Enzymatic synthesis and biological properties of flavonoid polymers

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Bioactive polyphenols are present in a variety of plants and used as important components of human and animal diets.215 Flavonoids are a broad class of low molecular weight secondary plant polyphenolics, which are benzo-7-pyrone derivatives consisting of phenolic and pyrane rings. Their biological and pharmacological effects including antioxidant, antimutagenic, anticarcinogenic, antiviral, and anti-inflammatory properties have been demonstrated in numerous human, animal, and in vitro studies. These properties are potentially beneficial in preventing diseases and protecting the stability of the genome. Many of these activities have been related to their antioxidant actions. Green tea is derived from Camellia sinensis, an evergreen shrub of the Theaceae family. Most of the polyphenols in green tea are flavanols, commonly known as catechins; the major catechins in green tea are (+)-catechin, (—)-epicatechin, (—)-epigallocatechin, (—)-epicatechin gallate, and (—)-epigallocatechin gallate (EGCG). Numerous biological activities have been reported for green tea and its contents; among them, the preventive effects against cancer are most notable.216

We have designed not only polymerized flavonoids but also flavonoid conjugates of various polyamines, in consideration of extension of the amplification of physiological properties of the flavonoids. Polymeric flavonoids were synthesized by the enzymatic oxidative coupling.217

The HRP-catalyzed polymerization of (+)-catechin was carried out in an equivolume mixture of 1,4-dioxane and buffer (pH 7) to give the polymer with molecular weight of 3.0 x 103 in 30% yield.218 Using methanol as co-solvent improved the polymer yield and molecular weight.219 In the polymerization of catechin by using laccase derived from Myceliophthora (ML) as catalyst, the reaction conditions were examined in detail.220 A mixture of acetone and acetate buffer (pH 5) was suitable for the efficient synthesis of soluble poly(catechin) with high molecular weight. The mixed ratio of acetone greatly affected the yield, molecular weight, and solubility of the polymer. The polymer synthesized in 20% acetone showed low solubility toward DMF, whereas the polymer obtained in an acetone content less than 5% was completely soluble in DMF. In the UV-vis spectrum of poly(catechin) in methanol, a broad peak centered at 370 nm was observed. In alkaline solution, this peak was red-shifted and the peak intensity became larger than that in methanol. In the ESR spectrum of the enzymatically synthesized poly(catechin), a singlet peak at g = 1.982 was detected, whereas the catechin monomer possessed no ESR peak.

Superoxide anion scavenging activity of the enzymatically synthesized poly(catechin) was evaluated. Poly(catechin), synthesized by HRP catalyst, greatly scavenged superoxide anion in a concentration-dependent manner, and almost completely scavenged at 200 jxM of a catechin unit concentration.218 The laccase-catalyzed synthesized poly(catechin) also showed excellent antioxidant property.219 Catechin showed pro-oxidant property in concentrations lower than 300 (xM. These results demonstrated that the enzymatically synthesized poly(catechin) possessed much higher potential for superoxide anion scavenging, compared with intact catechin.

EGCG is a major ingredient of green tea possessing powerful antioxi-dant activity and cancer-chemopreventive activity due to actions of radical scavenging, enzyme inhibition, and metal chelation. The polymer obtained by the laccase-catalyzed oxidative coupling of EGCG showed much higher superoxide anion scavenging activity than the EGCG monomer and enzymatically synthesized poly(catechin).221

Xanthine oxidase (XO) is not only an important biological source of ROS but also the enzyme responsible for the formation of uric acid associated with gout leading to painful inflammation in the joints. The XO inhibition effect by the enzymatically synthesized poly(catechin) increased as an increasing concentration of catechin units, while the monomeric catechin showed almost negligible inhibition effect in the same concentration range.219'220 This markedly amplified XO inhibition activity of poly(catechin) was considered to be due to effective multivalent interaction between XO and the condensed catechin units in the poly(catechin).

PolyEGCG also showed excellent inhibition effect for XO. The XO inhibition effect of EGCG monomer was quite low with inhibition less than about 5% over a range of tested concentrations. In contrast, polyEGCG showed greatly amplified XO inhibition effect in a concentration-dependent manner.221 Moreover, the inhibition of polyEGCG was higher than that of allopurinol, the frequently used commercial inhibitor for gout treatment. Thus, polyEGCG is expected as one of the leading candidates of therapeutic molecules against various diseases induced by free radicals and/or enzymes including gout. Kinetic analysis showed that polyEGCG is an uncompetitive inhibitor of XO.

Mutans streptococci are the major pathogenic organisms of dental caries in humans. The pathogenicity is closely related to production of extracellular, water-insoluble glucans from sucrose by glucosyltransferase and acid release from various fermentable sugars. Poly(catechin) obtained by HRP catalyst in a phosphate buffer (pH 6) markedly inhibited glucosyltransferase from Streptococcus sorbrinus 6715,222 whereas the inhibitory effect of catechin for this enzyme was very low.

