Communications

Amplification of Inhibitory Activity of Catechin againstDisease-Related Enzymes by Conjugation on Poly( E-lysine)

Noriko Ihara, Sarah Schmitz, Motoichi Kurisawa, Joo Eun Chung, Hiroshi Uyama,* andShiro Kobayashi*

Department of Materials Chemistry, Graduate School of Engineering, Kyoto University,Kyoto, 615-8510 Japan

Received March 25, 2004; Revised Manuscript Received May 28, 2004

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

Polypeptides and related artificial poly(amino acid)s havesignificantly become important due to their specific propertiessuch as biocompatibility and biodegradability. Poly(ε-lysine)(PL) is produced from culture filtrates fromStreptomycesalbulus1 and shows high water-solubility and biodegrad-ability.2 Furthermore, PL exhibited good antimicrobial activ-ity against Gram-positive and negative bacteria;3 thus, it iswidely used as an additive in the food industry.

Green tea catechins, belonging to the group of flavonoids,exhibit biological and pharmacological effects includingantioxidant, antimutagenic, anticarcinogenic, antimicrobial,and antiinflammatory properties in numerous human, animal,and in vitro studies.4 These properties might potentially bebeneficial in preventing disease and protecting the stabilityof the genome. Many of these activities have been relatedto their antioxidant actions.5 They are also known to be potentinhibitors for several enzymes closely related to diseases.6

However, such low-molecular weight flavonoids often donot possess physiological properties enough for biomedicalapplications.

In contrast, a relatively high-molecular fraction of ex-tracted plant polyphenols was reported to exhibit enhancedphysiological properties such as antioxidant and anticar-cinogenic activity.7 From these perspectives, we havedesigned not only polymerized flavonoids but also polymericflavonoid-conjugates in consideration of the amplificationof physiological properties of the flavonoids.8-10 Recently,we reported that poly(catechin) as one of the strategicmolecular designs was synthesized by peroxidase-catalyzedoxidative coupling and exhibited great improvement in

radical scavenging activity, protection effects against low-density lipoprotein oxidation, and inhibition effects onxanthine oxidase activity, comparing with a catechin mono-mer.8 Furthermore, laccase-catalyzed polymerization of rutinproduced a water-soluble flavonoid polymer exhibiting muchhigher scavenging activity of superoxide anion than the rutinmonomer.9

Enzymatic conjugation of phenols onto amine-containingpolymers has been developed. Tyrosinase catalyzed acoupling of several phenols including catechin with chitosanto produce functional materials based on the biopolymer.11,12

The formation of a Michael-type adduct and/or Schiff basewas proposed in the tyrosinase-catalyzed oxidation of cat-echin with chitosan.12 We have covalently conjugatedcatechin to poly(allylamine) by a similar enzymatic conjuga-tion technique, and succeeded in conferring antioxidantactivity to poly(allylamine) with slightly enhanced antioxi-dant activity against oxidation of human low-density lipo-protein (LDL), compared with unconjugate catechin.13

Furthermore, the enzymatically synthesized gelatin-catechinconjugate also showed a good antioxidant property againstLDL oxidation induced by a free radical.14 Furthermore, theenzymatic conjugation of catechin with amine-containingpolymer particles produced the antioxidant microspheresshowing good radical scavenging activity.15 In this study,we have developed a new enzyme inhibitor, a PL-catechinconjugate, by the conjugation of flavonoid on a polypeptide(Scheme 1). The synthesized PL-catechin conjugate showedhighly amplified inhibition effects against disease-relatedenzymes, collagenase, hyaluronidase, and xanthine oxidase.To our best knowledge, this is the first example demonstrat-ing high inhibitory activity of a polymeric conjugate offlavonoid for various disease-related enzymes.

* To whom correspondence should be addressed. Tel:+81-75-383-2460(H.U.); +81-75-383-2459 (S.K.). Fax:+81-75-383-2461. E-mail: uyama@mat.polym.kyoto-u.ac.jp (H.U.); kobayasi@mat.polym.kyoto-u.ac.jp (S.K.).

