenolic antioxidants on the rat liver microsomal glutathione S-transferase (MGST1) was investigated in vitro. When microsomes were incubated with various polyphenolic antioxidants, gallic acid (3,4,5-trihydroxybenzoic acid) markedly increased MGST1 activity and the increase was prevented in the presence of superoxide dismutase (SOD) or catalase. The MGST1 activity increased by gallic acid was decreased by further incubation with sodium arsenite, a sulfenic acid reducing agent, but was not with dithiothreitol, a disulfide bond reducing agent. The incubation of microsomes with gallic acid in the presence of the NADPH generating system which generates reactive oxygen species (ROS) through cytochrome P-450 system increased the MGST1activity in spite of scavenging the ROS and the increase was also depressed by SOD/catalase. The increase of MGST1 activity by gallic acid was prevented by co-incubation with a stable radical, 1,1-diphenyl-2-picrylhydrazyl or ferric chloride. These results suggest that the gallic acid acts as a pro-oxidant and activates MGST1 through oxidative modification of the enzyme.     

Novel echinocandin antifungals. Part 1: novel side-chain analogs of the natural product FR901379

A series of novel acylated analogs of the novel water-soluble echinocandin FR901379 have been prepared and evaluated for antifungal and hemolytic activity. A relationship between antifungal activity and lipophilicity of the acyl side chain, expressed as ClogP was demonstrated, and an analog (3c) with 5.5- to 8-fold superior in vivo activity relative to the previously disclosed 4-(n-octyloxy)benzoyl side chain analog, FR131535 obtained.     

Pestalotiopsis mangiferae isolated from cocoa leaves and concomitant tannase and gallic acid production

The enzyme tannase is of great industrial and biotechnological importance for the hydrolysis of vegetable tannins, reducing their undesirable effects and generating products for a wide range of processes. Thus, the search for new microorganisms that permit more stable tannase production is of considerable importance. A strain of P. mangiferae isolated from cocoa leaves was selected and investigated for its capacity to produce tannase enzymes and gallic acid through submerged fermentation. The assessment of the variables affecting tannase production by P. mangiferae showed that tannic acid, ammonium nitrate and temperature were the most significant (8.4 U/mL). The variables were analyzed using Response Surface Methodology - RSM (Box-Behnken design), with the best conditions for tannase production being: 1.9% carbon source, 1% nitrogen source and temperature of 23 °C. Tannase activity doubled (16.9 U/mL) after the optimization process when compared to the initial fermentation. A pH of 7.0 was optimal for the tannase and it presented stability above 80% with pH between 4.0 and 7.0 after 2h of incubation. The optimal temperature was 30 °C and activity remained at above 80% at 40–60 °C after 1 h. Production of gallic acid was achieved with 1% tannic acid (0.9 mg/mL) and P. mangiferae had not used up the gallic acid produced by tannic acid hydrolysis after 144 h of fermentation. A 5% tannic acid concentration was the best for gallic acid production (1.6 mg/mL). These results demonstrate P. mangiferae’s potential for tannase and gallic acid production for biotechnological applications.     

The Resistance of Gallic Acid and its Alkyl Esters to Attack by Bacteria able to Degrade Aromatic Ring Structures

Summary: Various bacteria capable of degrading aromatic ring structures were unable to utilize gallic acid, methyl, ethyl or propyl gallates as sole carbon sources for growth when tested in liquid and solid media. A bacterial isolate was obtained which degraded gallic acid but not methyl, ethyl or propyl gallates, although ellagic acid, a major spontaneous degradation product of the gallate esters, was utilized to a limited degree.     

Hypochlorous acid-induced heme degradation from lactoperoxidase as a novel mechanism of free iron release and tissue injury in inflammatory diseases

Lactoperoxidase (LPO) is the major consumer of hydrogen peroxide (H(2)O(2)) in the airways through its ability to oxidize thiocyanate (SCN(-)) to produce hypothiocyanous acid, an antimicrobial agent. In nasal inflammatory diseases, such as cystic fibrosis, both LPO and myeloperoxidase (MPO), another mammalian peroxidase secreted by neutrophils, are known to co-localize. The aim of this study was to assess the interaction of LPO and hypochlorous acid (HOCl), the final product of MPO. Our rapid kinetic measurements revealed that HOCl binds rapidly and reversibly to LPO-Fe(III) to form the LPO-Fe(III)-OCl complex, which in turn decayed irreversibly to LPO Compound II through the formation of Compound I. The decay rate constant of Compound II decreased with increasing HOCl concentration with an inflection point at 100 μM HOCl, after which the decay rate increased. This point of inflection is the critical concentration of HOCl beyond which HOCl switches its role, from mediating destabilization of LPO Compound II to LPO heme destruction. Lactoperoxidase heme destruction was associated with protein aggregation, free iron release, and formation of a number of fluorescent heme degradation products. Similar results were obtained when LPO-Fe(II)-O(2), Compound III, was exposed to HOCl. Heme destruction can be partially or completely prevented in the presence of SCN(-). On the basis of the present results we concluded that a complex bi-directional relationship exists between LPO activity and HOCl levels at sites of inflammation; LPO serve as a catalytic sink for HOCl, while HOCl serves to modulate LPO catalytic activity, bioavailability, and function.     

