Amplification of antioxidant activity and xanthine oxidase inhibition of catechin by enzymatic polymerization

To amplify the antioxidant activity, we synthesized poly(catechin) by the enzyme-catalyzed oxidative coupling using horseradish peroxidase as a catalyst. The poly(catechin) showed great improvement in antioxidant activity such as radical scavenging activity against the superoxide anion and inhibition effects against free radical induced-oxidation of low-density lipoprotein, compared with a catechin monomer. In addition, poly(catechin) showed very high inhibition effects on xanthine oxidase activity, whereas the catechin monomer showed very less inhibition effects. The amplified activities might offer a high potential as a therapeutic agent for prevention of various free radicals and/or enzyme-related diseases.     

Design,synthesis and bioactivity of catechin/epicatechin and 2-azetidinone derived chimeric molecules

A new class of chimeric molecules have been developed. These are based on polyphenols like catechin and epicatechin and monocyclic β-lactams. The two units are joined via a triazole linker using the ‘Click Chemistry’ conditions. The compounds showed good to weak antibacterial activity against Escherichia coli as well as moderate inhibition of RNase A.     

Catechin inhibition of mutagenesis and alteration of DNA binding of 2-acetyl-aminofluorene in rat hepatocytes

Bioflavonoids are naturally occurring plant products that have demonstrated inhibitory effects on chemically induced carcinogenesis or mutagenesis. The chemoprotective effects are either direct scavenging of reactive molecules or indirect effects, such as enzyme activity alteration. Exposure of cultures of isolated rat hepatocytes to catechin (0.01-1.0 mM), a plant phenolic flavonoid, and subsequent addition of 2-acetylaminofluorene (AAF) resulted in an enhanced binding of AAF metabolites to hepatocellular DNA. Incubations of hepatocytes with catechin and S. typhimurium demonstrated no mutagenicity of catechin. At 1.0 and 5.0 mM concentrations of catechin with AAF and 30-min incubation with hepatocytes prior to plating there was inhibition of AAF-induced mutagenicity. However, at 0.5 mM of catechin there was a significant enhancement in mutagenicity. The increase in DNA binding of AAF in the cultures of hepatocytes is due to the alteration of metabolism by exposure to catechin. Catechin increases both N-hydroxylation and deacetylation pathways in the hepatocytes producing increases in N-hydroxy-AAF and aminofluorene. Both of these metabolites are important in AAF intermediates binding with DNA. The short-term incubation of catechin, AAF, hepatocytes, and S. typhimurium in the mutagenesis assay is not sufficient for induction of metabolic pathways. However, previously reported inhibition of detoxification pathways and/or scavenging of the proximate carcinogen can occur to alter mutagenesis in a dose-dependent manner.     

Inhibition of β-amyloid–induced neurotoxicity by planar analogues of procyanidin B3

Reactive oxygen species (ROS) are known to be produced during the amyloid beta (Aβ) aggregation process. Both ROS production and Aβ fibril formation can result in nerve cell injury. Proanthocyanidins are oligomers of catechin that can act as inhibitors of Aβ aggregation. Procyanidin B3 (Cat-Cat), the dimer of (+)-catechin, can easily cross the blood-brain barrier. Previously, we synthesized two derivatives of Cat-Cat, namely Cat-PCat and PCat-PCat, in which the geometry of one or both catechin molecules in Cat-Cat was constrained to be planar. The antioxidative activities of Cat-PCat and PCat-PCat were found to be stronger than that of Cat-Cat, with PCat-PC at exhibiting the most potent activity. These compounds are predicted to protect against Aβ-induced neurotoxicity via inhibition of Aβ aggregation as well as by antioxidative effects toward Aβ-induced intracellular ROS generation. PCat-PCat exhibited the most potent neuroprotective effects against Aβ-induced cytotoxicity, which resulted from inhibition of β-sheet structure formation during the Aβ aggregation process. PCat-PCat may be a promising lead compound for the treatment of Alzheimer’s disease.     

