Interaction between Ester-Type Tea Catechins and Neutrophil

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Interaction between Ester-type Tea Catechins (ETC) and Neutrophil Gelatinase - Associated Lipocalin (NGAL): Inhibitory Mechanism Wei Zhang, Xiao Li, Fang Hua, Wei Chen, Wei Wang, Gang-Xiu Chu, and Guan-Hu Bao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05399 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 22, 2018

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Journal of Agricultural and Food Chemistry

Interaction between Ester-type Tea Catechins (ETC) and Neutrophil Gelatinase Associated Lipocalin (NGAL): Inhibitory Mechanism Wei Zhang⊥,†, Xiao Li⊥,†, Fang Hua†, Wei Chen§, Wei Wang†, Gang-Xiu Chu*,†, Guan-Hu Bao*,† †

Natural Products Laboratory, International Joint Lab of Tea Chemistry and Health

Effects, State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei, People’s Republic of China §

Department of Nephrology, Affiliated Anhui Provincial Hospital, University of

Science and Technology of China, Hefei, People’s Republic of China

*Corresponding author. Phone: +86-551-65786401. Fax: +86-551-65786765; E-mail: [email protected] (G.-H. Bao).

*Corresponding author. Phone: +86-551-65786217. Fax: +86-551-65786765. E-mail: [email protected] (G.-X. Chu).

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ACS Paragon Plus Environment


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Abstract: Tea is thought to alleviate neurotoxicity due to the antioxidative effect of

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ester-type tea catechins (ETC). Neutrophil gelatinase-associated lipocalin (NGAL)

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can sensitize beta-amyloid (Aβ) induced neurotoxicity and inhibitors of NGAL may

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relieve associated symptoms. As such, the interactions of ETC with NGAL were

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investigated by fluorescence spectrometry and molecular simulation. NGAL

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fluorescence is quenched regularly when being added with six processing types of tea

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infusion (SPTT) and ETC. Thermodynamic analyses suggest that ETC with more

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catechol moieties has a stronger binding capacity with NGAL especially in the

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presence of Fe3+. (-)-Epicatechin 3-O-caffeoate (ECC), a natural product isolated from

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Zijuan green tea, shows the strongest binding ability with NGAL (Kd = 15.21 ±

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8.68 nM in the presence of Fe3+). All ETC are effective in protecting nerve cells

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against H2O2 or Aβ1-42 induced injury. The inhibitory mechanism of ETC against

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NGAL supports its potential use in attenuation of neurotoxicity.

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Keywords: Tea, Ester-type catechins (ETC), Fluorescence spectrometry, Modeling

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simulation, Nerve cell protection, Chelator

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Introduction

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As the most consumed traditional and widespread beverage in the world, tea has

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attracted continuous academic interest towards its various health benefits, which are

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based on its rich content of chemical compounds such as polyphenols, alkaloids,

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saponins, amino acids, and polysaccharides.1 Tea catechins are considered to be one

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of the most effective dietary constituents, with intensive research conducted on its

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effects on different diseases such as cancer, cardiovascular, inflammatory, and

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neurodegenerative diseases.2 The neuroprotective effect of a green tea infusion was

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attributed to the excellent antioxidative activity and metal chelating effect of tea

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catechins.3 Green tea shows better neuroprotective effects than other types of teas

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processed from the leaves of Camellia sinensis, as green tea is a type of unfermented

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tea that has the highest amount of catechins, especially the ester-type catechins

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(ETC).4,5 The adjacent phenolic dihydroxyl (catechol) and/or trihydroxyl (pyrogallol)

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groups in the structure of ETC are excellent electron donors, effective free radical

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scavengers, and siderophores (metal chelators) in cells, which help protect against

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metal ions-induced cell oxidative stress, in turn inhibiting free radical-induced

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neuronal degeneration and apoptosis around the area damaged by brain injury.6 In

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addition to the antioxidative activity, green tea and ETC have also been reported to

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reduce beta-amyloid (Aβ) induced neurotoxicity.7

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Aβ, which are deposited in nerve cells to form related plaques, are the hallmark of

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Alzheimer’s disease (AD). Recently, researchers have tried to intervene AD at an 3



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earlier stage when AD symptoms are mild but already with the presence of Aβ plaques,

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and to discover “primary prevention” agents.8 To accomplish this, many challenges lie

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ahead. First, the identification and validation of suitable therapeutic targets are needed.

