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甲２の１／Style Kou 2-1》

学位論文の要旨
Abstract of Thesis
研究科
School専

攻Division

学生番号Student No.

氏名NameGraduate School of Environmental and Life Science
Agricultural and Life Science77425652
Zhe Liu

学位論文題目 Title of Thesis（学位論文題目が英語の場合は和訳を付記）

Tea catechins as potent non-competitive inhibitors of angiotensin converting enzyme（アンジオテンシン変換酵素の強力な非競合阻害剤としての茶カテキン）

学位論文の要旨 Abstract of Thesis

Renin-angiotensin system (RAS) is one of the major control systems for blood pressure,
fluid and electrolyte balance. Angiotensin converting enzyme (ACE) plays a crucial role in theRAS by converting angiotensin I to angiotensin II, a potent vasoconstrictor. The inhibition of
ACE is one of the most promising strategies for treatment of hypertension. Synthetic ACE
inhibitors, such as captopril and enalapril, are also used as the first-line drugs for the therapy ofcardiovascular diseases. However, these artificial ACE inhibitors are believed to have certain
side effects such as cough, skin rash, and angioneurotic edema. Thus, there is an urgent
requirement for the exploration and development of a new type of ACE inhibitors without
adverse effects. Accumulating epidemiological and intervention studies indicated that green tea
consumption is inversely associated with the risk of cardiovascular disease, which might belargely attributed to the presence of polyphenols. Tea catechins, including (-)-epicatechin (EC),(-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg) and (-)-epigallocatechin gallate(EGCg), are the primary class of polyphenols in green tea. Although green tea and tea catechinshave been shown to be promising as potential ACE inhibitors, the precise mechanisms
underlying ACE inhibition by catechins remain to be clarified. In this thesis, the molecularmechanisms involved in the ACE inhibition by tea catechins are investigated.

《H28様式甲２の２／Style Kou 2-2》

氏名 NameZhe Liu

In Chapter 2, the inhibitory effects of four tea catechins, including (-)-epicatechin (EC),(-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg) and (-)-epigallocatechin gallate(EGCg), on the enzymatic activity of ACE (purified from rabbit lung) were compared. Eachcatechin treatment significantly reduced the ACE activity with the order of potency being
EGCg > ECg > EGC = EC. The addition of 1 mM borate significantly recovered the reduced
ACE activities by tea catechins, suggesting that hydroxyl groups at B-ring or at a galloyl moiety
play an important role in the ACE-inhibitory mechanism. The covalent modification of ACE bytea catechins was also observed by a redox-cycling staining experiment. A Lineweaver-Burkplot indicated that EGC and ECg were non-competitive inhibitors. The galloylated catechinsmight more potently inhibit ACE activity in an allosteric manner through the hydrophobic andhydrogen-bonding interaction of the galloyl moiety with the non-catalytic site of ACE.
In Chapter 3, the molecular mechanisms involved in the ACE inhibition by EGCg, a major
tea catechin, were further investigated. The enzyme activity of recombinant human ACE wasinhibited by EGCg in a dose-dependent manner. Co-incubation with Zn2+ ion showed noinfluence on the ACE inhibition by EGCg, whereas it completely counteracted the inhibitory
effect of EDTA, a representative ACE inhibitor chelating Zn2+ at the active site of ACE.
Although a considerable amount of hydrogen peroxide was produced during the incubation ofEGCg, the treatment of ACE with hydrogen peroxide showed little effect on its enzymatic
activity. On the other hand, the co-incubation of EGCg with inhibitors of catechol oxidation,such as borate or ascorbic acid, significantly diminished the EGCg inhibition. A redox-cycling
staining experiment revealed that ACE was covalently modified by EGCg. Furthermore, aLineweaver-Burk plot analysis indicated that EGCg also inhibited the ACE activity in anon-competitive manner. These results strongly suggested that EGCg allosterically inhibits theACE activity through the oxidative conversion into an electrophilic quinone and subsequentbinding to the ACE.
3,4-Dihydoxyphenylacetic acid (DOPAC), having a catechol group, is one of the colonicmicroflora-produced catabolites of quercetin 4´-glucoside. A previous study indicated thatDOPAC is oxidized to form o-quinone, then covalently binds to sulfhydryls in GSH or proteinsdue to its catechol structure. In Chapter 4, the modulating effect of DOPAC on ACE activity was《H28様式甲２の３／Style Kou 2-3》

氏名 Name Zhe Liu

also studied. The co-incubation of DOPAC significantly inhibited the enzymatic activity of ACE
in a dose-dependent manner. Among the four tea catechins, DOPAC has the similar potency toinhibit ACE to that of EC. In addition, the covalent binding of DOPAC with ACE was detectedby a tag-free DOPAC probe with the azide labeled biotin and a horseradish peroxidase(HRP)-streptavidin complex. These findings suggested that (1) the catechol structure and its
oxidative conversion into electrophilic species are involved in the potential ACE inhibitory
activity; (2) ACE is one of the potential cellular targets of DOPAC.
In conclusion, this study indicates that: (1) tea catechins and DOPAC, both of which arederived from food stuffs, are potential ACE inhibitors; (2) Zn2+-chelation as well as hydrogen
peroxide-dependent mechanism are ruled out in the mechanisms of the ACE inhibition by tea
catechins; (3) the galloylated catechins (ECg and EGCg) allosterically inhibit the ACE activity
through the oxidative conversion into an electrophilic quinone and subsequent binding to theACE; (4) the presence of galloyl moiety might play an important role in the hydrophobic andhydrogen-bonding interaction with the non-catalytic site of ACE, associated with more potent
ACE inhibitory activity. The present findings encourage further study using not only theplausible structural model based on the docking simulation but also proteomic and reverse
genetic approaches to identify amino acid residues of ACE involved in the catechin-protein
interaction. It is likely that basic knowledge of the ACE-binding site of tea catechins will
provide a platform for the rational design of new domain-selective ACE inhibitors withimproved efficacy and pharmacological profiles. Taken together, this study suggests that thegalloylated catechins could be potential candidates to develop nutraceuticals against
hypertension and its related disease.