Role of green tea polyphenols in the inhibition of collagenolytic activity by collagenase

Inhibitory effect of green tea polyphenols viz., catechin and epigallocatechin gallate (EGCG) on the action of collagenase against collagen has been probed in this study. Catechin and EGCG treated collagen exhibited 56 and 95% resistance, respectively, against collagenolytic hydrolysis by collagenase. Whereas direct interaction of catechin and EGCG with collagenase exhibited 70 and 88% inhibition, respectively, to collagenolytic activity of collagenase against collagen and the inhibition was found to be concentration dependent. The kinetics of inhibition of collagenase by catechin and EGCG has been deduced from the extent of hydrolysis of (2-furanacryloyl-L-leucyl-glycyl-L-prolyl-L-alanine), FALGPA. Both catechin and EGCG exhibited competitive mode of inhibition against collagenase. The change in the secondary structure of collagenase on treatment with catechin and EGCG has been monitored using circular dichroism spectropolarimeter. CD spectral studies showed significant changes in the secondary structure of collagenase on treatment with higher concentration of catechin and EGCG. Higher inhibition of EGCG compared to catechin has been attributed to the ability of EGCG to exhibit better hydrogen bonding and hydrophobic interaction with collagenase.     

Enzymatic synthesis and antioxidant property of gelatin-catechin conjugates

Gelatin-catechin conjugate was synthesized by the laccase-catalyzed oxidation of catechin in the presence of gelatin. The conjugate had a good scavenging activity against superoxide anion radicals. Moreover, the conjugate showed an amplified inhibition effect on human low density lipoprotein oxidation initiated by 2,2'-azobis(2-amidinopropane)dihydrochloride as a radical generator.     

Synthesis and ribonuclease A inhibition activity of resorcinol and phloroglucinol derivatives of catechin and epicatechin: Importance of hydroxyl groups

The reported ribonuclease A inhibitory activity of the green tea extracts prompted us to synthesize novel catechin/epicatechin based conjugates with resorcinol and phloroglucinol with the aim to increase the number of phenolic OH groups. These are found to be more effective inhibitors of ribonuclease A as compared to catechin and epicatechin thus indicating the importance of number of phenolic OH groups for the inhibition of ribonucleolytic activity. Fluorescence studies have been carried out to evaluate the binding parameters. The protein–ligand docking studies are also performed to gain insight into the protein–polyphenols interactions. The epicatechin based polyphenols 1 and 2 also showed inhibition of angiogenin-induced angiogenesis, as determined by chorioallantoic membrane (CAM) assay.     

Electron-transfer capacity of catechin derivatives and influence on the cell cycle and apoptosis in HT29 cells

Galloylated and nongalloylated catechin conjugates with cysteine derivatives have been synthesized and evaluated for their capacity to scavenge free radicals and to influence crucial functions (cell cycle, apoptosis) in HT29 colon carcinoma cells. We show that the nonphenolic part of the molecule modified the capacity of catechins to donate hydrogen atoms and to transfer electrons to free radicals. Nongalloylated derivatives did not significantly influence either the cell cycle or apoptosis. Among the galloylated species, 4beta-[S-(O-ethyl-cysteinyl)]epicatechin 3-O-gallate, which showed a high electron-transfer capacity (5 e- per molecule), arrested the cell cycle and induced apoptosis as expected for galloylated catechins such as tea (-)-epigallocatechin 3-O-gallate. 4beta-[S-(N-Acetyl-O-methyl-cysteinyl)]epicatechin 3-O-gallate, which showed the highest hydrogen-donating capacity (10 H per molecule) while keeping the electron-transfer capacity low (2.9 e- per molecule), did not trigger any significant apoptosis. The gallate moiety did not appear to be sufficient for the pro-apoptotic effect of the catechin derivatives in HT29 cells. Instead, a high electron-transfer capacity is more likely to be behind this effect. The use of stable radicals sensitive exclusively to electron transfer may help to design molecules with either preventive scavenging action (high hydrogen donation, low electron transfer) or therapeutic pro-apoptotic activity (high electron transfer).     