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Accumulated evidences suggest that neutrophil gelatinase-associated lipocalin

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(NGAL) or lipocalin-2 (LCN2) could be an earlier biomarker in multiple

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ageing-related diseases and also a therapeutic target for brain injury.9,10 NGAL is an

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acute phase protein with a primarily bacteriostatic function. It can finely tune the

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intracellular iron content through a catechol complex in a non-transferrin iron delivery

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pathway.11-13 Through tuning the content of iron, NGAL regulates mammalian

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neurogenesis and controls the maintenance of neural stem cells.14 Aβ toxicity to

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astrocytes can also be sensitized by NGAL through silencing tumor necrosis factor

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receptor 2 (TNFR2)-mediated neuroprotection, which suggests that NGAL is a

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modulator of neuro-inflammation at an earlier stage of AD.15,16 Therefore, NGAL is

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expected to be a therapeutic target for primary prevention of ageing-related diseases,

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and development of small-molecule inhibitors or neutralizing antibodies to target

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NGAL could be a related approach for the early intervention of the diseases.

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The main ligands of NGAL are structurally distinct, with a catechol moiety in their

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structures.17 Strong binding between ligands and NGAL occurs in the presence of Fe3+.

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Catechol (CAT) and enterobactin are considered to be two typical ligands of NGAL.

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(-)-Epigallocatechin gallate (EGCG), the major tea polyphenol, is also a ligand of

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NGAL.18 Just like EGCG, most ETC have catechol moiety (moieties) in their 4


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structures. Therefore, we hypothesize that ETC can act as inhibitors of NGAL and

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play roles in prevention of ageing-related diseases.

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A new type of ETC called hydroxycinnamoylated catechins (HCCs, Figure 1) have

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been identified from tea recently. These HCCs including (-)-epigallocatechin

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3-O-ferulate,

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3-O-caffeoate (ECC), were found to be strong acetylcholinesterase inhibitors.19 As the

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most active one in this category, ECC is also the major subject studied in this study.

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Previous studies on ETC suggested that its neuroprotective effect was attributed to its

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antioxidant property. However, ETC is easily oxidized and can’t be stably delivered.

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Therefore, how ETC stably travel to the target area and exert neuroprotective effects

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remains uncertain. Based on the studies on NGAL’s roles in ageing-related diseases,

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we hope to explain the neuroprotective mechanism of a green tea infusion through the

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interaction between ETC and NGAL. As such, fluorescence quenching was used.20

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The strength and types of binding were detected in the presence or absence of Fe3+.

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Binding model was established by molecular docking technique to elucidate both the

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binding sites between ETC and NGAL, and their binding mechanisms as well.21

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Neuroprotective effects of ETC were assayed in an H2O2 or Aβ1-42 injured nerve cells

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model.22 Besides, measurement of NGAL expression in culture medium and nerve

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cells explains the mechanism by which ETC may protect nerve cells through the

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interaction with NGAL.

(-)-epigallocatechin

3-O-p-coumaroate,

5


and

(-)-epicatechin


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Materials and Methods

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Chemicals. NGAL was expressed in BL-21 bacteria as reported.11 FeCl3·6H2O were

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bought from Sinopharm (Shanghai, China, purity > 99%) and catechol (CAT) were

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bought from Shanghai Zhongqin Chemical Reagent Co. Ltd (Shanghai, China).

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Phosphate buffered saline (PBS) was purchased from Solar-bio (Beijing, China).

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Deionized water (Watsons, Guangzhou, China) was used throughout the whole study.

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Small

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Sigma-Aldrich, St Louis, MO, reagent grade). Methanol (HPLC grade), acetonitrile

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(HPLC grade), and formic acid (LC-MS grade) were purchased from Duksan

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(Ansansi, Korea). Thermo-Fisher provided the DMEM/F12 and fetal bovine serum

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(Grand Island, NY, USA). The SH-SY5Y cells were bought from ATCC (New York,

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NY, USA). GL Biochem Ltd (Shanghai, China) provided Aβ1-42 protein. Small

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molecular ligands used in the experiment were isolated from our laboratory and the

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purity of these compounds was ≥ 98% confirmed by HPLC or UPLC analysis.5,19,23

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All materials were stored in refrigerator before use.

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Tea Material and Extraction for UPLC Analysis. The cultivar Longjingchangye (C.

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sinensis var. sinensis) was processed to give six major processing types of tea (SPTT)

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samples (green, yellow, white, oolong, dark, and black teas) with corresponding tea

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manufacturing methods.5 In May 2015, tea leaves were plucked from the tea base of

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Anhui Agricultural University (Hefei, Anhui, China). Ground tea powder 0.5 g was

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extracted in 20 mL 70% aqueous methanol by ultrasonic extraction. The tea extract

molecular

ligands

were

dissolved

in

dimethyl

6


sulfoxide

(DMSO,

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were centrifuged at 10,000 rpm and then run through a 0.22 µm filter to store the

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supernatant.