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10.1021/bm049823x CCC: $27.50 © 2004 American Chemical SocietyPublished on Web 08/04/2004

Matrix metalloproteinases (MMPs), typically collagenaseand gelatinase, are a family of zinc containing enzymeswhich degrade and remodel structural proteins in the extra-cellular matrix (ECM). Since MMPs play an essential rolein the homeostasis of ECM, an imbalance in their expressionor activity may have important consequences in variouspathologies. Thus, MMPs have recently become interestingtargets for drug design in the search of novel anticancer,antiarthritis, and other pharmacological agents useful in themanagement of inflammatory processes.16 Like MMPs,bacterial collagenase fromClostridium histolyticum(ChC)has also been extensively investigated. This enzyme hydro-lyzes the triple helical region of collagen under physiologicalconditions and was reported to be involved in the pathoge-nicity related to degradation of a connective tissue. So far,several inhibitors of ChC have been developed and thequantitative structure-activity relationship was demon-strated.17 Herein, we examined the ChC inhibitory activityof the PL-catechin conjugate according to the literaturemethod.18 The conjugate was prepared by the laccase-catalyzed reaction of catechin with PL, a slightly modifiedmethod for the synthesis of poly(allylamine)-catechinconjugate.13,19

The conjugate showed greatly amplified concentration-dependent inhibition activity against ChC on the basis of

the catechin unit (Figure 1), which is considered to be dueto effective multivalent interaction between ChC and thecatechin unit in the conjugate.8,10 These data clearly indicatethat the conjugation of catechin on PL greatly amplifies theinhibitory activity of catechin against ChC.

The type of inhibition by the conjugate was analyzed byLineweaver-Burk plots in the steady state (Figure 2). Thereciprocal plots of the substrate concentration and hydrolysisrate with different concentrations of the conjugate intersectedat the left side of the 1/velocity axis above the 1/substrateaxis. This result suggests that the conjugate is a mixed-typeinhibitor.20 The Ki value is estimated as 23µM.

Hyaluronidase is an enzyme which catalyzes hydrolysisof hyaluronic acid and is often involved in a number ofphysiological and pathological processes. Potent hyalu-ronidase inhibitors have antiallergic effects, which may leadto development of new antiallergic agents.21,22 Here, weevaluated antihyaluronidase activity of the conjugate ac-cording to the literature.21 The efficient inhibition activityof the conjugate was found (Figure 3), whereas the mono-meric catechin showed almost negligible inhibition effectover a range of tested concentrations. No inhibition activityof PL toward hyaluronidase was found. Procyanidine, acatechin oligomer, was reported to exhibit the good inhibitioneffect against hyaluronidase;18 however, the inhibition activityof procyanidine was inferior to that of the present conjugate.

Scheme 1

Figure 1. Inhibition activity of catechin and poly(ε-lysine)-catechinconjugate (catechin content ) 3.4%) against collagenase, n ) 3.

Figure 2. Lineweaver-Burk plots for the inhibition of collagenaseby poly(ε-lysine)-catechin with different concentration: ([) 0 µM; (9)10 µM; (2) 20 µM; (b) 50 µM.

1634 Biomacromolecules, Vol. 5, No. 5, 2004 Communications

These data suggest that the conjugation on a polymer moreefficiently amplified the inhibitory action of catechin againsthyaluronidase.