Amplification of inhibitory activity of catechin against disease-related enzymes by conjugation on poly(epsilon-lysine)

A new inhibitor against disease-related enzymes, collagenase, hyaluronidase, and xanthine oxidase, has been developed by the laccase-catalyzed conjugation of catechin on poly(epsilon-lysine). The resulting poly(epsilon-lysine)-catechin conjugate showed greatly improved inhibition effects on activity of these enzymes, whereas the catechin monomer showed very low inhibition activity. The kinetic analysis on the inhibition of collagenase exhibited that the conjugate was a mixed-type inhibitor. The amplified activities might offer high potential as a therapeutic agent for prevention of various enzyme-related diseases.     

Enhanced radical scavenging activity of a procyanidin B3 analogue comprised of a dimer of planar catechin

Proanthocyanidins are oligomers of catechins that exhibit potent antioxidative activity and inhibit binding of oxidized low-density lipoprotein (OxLDL) to the lectin-like oxidized LDL receptor (LOX-1), which is involved in the onset and development of arteriosclerosis. Previous attempts aimed at developing proanthocyanidin derivatives with more potent antioxidative activity and stronger inhibition for LOX-1 demonstrated the synthesis of a novel proanthocyanidin derivative (1), in which the geometry of one catechin molecule in procyanidin B3 was constrained to a planar orientation. The radical scavenging activity of 1 was 1.9-fold higher than that of procyanidin B3. Herein, we synthesized another procyanidin B3 analogue (2), in which the geometries of both catechin molecules in the dimer were constrained to planar orientations. The radical scavenging activity of 2 was 1.5-fold higher than that of 1, suggesting that 2 may be a more effective candidate than 1 as a therapeutic agent to reduce oxidative stress induced in arteriosclerosis or related cerebrovascular disease.     

Peroxidase-catalyzed generation of catechin oligomers that inhibit glucosyltransferase from Streptococcus sobrinus

Abstract Oolong tea extract (OTE) and the purified polymeric polyphenols from OTE have been found to inhibit glucosyltransferase (GTase) of mutans streptococci. In view of the partial fermentation characteristic of oolong tea, we describe here an in vitro model reaction system to produce partially fermented products of d-(+)-catechin or green tea extract (GTE) using horseradish peroxidase. A dimeric catechin molecule was identified as dehydro-dicatechin A by instrumental analyses. The molecular size of some oligomeric catechins was estimated by the elution profile with HPLC. These catechin oligomers markedly inhibited GTase from Streptococcus sobrinus 6715. As the degree of polymerization of catechin or GTE increased, GTase was inhibited more effectively. These results suggest that polymeric polyphenols found in OTE are synthesized by partial fermentation due to oxidases/peroxidases present in tea leaves.     

1-Oxo-3-substitute-isothiochroman-4-carboxylic acid compounds: Synthesis and biological activities of FAS inhibition

A new series 1-oxo-3-substitute-isothiochroman-4-carboxylic acid compounds have been designed and synthesized. Screening of these molecules for FAS inhibition in vitro has indicated that compounds 2c and 2d showed more effective FAS inhibition activities and higher therapeutic index than C75.     

1-Toluene-sulfonyl-3-[(3'-hydroxy-5'-substituted)-gamma-butyrolactone]-indoles: synthesis, COX-2 inhibition and anti-cancer activities

Indoles carrying a cyclic ester (gamma-butyrolactone) at C-3 position have been synthesized by the allylation of 3-indoleglyoxylate followed by iodocyclisation and the nucleophilic replacement of the iodo-group. Screening of these molecules for COX-2 inhibition and anti-cancer activities has identified compounds 10 and 11 as highly potent and selective for COX-2 as well as showing remarkable anti-cancer activities (better than that of indomethacin).     

Synthesis of nitrated indenoisoquinolines as topoisomerase I inhibitors

Indenoisoquinolines and dihydroindenoisoquinolines have been synthesized possessing a nitro-substituted isoquinoline ring in an effort to explore the effects of electron-withdrawing substituents on biological activity. The in vitro anticancer activities of these molecules have been tested in the National Cancer Institute's screen of 55 cell lines. The compounds have also been tested for topoisomerase I (top1) inhibition. The results indicate that these substances are a potent class of top1 inhibitors with sub-micromolar cytotoxicity mean graph midpoints (MGM) and top1 inhibition equal to camptothecin.     