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Quantification of Catechins in SPTT by UPLC Analysis. The gradient elution of

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mobile phase A was 0.17% aqueous acetic acid and mobile phase B was acetonitrile in

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UPLC (Waters, USA) analysis. Details about UPLC method can be found at our

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previous paper.23 The UPLC method was set as follows (A%): 94%, 0-1.08 min; from

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94% to 85%, 1.1-3.0 min; from 85% to 80%, 3.0-7.5 min; from 80% to 76%, 7.5-10.0

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min; from 76% to 72%, 10.0-11.5 min; form 72% to 40%, 11.5-17.0 min; from 40%

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to 94%, 17.0-19.0 min; keep 94%, 19.0-22.0 min. The injection volume was 1 µL.

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The flow rate was 0.22 mL/min and the wavelength was set at 274 nm. Each sample

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was repeated trice. We calculated the regression equation, correlation coefficient,

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relative standard deviation (RSD, including repeatability and reproducibility), limit of

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detection (LOD), limit of quantification (LOQ), and the recovery ratio as literature.5

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SPTT Solutions Preparation for Fluorescence Quenching. To detect NGAL

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binding ability of a tea infusion in different degree of fermentation, the SPTT

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infusions were diluted 100 times by DMSO as tested ligands. A standard TBS (the tris

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buffered saline, pH = 7.4) buffer was prepared with an experimental volume of 3 mL

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containing 5% DMSO. The experimental volume was added with 10 µL ligands. In

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fluorescence quenching experiment, the final reaction solution contained 0.0034%

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SPTT infusion.

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Complex

(Chelator)

Solutions

Preparation

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ETC-iron

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Quenching. Previous studies on EGCG suggested that NGAL: EGCG: Fe3+ = 1:3:1 is

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the best stoichiometric condition for their binding.18 So, chelator solution was

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prepared as follows: ETC (0.15 mM, 0.5 mL) and FeCl3·6H2O (0.05 mM, 0.5 mL)

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were mixed, vigorously shaken, and diluted with DMSO (no other metal added for the

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titrations of ligand).

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Fluorescence Quenching Experiment of NGAL. To detect NGAL binding ability of

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different ETC in absence or presence of Fe3+, the concentration of NGAL was 100 nM

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except for the blank group. Different concentrations of ligands were added to shake

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and react at room temperature for 12 h. Fluorescence quenching of NGAL was

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measured at three temperatures (277 K, 293 K, and 310 K) on a Cary Eclipse

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fluorescence spectrophotometer (Agilent Inc., Santa Clara, USA). A 10 nm slit

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band-pass was used for excitation and emission with a high-voltage detector. The

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excitation was set at 281 nm and emission spectra at wavelengths of 300 - 500 nm

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were collected.24

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Type of Fluorescence Quenching of NGAL by ETC. Fluorescence quenching

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includes dynamic quenching (collision, diffusion-limited) and static quenching

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(diffusion-independent).25 The quenching can be identified by different trends in the

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excited state lifetime fluctuations with temperature.26 The Stern-Volmer equation was

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used to describe the dynamic fluorescence quenching process of small molecules and

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proteins (Figure 2).27 8


for

Fluorescence

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Binding Constants KA and Dissociation Constant Kd. For a single type of

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fluorescence quenching experiment, the Line-weaver-Burk formula can be used to

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calculate the binding constant KA (Figure 3).26 For chelators, the dissociation constant

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Kd is a necessary parameter for describing the binding system and providing relative

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affinity information between ligands and proteins.

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Binding Forces between NGAL and ETC. Proteins and small molecules usually

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form supramolecular complexes by classical forces: hydrophobic, hydrogen bonds,

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vander Waals, and other non-covalent bond forces. The thermodynamic parameters of

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the reaction are important evidences for binding forces. And the parameters between

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small molecules and proteins can be calculated by the formula (Figure 4).28

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Molecular Modeling Interaction between ETC and NGAL. The crystal structure of

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NGAL protein was obtained from the PDB database (PDB ID CODE: 1L6M).11 The

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structure of protein was optimized using the molecular docking software AutoDock

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4.2 (http://autodock.scripps.edu/).29 ETC chelating ferric iron are considered to be

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ETC-iron complex formed by the coordination bond. ETC-iron complex were

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constructed and minimally optimized using the software GROMACS 3.3.1

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(www.gromacs.org).30

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Ligand and NGAL interaction were investigated by AutoDock 4.2 software.29 The

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default is a semi-flexible docking to maintain the rigid structure of the protein and the

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flexible structure of small molecules during molecular docking process. The target

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binding sites of protein were determined according to the relevant report.11,24 We

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performed multiple sets of dockings and selected the optimal values.