The superoxide radical is a reactive oxygen species, whichis formed during normal aerobic metabolism and by activatedphagocytes.23 Reduction of molecular oxygen to superoxideby xanthine oxidase (XO), generating hydroxyl radicals anduric acid, is an important physiological pathway. However,an excess of superoxide radicals damages biomacromoleculesboth directly and indirectly by forming hydrogen peroxideor highly reactive hydroxyl radicals. Thus, XO is animportant biological source of reactive oxygen species; XOis an enzyme responsible for the formation of uric acidassociated with gout leading to painful inflammation in thejoints.24

Figure 4 showed XO inhibitory activity assessed byevaluating uric acid formation from XO.25 The XO inhibitioneffect of catechin was negligible at a concentration below300 µM, on the other hand, the conjugate exhibited anincrease in XO inhibitory activity as an increasing concentra-tion of the catechin unit. PL exhibited no inhibition activitytoward XO. Poly(catechin), prepared by the enzymaticoxidative coupling, also showed high inhibition activityagainst XO; however, the inhibition activity of the conjugatewas superior to that of poly(catechin).8 These data suggest

that the XO inhibitory activity of catechin is greatly amplifiedby the enzymatic reaction leading to the polymeric fla-vonoids.

In conclusion, a new disease-related enzyme inhibitor, thepoly(ε-lysine)-catechin conjugate, was developed. Theconjugate showed great amplification of inhibitory activityagainst collagenase, hyaluronidase, and xanthine oxidase onthe basis of the catechin unit, compared to intact catechin.We believe that the present conjugate may be useful for atherapeutic agent to offer protection against a wide range ofenzyme-related diseases including cardiovascular diseases,cancer invasion and metastasis, arthritis, atherogenesis, andgout. Bioavailability and in vivo and in vitro toxicity of theconjugate should be examined for the application of therapicagents. Moreover, we preliminarily found that the PL-catechin conjugate exhibited good antimicrobial activity,comparable with unmodified PL. Further investigationincluding applications of the present conjugate and relatedpolymeric flavonoids for biomedical purposes is under wayin our laboratory.

Acknowledgment. This work was supported by Programfor the Promotion of Basic Research Activities for InnovativeBioscience. We acknowledge the gift of PL and laccase fromChisso Co. and Novozymes Japan Ltd., respectively. S.S. isgrateful to the DAAD program, Germany, for the fellowship.

References and Notes

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(8) (a) Kurisawa, M.; Chung, J. E.; Kim, Y. J.; Uyama, H.; Kobayashi,S. Biomacromolecules2003, 4, 469. (b) Kurisawa, M.; Chung, J.E.; Kim, Y. J.; Uyama, H.; Kobayashi, S.Macromol. Biosci.2003,3, 758. (c) Kurisawa, M.; Chung, J. E.; Uyama, H.; Kobayashi, S.Chem. Commun.2004, 294.

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Figure 3. Inhibition activity of catechin and poly(ε-lysine)-catechinconjugate (catechin content ) 3.4%) against hyaluronidase, n ) 3.

Figure 4. Inhibition activity of catechin and poly(ε-lysine)-catechinconjugate (catechin content ) 3.1%) against xanthine oxidase, n )3.

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(13) Chung, J. E.; Kurisawa, M.; Tachibana, Y.; Uyama, H.; Kobayashi,S. Chem. Lett.2003, 32, 620.

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(19) A typical procedure of the conjugate production is as follows. PL(1.65 g, 10 mmol of monomer unit) was dissolved in 40 mL of water.The pH of the solution was adjusted at 7 or 8 by adding 2 N NaOH.Catechin (0.15 g, 0.50 mmol) in 4 mL of methanol was added to thesolution. The reaction was started by the addition of laccase solution(4 µL, 4 units). After the reaction, the mixture was acidified by theaddition of 6N HCl and subjected to purification by dialysis (cutoff

molecular weight) 5 × 102) four times. The remaining solutionwas lyophilized to give the conjugate. The conjugate formation wasconfirmed by UV spectroscopy; a broad peak around 430 nm newlyappeared by the conjugation.12 The catechin content in the conjugatewas determined by elemental analysis and the enzyme inhibition wasmeasured on the basis of the catechin unit.

(20) Yokochi, N.; Morita, T.; Yagi, T.J. Agric. Food Chem.2003, 51,2733.

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