Rutin is one of the most commonly found flavonol glycosides identified as vitamin P with quercetin and hesperidin. An oxidative polymerization of rutin using ML as catalyst was examined in a mixture of methanol and buffer to produce a flavonoid polymer.223 Under selected conditions, the polymer with molecular weight of several thousands was obtained in good yields. The resulting polymer was readily soluble in water, DMF, and DMSO, although rutin monomer showed very low water solubility. UV measurement showed that the polymer had broad transition peaks around 255 and 350 nm in water, which were red-shifted in an alkaline solution. ESR measurement showed the presence of a radical in the polymer. Quercetin and kaempferol were polymerized by laccase and tyrosinase, and the morphology of the resulting aggregate was examined.224

The antioxidant activity of rutin was greatly amplified by the laccase-catalyzed oxidative coupling; poly(rutin) also showed much higher scavenging capacity toward superoxide anion than rutin.223 We examined the protection effects of rutin and poly(rutin) against endothelial cell damage caused by 2,2'-azobis(2-amidinopropane)dihydrochloride. Poly(rutin) enhanced cell viability with higher protection effects against the oxidative damage than that of the rutin monomer at low concentration. In particular, in high concentration, the polymer exhibited further raised protection relating to a concentration increase. In contrast, the monomer induced fatal cytotoxicity by itself at the same concentration. These results imply that poly(rutin) is a more potent chain-breaking antioxidant when scavenging free radicals in an aqueous system than the monomer.

Enzymatic conjugation of (+)-catechin on biopolymers as well as synthetic polymers has been developed. Poly(e-lysine) (PL) is a biopolymer produced from culture filtrates of Streptomyces albulus and shows good antimicrobial activity against Gram-positive and negative bacteria and hence widely used as an additive in food industry. A new inhibitor against disease-related enzymes, collagenase, hyaluronidase, and xanthine oxidase, was developed by the conjugation of catechin on poly(e-lysine) by using ML as catalyst (Scheme 30).225

The PL-catechin conjugate showed greatly amplified concentration-dependent inhibition activity against bacterial collagenase (ChC) on the basis of the catechin unit, which is considered to be due to effective multivalent interaction between ChC and the catechin unit in the conjugate. The kinetic study suggests that this conjugate is a mixed-type inhibitor for ChC. Hyaluronidase is an enzyme which catalyzes hydrolysis of hyaluronic acid and is often involved in a number

Catalase And Lead Inhibition
Scheme 30

of physiological and pathological processes. Potent hyaluronidase inhibitors have antiallergic effects, which may lead to development of new antiallergic agents. The efficient inhibition activity of the PL-catechin conjugate was found, while the monomeric catechin showed almost negligible inhibition effect.225 The PL-catechin conjugate also showed good inhibition effects for XO.

The conjugation of catechin on poly(allylamine) using ML as catalyst was examined under air.226 During the conjugation, the reaction mixture turned brown and a new peak at 430 nm was observed in the UV-vis spectrum. At pH 7, the reaction rate was the highest. The conjugation hardly occurred in the absence of laccase, indicating that the reaction proceeded via enzyme catalysis.

Gelatin is the most widespread water-soluble protein in the body, resulting from partial degradation of water-insoluble collagen. Gelatin has widely been used in food, pharmaceutical, and photographic industries. Gelatin-catechin conjugate was synthesized by the laccase-catalyzed oxidation of catechin in the presence of gelatin, in which the lysine residue of gelatin was used for grafting of catechin.227 Antioxidant properties of the gelatin-catechin conjugate were evaluated. The conjugate possessed scavenging activity of superoxide anion, whereas gelatin was not active for the scavenging.

Polyhedral oligomeric silsesquioxane (POSS) has been extensively studied as starting substrate to construct nanocomposites with precise control of nanoar-chitecture and properties. Octahedral derivatives are the most representative ones of this family. It was reported that the HRP-catalyzed conjugation of catechin on amine-substituted octahedral silsesquioxane amplified the beneficial physiological property of flavonoids.228 The POSS-catechin conjugate exhibited great improvement in scavenging activity against superoxide anion, compared with intact catechin. In addition, the conjugate showed high inhibitory effect on XO activity, while the inhibition effect of catechin was very low. These unique properties of the conjugate may be derived from the POSS structure.

Catechin-immobilizing polymer particles were prepared by laccase-catalyzed oxidation of catechin in the presence of amine-containing porous polymer particles.229 The resulting particles showed good scavenging activity toward stable free 1,1-diphenyl-2-picryl-hydrazyl radical and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical cation. These particles may be applied for packed column systems to remove radical species such as reactive oxygen closely related to various diseases.

Polyphenol oxidase (PPO) was reported to catalyze the conjugation of cat-echin on chitosan. The formation of a Michael-type adduct and/or Schiff base was proposed during the PPO-catalyzed conjugation of catechin with chitosan.230 Rheological measurement demonstrates that the resulting conjugate behaves as an associative thickener. This PPO-catalyzed modification of chitosan has been applied for various phenols. For example, the enzymatic treatment of chitosan in the presence of chlorogenic acid produced the modified chitosan soluble under both acidic and basic conditions.231 The PPO-catalyzed reaction of chitosan and 3,4-dihydroxyphenylethylamine (dopamine) provided water-resistant adhesive properties to chitosan.232 A chitosan derivative modified with hydroxy or dihy-droxybenzaldehyde was crosslinked by PPO to give stable and self-sustaining gels.233

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