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SH-SY5Y Cell Culture. In 5% CO2, at 37 °C, cells were cultured in DMEM/F12

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with 10% fetal bovine serum, 100 units / mL of penicillin and 100 µg / mL

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streptomycin. After being cultivated for 48 h, the cells were transferred to 6- or

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96-well plates.

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Neuroprotective Effects Assay of ETC.22 The medium containing 10 µM of ETC

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was added and cultured for 24 h. The cells were treated with Aβ1-42 (5 µM) or H2O2

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(200 µM) for 24 h. MTT assay was performed to study the survival rate of cells. The

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medium was replaced and 20 µL 5 mg / mL MTT was added and kept for 4 h. The

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cells were washed with PBS and then added DMSO. Absorbance was measured at 490

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nm using a micro-plate reader. The experiment was repeated thrice. Cell viability was

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expressed as a percentage relative to untreated cells as a control group and positive

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control was set.31

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NGAL Level Detected by Human NGAL Elisa Kits. 32 Cells were cultured in 6-well

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plates, 10 µM EGCG, ECC, vitamin-E, and huperzine-A were added to 2 mL culture

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medium, respectively, and then Aβ1-42 protein was added for further culturing after 24

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h. The control group and model group were set up. After culturing for 24 h, the cell

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culture fluid was collected in a sterile tube and the supernatant was centrifuged. The

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trypsinized cells were suspended in PBS at one million / mL. Cells were repeatedly

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frozen and thawed, and the supernatant was centrifuged.

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NGAL protein levels were quantified using the Human NGAL Elisa Kits (Senbeijia

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Nanjing Biotechnology Co., Ltd, Nanjing, China). All experiments were performed

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according to the steps of manufacturer's protocol.

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Statistical Analysis. All bioassays were repeated thrice and the values are presented

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as the mean ± SD, unless otherwise specified. One way ANOVA with Turkey tests

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was applied to determine significant differences (*P < 0.05, **P < 0.01, ***P
0 can be considered to be

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hydrophobic and ∆H < 0 can be considered to be the electrostatic interaction between

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ions. The binding forces between protein and small molecules are not a single force, 16


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but the role of several forces together according to the complex structure. The binding

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process of a ligand and NGAL is a spontaneous one with an increase in entropy and a

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decrease in free energy. Thus, from the thermodynamic theory, we deduced that the

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interaction between NGAL and ETC is mainly composed of hydrophobic and

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electrostatic interactions. In addition, the process also includes hydrophilic interaction

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and dipole-dipole interactions since ligands have polyphenol structure.

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Binding sites Decided by Molecular Modeling of EIC-NGAL Complex. The

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binding of ligand and NGAL is a dynamic process. Binding sites of NGAL were

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determined from the amino acid residues. At the binding sites, the conformation

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with the lowest energy of ligand was determined to bind NGAL. We deduced the

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binding sites of other ligands at NGAL based on those of CAT.11

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The hydrogen bond was the most important force and the existing distance was less

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than 0.35 nm. CAT formed hydrogen bonds with Arg81, Tyr106, Lys125 and Lys134

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of NGAL protein. EGCG formed hydrogen bonds with Asn39, Glu44, Gln49, Try52,

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Arg72, Ser99, Lys125, Phe133 and Lys134. ECC formed hydrogen bonds with Lys50,

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Pro101, Tyr106, Phe123 and Phe133 (Figure 10). Besides hydrogen bonds,

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hydrophobic contact was also important (Figure 11). We listed the best docking

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results about NGAL-EIC complex comparable to those of CAT (Table 3).

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Lys125 and Lys134 are the key binding sites for NGAL protein to sequester

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iron-siderophore complex.11 As discussed above, CAT and EGCG formed hydrogen

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bonds with Lys125 and Lys134 residues. The structural characteristic of ECC makes

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it impossible to form hydrogen bonds with these two residues, but it can interact with

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them through hydrophobic forces.

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Usually, in search for small molecules with bioactivity, we tend to focus on the

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molecules with smaller molecular weight, which can effectively reduce the steric

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hindrance to bind target protein. Catechins don’t conform to this practice. Compared

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with CAT, the molecular weight of EIC was huge, which resulted in a greater steric

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hindrance during the binding process (torsional free energy = 8.05 kcal/mol). But it

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still bound NGAL strongly because more hydrophilic and hydrophobic interactions

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were obtained during its binding process with NGAL (Figure 11).

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Energy Effect of the Interaction between EIC and NGAL. To study the

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intermolecular interactions, Gibbs free energy is a very important parameter.