Anti-HSV-1 and anti-HIV-1 activity of gallic acid and pentyl gallate

The synthetic n-alkyl esters of gallic acid (GA), also known as gallates, especially propyl, octyl and dodecyl gallates, are widely employed as antioxidants by food and pharmaceutical industries. The inhibitory effects of GA and 15 gallates on Herpes Simplex Virus type 1 (HSV-1) and Human Immunodeficiency Virus (HIV-1) replication were investigated here. After a preliminary screening of these compounds, GA and pentyl gallate (PG) seemed to be the most active compounds against HSV-1 replication and their mode of action was characterized through a set of assays, which attempted to localize the step of the viral multiplication cycle where impairment occurred. The detected anti-HSV-1 activity was mediated by the inhibition of virus attachment to and penetration into cells, and by virucidal properties. Furthermore, an anti-HIV-1 activity was also found, to different degrees. In summary, our results suggest that both compounds could be regarded as promising candidates for the development of topical anti-HSV-1 agents, and further studies concerning the anti-HIV-1 activity of this group of molecules are merited.     

The Degradation of Gallic Acid and its Alkyl Esters by Pullularia pullulans

By elective techniques an isolate of Pullularia pullulans was obtained which possessed the ability to utilize gallic acid (3,4,5-trihydroxybenzoic acid) and its alkyl esters together with other aromatic substrates as the sole carbon source for growth in a mineral salts medium. Quantitative measurements were made of the disappearance of the gallates from culture media and of their metabolism by washed suspensions by the Warburg technique. The pattern of oxygen uptake with various substrates by suspensions adapted to gallic acid was examined in the light of Stanier's simultaneous adaptation postulates in an attempt to identify intermediates of gallic acid metabolism. As a result, β-ketoadipic acid was suspected of being an intermediate. A keto compound was isolated as its 2,4-dinitrophenylhydrazone during gallic acid metabolism but it was not β-ketoadipic acid or levulinic acid.     

Comparative in vitro and in vivo studies on long-wavelength photosensitizers derived from bacteriopurpurinimide and Bacteriochlorin p6: fused imide ring enhances the in vivo PDT efficacy

In situ conversion of bacteriochlorophyll-a, present in Rhodobacter sphaeroides (Rb. sphaeroides) gave bacteriopurpurin-18 in modest yield, which in a sequence of reactions was converted into two series of bacteriochlorins: bacteriopurpurinimide and bacteriopurpurin p6 with and without a fused imide ring system, respectively. To determine the effect of overall lipophilicity in photosensitizing efficacy, these bacteriochlorins were independently reacted with HBr gas and subsequently treated with various alkyl alcohols to afford the corresponding alkyl ether derivatives as diastereomeric mixtures (the R- and S-isomers were obtained in almost equal ratios). Between the two series of bacteriochlorins, the bacteriopurpurinimides containing a fused imide ring system were found to be more effective in vivo (C3H mice bearing RIF tumors). To investigate the effect of the presence of the chiral center at position 3 of the most effective purpurinimide 9 [3(1'-heptyloxy)ethyl-3-deacetyl-bacteriopurpurin-18-N-hexylimide propyl ester], the acetyl group was replaced with a hydroxymethyl substituent and converted into 3(1'-decyloxy)methyl-3-deacetyl-purpurin-18-N-hexylimide methyl ester 26 with a similar lipophilicity. Interestingly, compared to 26, the bacteriopurpurinimide 9 was found to be more effective, suggesting that the chiral center at position 3 certainly plays an important role in photosensitizing activity. Among a series of alkyl ether analogues, between the PDT efficacy and the lipophilicity (log P and log D) calculated by computational methods (PALLAS program), a parabolic relationship was observed to some extent. However, it was limited to a particular series, e.g., compounds with similar log P values between bacteriopurpurinimides and bacteriochlorin e6 did not produce similar in vivo efficacy. As expected, within a series, a linear relationship was observed between the log P values and the HPLC retention times of the photosensitizers. Some of the mitochondrial localized photosensitizers showed a significant peripheral benzodiazepine binding (PBR) affinity. However, limited correlation between PBR binding affinity and in vivo PDT efficacy was observed. Compared to the naturally occurring bacteriochlorophyll-a, the bacteriopurpurinimides with fused imide ring system showed higher in vitro/in vivo stability. In contrast to methyl pyropheophorbide-a, the ester functionalities in bacteriopurpurinimide did not convert into the corresponding carboxylic acid by the enzyme esterases.     

