Workshop Argentina-Japan Bioscience and Biotechnology for the Promotion of Agriculture and Food Production August 4 th 2009 Chemical Biology of Tea Catechins Tsutomu NAKAYAMA Laboratory of Molecular Food Engineering and Global CE Program, School of Food and Nutritional Sciences, University of Shizuoka
What are tea catechins? Tea catechins are present in green tea and black tea. EC 6%, EGC 19%, ECg 14%, EGCg 59% In green tea. Epicatechin [EC] Epigallocatechin [EGC] Antioxidant activity Antibacterial effect Antimutagenicity Antihypercholesterolemia etc A C B The four analogues show substantially different activities in assays in vitro. Epicatechin gallate [ECg] galloyl Epigallocatechin gallate [EGCg] Inactivation of influenza virus ECg > EGCg >> EGC Antibacterial effect EGCg > ECg > EGC > EC
Part 1 Interaction between catechins and proteins (albumin)
1-1. 1. Detection of EGCg-binding proteins with redox cycling staining H nitroblue tetrazolium (NBT) electrophoresis RS PVDF membrane RS (superoxide) R Glycine, alkaline ph nitroblue formazan PVDF membrane
Conclusions obtained by the redox cycling staining 1. Tea catechins bind covalently human serum albumin, but only under oxidative conditions. 2. Under these conditions, human serum albumin is also oxidized. 3. A LC-MS study conducted in our laboratory revealed that the covalent binding occurred between EGCg and cystein of human serum albumin. Mol. Nutr. Food Res., 53, 709-715, 2009 Albumin stabilizes (-)-epigallocatechin gallate in human serum: Binding capacity and antioxidant property
1-2. Dynamic analysis of non-covalent binding of catechins to proteins by Quartz-Crystal Microbalance (QCM) HSA Catechins injection QCM cells AT-cut Quartz Gold electrode Binding amount Time (min) 1/τ = Kon[Y]+Koff Host X + Ka = Guest Y [XY] [X] [Y] Kon Koff = Kon/Koff Complex XY 1/τ Conc. of catechins
Binding constants of catechins to human serum albumin (HSA) Catechins K on [M -1 S -1 ] K off [S -1 ] K a [M -1 ] EC 5.9 10 0 2.8 10-3 5.8 10 3 EGC 8.5 10 0 3.0 10-3 3.4 10 3 ECg 5.0 10 2 3.6 10-3 4.3 10 5 EGCg 2.2 10 2 8.9 10-4 4.3 10 5 H H H H EC EGC ECg EGCg
1-3. Affinity of catechins to HSA analyzed by HPLC with HSA column Si N H NH HSA Si N H NH HSA 3-aminopropyl silica-gel K HSA = (t R - t 0 )/t 0 t R : retention time of a catechin (min) t 0 : retention time of unretained molecules (i.e., citric acid) (min) HPLC condition Column: SUMICHIRAL HSA (4.0 150 mm) Mobile phase: 20% acetonitrile in 0.1 M sodium phosphate buffer ph 5.0 Flow rate: 0.9 ml/min Injection volume: 10 μl UV detection: 200 nm
HPLC chromatogram of catechins with HSA column 100 2.89 KHSA EC 0 0.65 100 3.12 0 EGC 0.78 100 16.90 ECg 8.65 0 100 20.82 EGCg 0 0 20 40 60 10.89 Retention time (min)
Summary of Part 1 Structural factors governing affinity of catechins for HSA Number of hydroxy group of the B-ring contributing hydrogen bonding H Hydrophobic region The presence of galloyl moiety producing hyrophobic region
Part 2 Interaction between catechins and phospholipids investigated by HPLC and NMR
How do tea catechins interact with lipid membranes? Tea catechins Phospholipids
2-1. Immobilized Artificial Membrane (IAM) column HPLC chromatogram with an IAM column t R : The retention time of the compound t 0 : The time for unretained molecules (i.e., citric acid)
Phospholipophilicity of tea catechins The K IAM values correlated well with the amounts incorporated into the liposomes and with the partition coefficient obtained from n-octanol/pbs system.
2-2. Solution NMR study B 0 DMPC DHPC isotropic bicelle Isotropic bicelle solutions were prepared by the following conditions: DMPC : DHPC = 1 : 2 tea catechins : DMPC = 0.24 : 1 final lipid concentration: 8% w/v (D 2 )
1 H NMR spectra (ECg) 1 H NMR (400 MHz, D 2 ) galloyl A C galloyl B ECg (free) B-ring ECg + bicelles The B-ring and galloyl moiety interact with phospholipid membranes.
Comparison of 1 H chemical shift change values (bicelles) 0.04 [ppm] 0.02 0.00 0.02 0.04 γ β α G3 G2 G1a G1b C2 C3 (CH 2 ) (CH 3 ) 0.06 0.08 0.10 0.12
1 H spin-lattice relaxation times (T 1 ) T 1 [sec] 2 Inversion recovery method (PD: 40 s) Catechins (free) 3 Catechins + bicelles 4a verlapped 4b FAST SLW 6/8 τ c 2 5 B ring Molecular motion of B-ring and galloyl moiety was restricted in the presence bicelles. 6 2, 6 galloyl 0.0 1.0 2.0 3.0 4.0 5.0 6.0 T 1 [sec]
Location of ECg in the model membrane clarified by solution NMR H in aqueous solution (H 3 C) 3 N P catechin (ECg) C 12 H 25 C 10 H 21 intermolecular NE interacting with bicelles
Summary of Part 2 The molecular-level interactions of tea catechins with lipid bilayers have been clarified based on: HPLC IAM column Affinity of tea catechins for phospholipid membranes Biosci. Biotechnol. Biochem. 72, 3289 3292 (2008). Solution NMR Chemical shifts ( 1 H NMR) T 1 relaxation times ( 1 H NMR) B-ring and galloyl moiety of ECg and EGCg, and phospholipids γ-h, are involved in the interaction. NE effects ( 1 H 1 H, 1 H 13 C) B-ring and galloyl moiety are closely located to γ-h of phospholipids J. Agric. Food Chem. 55, 9986 9992 (2007).
1. Affinity for proteins Conclusions EGCg > ECg >> EGC > EC Presence of galloyl moiety is the most decisive factor. Presence of hydroxy moiety in the B ring increases the affinity. Catechins interact with proteins both by hydrophobic bonding and hydrogen bonding. 2. Affinity for phospholipids ECg > EGCg >> EC > EGC Presence of galloyl moiety is the most decisive factor. Presence of hydroxy moiety decreases the affinity. Catechins interact with phospholipids in the surface of membranes only by hydrophobic bonding. 3. These results should be useful to clarify the mechanisms of the functions of tea catechins.