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Generally, selection of binding mode with the lowest energy is carried out for

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molecular docking.38 In fluorescence experiment, although the lowest combination of

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energy is the most stable one, but each combination to achieve the lowest energy is

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impossible. Usually, the complexity of binding processes leads to better results from

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molecular docking than those of the fluorescence experiments as docking results are

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got from an ideal condition. However, the free energy obtained by molecular docking

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experiments (Table 3) was higher than the free energy obtained by fluorescence

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quenching experiments (Table 1) in this study. We speculate that the flexibility of

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small molecules is weakened and the steric hindrance is increased due to the artificial 18


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construction of EIC in the process of molecular docking. In addition, coordinate

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covalent bonds are also important factors which affect the docking results.

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Nerve Cells Protection by ETC. In vitro neuroprotective assays showed that all ETC

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(10 µM) exhibited significantly protective effects on SH-SY5Y cells against H2O2 or

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Aβ1-42 injury (Table 4).

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As EGCG was reported to have neuroprotective activity against Aβ1-42-induced cell

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death,39 the neuroprotective effect of ETC on human neuroblastoma SH-SY5Y cells

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was evaluated here using MTT method. As shown in Table 4, ETC have significant

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neuroprotective effect against H2O2- or Aβ1-42 induced cytotoxicity in SH-SY5Y cells.

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Among them, ECC showed more neuroprotective effects than epigallocatechin

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3-O-ferulate and epigallocatechin 3-O-p-coumaroate. The bioassay data confirmed

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that ETC possessed potential neuroprotective effects.

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ETC Can Inhibit NGAL Level in Nerve Cells. Data in Figure 12 illustrate the

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influence of Aβ1-42 without or with addition of EGCG or ECC on NGAL expression in

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nerve cells and cell culture medium. The increase of NGAL level in culture medium

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was observed with Aβ1-42 treatment. In the presence of Aβ1-42, ETC decreased the level

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of NGAL expression in cells significantly (P < 0.05) while the effect of huperzine-A

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and vitamin-E were not obvious.

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Tea can be classified into six major types (green, yellow, white, oolong, dark, and

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black) based on the different processing methods and the degree of fermentation.1,5 In 19



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this paper, we processed SPTT from the same tea leaves, quantified major catechins

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(EGCG, ECG, GCG, EGC, EC, GC), and first tested their NGAL binding activity by

365

fluorescence quenching method. The results showed that NGAL binding activity of

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SPTT was positively correlated with the content of ETC (esp. EGCG and ECG)

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(Figure 5). Subsequent experiment showed that ETC can bind NGAL more tightly

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than NETC (P = 2.8 × 10-8) (Figure 6A). Then, the fluorescence quenching of NGAL

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by different ETC found ECC was the strongest one (Kd = 15.21 ± 8.68 nM, Table 1).

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ECC is better than ECG (Kd = 41.96 ± 17.49, Table 1), which means a caffeoyl

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substitute is better than a galloyl one. Interestingly, caffeic acid itself had no NGAL

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binding activity (Kd > 300 nM, Table 1) and the skeleton compound EC was even

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worse than caffeic acid, which further confirmed the importance of the caffeoyl

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substitute in a catechin skeleton. Further study showed that ETC bound NGAL in a

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static type while EIC in a combined one. The in vitro assay confirmed that ETC can

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prevent nerve cell injury and ECC was the best one (P < 0.0027) (Table 4). The

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inhibition of EGCG or ECC against NGAL level in the nerve cells and the culture

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medium suggested that ETC may protect the nerve cells from injury through the

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interaction with NGAL. Huperzine A and vitamin E cannot inhibit NGAL level,

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suggesting that they play protective roles in different mechanisms. Besides able to

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bind NGAL tightly, ECC is also an excellent siderophore, which is able to chelate

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iron and may act in iron homeostasis in nerve cells. Additionally, ECC was found to

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be an effective acetylcolinesterase inhibitor too.19 Above evidences suggest that ECC

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could be a multi-target agent against ageing-related diseases, and hopefully be

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developed as a primary prevention agent for intervention of related diseases at an

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earlier stage. However, ECC is a minor component in tea. For its quantification and

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further in vitro or in vivo study, synthesis of ECC is highly needed. Neuro-prevention

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effect of tea consumption may be related to catechins association with NGAL and

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iron in following reasons. (1) Tea catechins esp. ETC are effective antioxidants, and

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antioxidants prevention is an effective neuroprevention approach.4 (2) EGCG can be

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detected in its free form in vivo while EC and EGC was conjugaged, methylated or

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even decomposed, and all the catechins were cleared in 12 h.40 Free catechins and iron