Novel 3-O-acyl mesquitol analogues as free-radical scavengers and enzyme inhibitors: synthesis,biological evaluation and structure-activity relationship

A new isomer of mesquitol (2,3-trans-3',4',7,8-tetrahydroxyflavan-3-ol) was isolated from Dichrostachys cinerea in excellent yields. It has shown free-radical scavenging property and alpha-glucosidase inhibitory activities but, it could not display xanthine oxidase inhibitory property. However, it was observed that acylation of 3-OH group significantly enhanced the alpha-glucosidase inhibition and displayed xanthine oxidase inhibitory potential. The structure activity relationship revealed that the degree of lipophilicity played a major role in improving enzyme inhibitory activities. A positive correlation was observed between enzyme inhibitory potential and acyl chain length (upto C-16) of aliphatic esters.     

Prooxidant toxicity of polyphenolic antioxidants to HL-60 cells: description of quantitative structure-activity relationships

Polyphenolic antioxidants exhibited a dose-dependent toxicity against human promyelocytic leukemia cells (HL-60). Their action was accompanied by malondialdehyde formation, and was partly prevented by desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine. This points to a prooxidant character of their cytotoxicity. A quantitative structure-activity relationship (QSAR) has been obtained to describe the cytotoxicity of 13 polyphenolic antioxidants belonging to three different groups (flavonoids, derivatives of gallic and caffeic acid): log cL50 (microM) = (2.7829+/-0.2339)+(1.2734+/-0.4715) Ep/2 (V)-(0.3438+/-0.0582) log P (r2 = 0.8129), where cL50 represents the concentration for 50% cell survival, Ep/2 represents the voltammetric midpoint potential, and P represents the octanol/water partition coefficient. Analogous QSARs were obtained using enthalpies of single-electron oxidation of these compounds, obtained by quantum-mechanical calculations. These findings clearly point to two important characteristics determining polyphenol cytotoxicity, namely their ease of oxidation and their lipophilicity.     

Comparative cytotoxicity of alkyl gallates on mouse tumor cell lines and isolated rat hepatocytes

Alkyl esters of gallic acid inhibited the respiration rate of mouse sarcoma 786A and mouse mammary adenocarcinoma TA3 cell lines and its multiresistant variant TA3-MTX-R more effectively than gallic acid, both in the absence and in the presence of the uncoupler CCCP. The order of inhibition of the respiration rate by gallates in intact cells was n-octyl- approximately iso-amyl- approximately n-amyl- approximately iso-butyl->n-butyl->iso-propyl->n-propyl-gallate>gallic acid. Sarcoma 786A was significantly more susceptible to all seven esters than the TA3 cell line. Respiration rates of the TA3-MTX-R cell line showed almost the same sensitivity to these esters as the TA3 cell line. However, hepatocytes were significantly less sensitive than all tumor cells tested. These alkyl gallates blocked mitochondrial electron flow, mainly at the NADH-CoQ segment, preventing ATP synthesis, which would lead to cellular death. These esters also inhibited, in the same order of potencies as respiration, the growth of 786A, TA3 and TA3-MTX-R cells in culture. In mice carrying TA3 or TA3-MTX-R tumor cells, an important decrease of the tumor growth rate and an increase of survival were observed when mice were treated with iso-butyl gallate alone or in combination with doxorubicin. These results indicate that alkyl gallates are selectively cytotoxic to tumor cells, which may be due to the mitochondrial dysfunctions of these cells.     

Inhibition of vertebrate squalene epoxidase by isoprenyl gallates and phenylalkyl gallates

Galloy esters with 'substrate-like' isoprenoid or phenylalkyl side chains were newly synthesized and tested for the enzyme inhibition activities toward recombinant rat squalene epoxidase. Isoprenyl gallates (4-6) showed good inhibition (IC50 = 1.5 5.1 microM), as potent as previously reported substrate analogues, while phenylalkyl gallates (7-10) were moderate inhibitors of the enzyme (IC50 = 12-61 microM).