Synthesis of 1,2,4-triazole-5-on derivatives and determination of carbonic anhydrase II isoenzyme inhibition effects

Carbonic anhydrase (CA) II plays major roles in pH regulation of body, protection of electrolyte balance, transportation of water and some metabolic pathways. Therefore, CA II inhibitors are very important molecules for drug design and have many pharmacological applications. CA II as a target molecule is also important for eliminating some pathological conditions such as glaucoma, cancer, epilepsy, ulcer and obesity. In this study, some 1,2,4-triazole derivatives were synthesized and CA II inhibition potentials of these molecules were examined. It has been found that molecule 7c was the most potent inhibitor with the lowest IC₅₀ value at micromolar level among the examined molecules. The inhibition in the range of 18.41–64.97% was seen in the presence of newly synthesized molecules at their reachable maximum concentration in the reaction mixtures. Kinetic studies showed that the inhibition mechanism of compound 7c on carbonic anhydrase activity was reversible and uncompetitive. Molecular docking studies also indicated that compound 7c could bind to the active site of the enzyme by weakly interacting with especially Gln102, Leu240, Ala241 and Trp243. ADME properties of these newly synthesized (3a-e, 6, 7a-e) were also studied and showed good oral drug candidate like properties.     

儿茶素对棉枯萎病菌胞壁降解酶的抑制及在棉花抗病性中的作用

在含有儿茶素的培养液中棉枯萎病菌(Fusarium oxysporum f. sp. vasinfectum)的多聚半乳糖醛酸酶(PG)和果胶裂解酶(PL)活性明显降低。儿茶素可明显抑制初步纯化的PG和PL活性以及它们对棉苗组织的浸软作用。对棉苗组织中儿茶素含量的测定结果表明:抗病品种棉苗组织中儿茶素含量较高;氟乐灵处理可诱发棉苗产生对枯萎病的诱导抗性,同时也提高棉苗组织中的儿茶素的含量;枯萎病菌侵染后棉苗组织中儿茶素含量明显升高,以抗病品种棉苗和氟乐灵诱发处理棉苗组织中儿茶素含量的增加更为明显。棉苗组织提取液中的酚类物质可抑制PG和PL的活性,且证明这种抑制作用主要是由儿茶素引起。提取液对PG和PL活性的抑制作用与棉苗组织中儿茶素的含量呈直接的正相关关系。因此,作者认为棉苗组织中的儿茶素可能通过对病菌PG和PL等胞壁降解酶的抑制而与棉花对枯萎病的抗病性及氟乐灵诱发的诱导抗性有关。     

Dietary green and white teas suppress UDP-glucuronosyltransferase UGT2B17 mediated testosterone glucuronidation

The anabolic steroid testosterone can be used by athletes to enhance athletic performance and muscle growth. UDP-glucuronosyltransferase (UGT2B17) is the key enzyme involved in the glucuronidation of testosterone to testosterone glucuronide, which also serves as a marker for the testosterone/epitestosterone (T/E) ratio used to detect testosterone abuse in sport. Inhibitors of testosterone glucuronidation could have an impact on circulating testosterone levels, thus aiding performance, as well as potentially affecting the urinary T/E ratio and therefore masking testosterone abuse. Previous reports have revealed that non-steroidal, anti-inflammatory drugs, diclofenac and ibuprofen, inhibit the UGT2B17 enzyme. The aim of this study is to analyse dietary tea samples for inhibition of testosterone glucuronidation and, where inhibition is present, to identify the active compounds. Analysis of testosterone glucuronidation was conducted by performing UGT2B17 assays with detection of un-glucuronidated testosterone using high performance liquid chromatography. The results from this study showed that testosterone glucuronidation was inhibited by the green and white tea extracts, along with specific catechin compounds, notably: epicatechin, epigallocatechin gallate (EGCG) and catechin gallate. The IC50 inhibition value for EGCG was determined, using a Dixon plot, to be 64 μM, equalling the most active NSAID inhibitor diclofenac. Thus, common foodstuffs and their constituents, for the first time, have been identified as inhibitors of a key enzyme involved in testosterone glucuronidation. Whilst these common compounds are not substrates of the UGT2B17 enzyme, we showed that they inhibit testosterone glucuronidation which may have implications on current doping control in sport.     