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are reactive and hard for delivery. Based on related behavior from the

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NGAL-catechol-iron complex in vivo, tea catechins and iron could also be stably

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delivered to target area through the relative stable NGAL-catechins-iron complex,

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since they all have and use the catechol moiety (moieties) to catch NGAL protein, and

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tea catechins can inhibit the reactive iron through the complex.11,18 However, further

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in vivo experiments are needed to confirm the NGAL-catechins-iron behavior. (3)

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Iron homeostasis is also an important factor to keep nerve cells from iron caused

400

toxicity. As natural iron chelators and effective antioxidants, tea catechins can help to

401

keep iron homeostasis in vivo, which is helpful in the treatment of neurodegenerative

402

disorders.3,18 Green tea drinking can provide ETC and may exert neuroprotection

403

through inhibiting NGAL ability to silence a TNFR2 mediated neuroprotective

404

pathway and thus to sensitize Aβ toxicity to nerve cells, which is also a valuable topic

21



405

for further study.15,16

406

Conclusively, green tea infusion is the most active one to bind the proinflammatory

407

protein NGAL among all the six processing types of teas (P < 0.0017), and the

408

binding activity is positively correlated with the content of ETC. NGAL can bind

409

ETC tightly in a static way while it can bind ETC-iron complex in a combined way.

410

Among different ETC, ECC is the most active one for binding NGAL and protecting

411

nerve cells against H2O2 or Aβ1-42 induced injury. ECC and EGCG can inhibit Ngal

412

level in Aβ1-42 induced nerve cells.

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Author Contributions: W. Zhang and X. Li contribute equally to the paper

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Supporting Information

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Supplementary data can be found in the online version. These data include NGAL

416

protein purification results (Figure S1), three-dimensional fluorescence contour and

417

the corresponding projections spectra of NGAL (Figure S2), validation data for

418

quantification of catechins in different tea (Table S1-3), correlation between catechins

419

content and NGAL fluorescence intensity by Pearson correlation method (Table S4)

420

and the original PDB file for modeling analysis.

421

References

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(1) Yue, Y.; Chu, G. X.; Liu, X. S.; Tang, X.; Wang, W.; Liu, G. J.; Yang, T.; Ling, T.

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J.; Wang, X. G.; Zhang, Z. Z.; Xia, T.; Wan, X. C.; Bao, G. H.

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BMC Plant Biol. 2014, 14, 243.

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Am. Coll. Nutr. 2006, 25, 79–99.

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(3) Mandel, S.; Amit, T.; Reznichenko, L.; Weinreb, O.; Youdim, M. B. H. Green tea

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catechins as brain-permeable, natural iron chelators-antioxidants for the treatment of

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547

Blood and urine levels of tea catechins after ingestion of different amounts of green

548

tea by human volunteers. Cancer Epidemiol. Biomarkers Prev. 1998, 7, 351-354.

549

Note of Acknowledgement

550

The authors declare no competing finacial interest

551

Financial assistances were received with appreciation from the Natural Science

552

Foundation of Anhui Provincial Department of Education KJ2016A217, the National

553

Natural Science Foundation of China 81170654/H0507, Anhui Agricultural

554

University Talents Foundation (YJ2011-06), Anhui Agricultural University graduate

555

student innovation project 2017yjs-24, The Open Fund of State Key Laboratory of

556

Tea Plant Biology and Utilization SKLTOF20150106.

29



Page 30 of 51

557

Figure captions

558

Figure

559

(-)-epigallocatechin gallate (EGCG)

560

Figure 2 The Stern-Volmer equation. F0 and F are the fluorescence intensities of

561

NGAL in the absence and presence of ligands. KSV is dynamic quiescent constant and

562

Kq is diffusion collision quenching rate constant in dynamic fluorescence quenching.

563

The τ0 represents the average life time of the fluorescent molecules when the quencher

564

is absent and the value was taken as 10-8 s.

565

Figure 3 The line-weaver-Burk formula. The least squares curve fitting of these

566

fluorescence data was used to calculate Kd. Analysis process used DynaFit

567

(http://www.biokin.com/dynafit/). And KA was calculated by Kd: KA = 1/Kd.

568

Figure 4 The formula used to calculate the thermodynamic parameters of the reaction

569

between small molecules and proteins. KA is the binding constant for the

570

Line-weaver-Burk formula at different temperatures. R is the gas constant (R = 8.314

571

J·mol-1K-1).

572

Figure 5 UPLC analysis of (-)-epigallocatechin gallate (EGCG), epicatechin-3-gallate

573

(ECG), (-)-epicatechin (EC), (-)-epigallocatechin (EGC), gallocatechin (GC),

574

(-)-gallocatechin-3-gallate (GCG) in six processing types of tea samples (Green,

575

Yellow, White, Oolong, Black and Dark tea, SPTT) and the increase in the relative

576

NGAL fluorescence intensity at 340 nm (Fl 340 nm) with the decrease in amount of

1

Structure

of

hydroxycinnamoylated

30


catechins

(HCC)

and

Page 31 of 51


577

ester-type catechins (ETC, maily EGCG and ECG) in SPTT. Fluorescence test

578

solution contains 100 nM NGAL and 0.0034% tea infusion (T = 293 K, pH = 7.40, λex

579

= 281 nm and λem = 340 nm).

580

Figure 6 Relative fluorescence intensity of NGAL at 340 nm quenched by ETC and

581

ETC-iron complex (T = 293 K, pH = 7.40). (A). Determination of the affinity of

582

different catechins (EGCG, ECG, GCG, EGC, EC, and GC) in complex with NGAL.

583

(B). Determination of the affinity of chelators in complex with NGAL. Chelators are

584

different ligands complexed with Fe3+ including EGCG, ECG, GCG, (-)-epicatechin

585

3-O-caffeoate

586

3-O-p-coumaroate, caffeic acid, and the positive control catechol (CAT).

587

Figure 7 Fluorescence quenching of NGAL by ester-type catechins (ETC) and

588

ETC-iron chelator (EIC) at λex = 281 nm (T = 293 K, pH = 7.40).

589

(A) Effect of EGCG on fluorescence spectra of NGAL. C (EGCG) / (nM): 0, 100, 200,

590

300, 400, 500, 600, 700 and 800, respectively (From spectra 1 to 9).

591

(B) Effect of EGCG-iron chelator on fluorescence spectra of NGAL. C (EGCG-iron

592

chelator) / (nM): 0, 50, 100, 150, 200, 250, 300, 350 and 400, respectively (From

593

spectra 1 to 9).

594

(C) Effect of ECC on fluorescence spectra of NGAL. C (ECC) / (nM): 0, 100, 200,

595

300, 400, 500, 600, 700 and 800, respectively (From spectra 1 to 9).

(ECC),


3-O-ferulate,

31




596

(D) Effect of ECC-iron chelator on fluorescence spectra of NGAL. C (ECC-iron

597

chelator) / (nM): 0, 50, 100, 150, 200, 250, 300, 350 and 400, respectively (From

598

spectra 1 to 9).

599

Figure 8 Stern–Volmer plots for the quenching of NGAL by ETC and EIC at

600

different temperature (λex = 281 nm and λem = 340 nm). (A) EGCG. (B) EGCG-iron

601

chelator. (C) ECC. (D) ECC-iron chelator.

602

Figure 9 Line-weaver-Burk curves for quenching of NGAL with ETC at different

603

temperatures (λex = 281 nm and λem = 340 nm). (A) EGCG. (C) ECC.

604

Figure 10 Ligands formed hydrogen bonds with NGAL protein. (A) Comparison of CAT and EGCG. (B) Comparison of CAT and ECC.

605

606

Figure 11 Ligands formed hydrophobic contact with NGAL protein. (A) CAT. (B)

607

EGCG. (C) ECC The red half circle represents the amino acid residue that interacts

608

with the ligands. The green short line represents the hydrogen bond, and the number

609

indicates the length of the hydrogen bond.

610

Figure 12 Effect of ETC on NGAL protein expression following Aβ1-42 treatment.

611

#

P < 0.05 vs. control. * P < 0.05, ** P < 0.01 vs. model group

32


Page 32 of 51

Page 33 of 51


Table 1 Calculated Dissociation Constant (Kd), Binding Constants (KA) and Free-energy Change (∆G) of Chelators in Complex with NGAL (293K)

a

b

Ligands

Kd (nM)

10-7KA (L.mol-1)

△G (KJ.mol-1)

epigallocatechin3-O-ferulate

>300a

-b

-

epigallocatechin3-O-p-coumaroate

>300 a

-

-

caffeic acid

>300 a

-

-

Catechol (CAT)

1.91 ± 0.95

52.62

- 48.92

(-)-epicatechin 3-O-caffeoate (ECC)

15.21 ± 8.68

6.67

- 43.89


38.12 ± 10.85

2.63

- 41.62

epicatechin-3-gallate (ECG)

41.96 ± 17.49

2.38

-41.38

(-)-gallocatechin-3-gallate (GCG)

32.06 ± 9.79

3.13

-42.04

The minimum quenching value is not available at a concentration of 0 nM to 400 nM.