Degree of crosslinking of collagen at interfaces: adhesion and shear rheological indicators

Work of adhesion (ΔW) and surface rheology at solid/air and solution/air interface have been used as indicators to study the stabilization of collagen by different crosslinking agents like basic chromium sulfate (BCS), tannic acid, catechin and formaldehyde. The results show that an increase in rate of ΔW would promote adsorption while a decrease leads to hindered adsorption. Shear rheological studies on collagen demonstrate an increase in both shear viscosity and elasticity with time while for collagen with polyphenols like catechin and tannic acid there is an unusual breakdown of these values. A correlation between the rheological properties and the work of adhesion suggests that the time frame in which the viscoelastic behavior is initiated for collagen with different crosslinking agents determines the final macroscopic property of the protein. The study attempts to quantify the degree of crosslinking of collagen through the dynamics and strength of the water molecules in the assembly of hydrated protein and the crosslinking agents.     

Superoxide anion scavenging and xanthine oxidase inhibition of (+)-catechin-aldehyde polycondensates. Amplification of the antioxidant property of (+)-catechin by polycondensation with aldehydes

In this study, the antioxidant property of (+)-catechin-aldehyde polycondensates has been examined. Superoxide anions are one of the most typical reactive oxygen species (ROS) and generated by xanthine oxidase (XO). The measurements of the superoxide anion scavenging and XO inhibition activity showed that catechin had pro-oxidant properties in lower concentrations and little XO inhibition. On the other hand, the polycondensates exhibited much higher effects compared to the catechin monomer, and their physiological activities were greatly affected by the structure of polycondensates. Steady-state analysis of the inhibition against XO showed that the inhibition type of the polycondensate was uncompetitive. Furthermore, the results of the circular dichroism and UV-visible measurements of a mixture of the polycondensate and XO were in good agreement with that of the steady-state analysis; the spectral changes due to the chelation of the polycondensate onto the Fe/S and/or the FAD center of XO were observed. These data strongly suggest that the polycondensates possess a great potential as antioxidant for various applications.     

Interaction of epicatechin gallate with phospholipid membranes as revealed by solid-state NMR spectroscopy

Epicatechin gallate (ECg), a green tea polyphenol, has various physiological effects. Our previous nuclear Overhauser effect spectroscopy (NOESY) study using solution NMR spectroscopy demonstrated that ECg strongly interacts with the surface of phospholipid bilayers. However, the dynamic behavior of ECg in the phospholipid bilayers has not been clarified, especially the dynamics and molecular arrangement of the galloyl moiety, which supposedly has an important interactive role. In this study, we synthesized [13C]-ECg, in which the carbonyl carbon of the galloyl moiety was labeled by 13C isotope, and analyzed it by solid-state NMR spectroscopy. Solid-state 31P NMR analysis indicated that ECg changes the gel-to-liquid-crystalline phase transition temperature of DMPC bilayers as well as the dynamics and mobility of the phospholipids. In the solid-state 13C NMR analysis under static conditions, the carbonyl carbon signal of the [13C]-ECg exhibited an axially symmetric powder pattern. This indicates that the ECg molecules rotate about an axis tilting at a constant angle to the bilayer normal. The accurate intermolecular-interatomic distance between the labeled carbonyl carbon of [13C]-ECg and the phosphorus of the phospholipid was determined to be 5.3±0.1 ? by 13C-(31)P rotational echo double resonance (REDOR) measurements. These results suggest that the galloyl moiety contributes to increasing the hydrophobicity of catechin molecules, and consequently to high affinity of galloyl-type catechins for phospholipid membranes, as well as to stabilization of catechin molecules in the phospholipid membranes by cation-π interaction between the galloyl ring and quaternary amine of the phospholipid head-group.