-No value

33



Page 34 of 51

Table 2 Calculated Quenching Constants (Ksv), Diffusion Collision Quenching Rate Constant (Kq), Binding Constants (KA) and Thermodynamic Parameters of Interacting ETC with NGAL.

T (K)

EGCG

ECC

a

Ra2

Equation

10-6Ksv (L.mol-1)

10-14Kq (L.mol-1)

b

Rb2

10-6KA

∆H

∆G

∆S

(L.mol-1)

(KJ.mol-1)

(KJ.mol-1)

(J.(mol.K)-1)

277

0.9908

F0 / F = 0.7482+4.1 × 106[Q]

3.95 ± 0.11

3.95 ± 0.11

0.9915

2.64

293

0.9926

F0 / F = 0.6785+3.7 × 106[Q]

3.51 ± 0.15

3.51 ± 0.15

0.9932

310

0.9928

F0 / F = 0.7172+3.2 × 106[Q]

3.03 ± 0.13

3.03 ± 0.13

277

0.9945

F0 / F = 0.99+3.9 × 106[Q]

3.93 ± 0.01

293

0.9911

F0 / F = 0.9+3.5×106[Q]

310

0.9933

F0 / F = 0.98+2.9×106[Q]

-34.05

79.13

1.98

-35.32

79.13

0.9919

1.51

-36.67

79.15

3.93 ± 0.01

0.9973

3.81

- 34.92

83.04

3.41 ± 0.05

3.41 ± 0.05

0.9953

2.89

- 36.25

83.96

2.87 ± 0.02

2.87 ± 0.02

0.998

2.2

- 37.68

83.53

34


-12.12

- 11.78

Page 35 of 51


a

Ra2 is the coefficient of decision in Stern-Volmer equation fitting.

b

Rb2 is the coefficient of decision in Line-weaver-Burk formula

35



Page 36 of 51

Table 3 Best Docking Results About the Chelator Combine with NGAL (298K)a.

∆G

Estimated Free

Estimated Inhibition

Final Intermolecular

Final Total

Torsional

Energy of Binding

Constant Ki

Energy

Internal Energy

Free Energy

CAT

-10.76 kcal/mol

12.99 nM

-10.76 kcal/mol

0.00 kcal/mol

0.00 kcal/mol

- 45.14

EGCG

-9.13 kcal/mol

202.69 nM

-17.18 kcal/mol

-12.81 kcal/mol

8.05 kcal/mol

-38.18

ECC

-9.94 kcal/mol

51.35 nM

-18.00 kcal/mol

-6.25 kcal/mol

8.05 kcal/mol

- 41.74

a

Cluster analysis of 100 docking runs and select the optimal value.

36


(KJ.mol-1)

Page 37 of 51


Table 4 Neuroprotective Effects of ETC Against Hydrogen Peroxide (H2O2 200 µM) and Amyloid-β (Aβ1-42 5 µM) Induced Human Neuroblastoma SH-SY5Y Cells Cytotoxicity.a

Aβ1-42 (5 µM)

H2O2 (200 µM)

Cell survival rate (%

Group

P value

Cell survival rate (%

P value

of control)

of control)

Control

100 ± 1.14

100 ± 1.12

Model

70.65 ± 2.64 ###

< 0.001

66.48 ± 6.96 ###

< 0.001

vitamin-E b

84.15 ± 2.68 **

0.002

80.19 ± 6.16 *

0.023

huperzine-A b

97.04 ± 1.37 ***

< 0.001

89.36 ± 6.96 **

0.002

(-)-epicatechin 3-O-caffeoate (ECC)

90.52 ± 1.57 ***

< 0.001

84.55 ± 6.26 **

0.007


87.82 ± 2.63 ***

< 0.001

82.61 ± 5.99 *

0.013

epigallocatechin3-O-ferulate

86.32 ± 2.85 **

0.001

80.66 ± 5.32 *

0.012

epigallocatechin3-O-p-coumaroate

85.63 ± 3.42 **

0.003

80.84 ± 5.93 *

0.018

a

All the compounds were tested at 10 µM.

b

Positive control.

###

P < 0.001 vs. control. Data (cell

viability (%) assessed using MTT assay) are expressed as the mean ± SEM based on three independent experiments. * P < 0.05,

**

P < 0.01,

***

P < 0.001 vs. model group. One-way analysis of variance was

used, n = 3. 37



Figure 1

38


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Figure 2

39



Figure 3

40


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Figure 4

41



Figure 5

42


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Figure 6

43



Figure 7

44


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Figure 8

45



Figure 9

46


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Figure 10

47



Figure 11

48


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49



Figure 12

50


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Graphic for table of contents

51


Interaction between Ester-Type Tea Catechins and Neutrophil

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