Synergistic Application of Black Tea Extracts and Lactic Acid Bacteria

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Synergistic application of black tea extracts and lactic acid bacteria in protecting human colonocytes against oxidative damage Nagendra P Shah, and Danyue Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05742 • Publication Date (Web): 21 Jan 2016 Downloaded from http://pubs.acs.org on February 8, 2016

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Synergistic application of black tea extracts and lactic acid bacteria in protecting

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human colonocytes against oxidative damage

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Danyue Zhao and Nagendra P. Shah*

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Food and Nutritional Science - School of Biological Sciences, The University of Hong

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Kong, Pokfulam Road, Hong Kong.

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*Corresponding

author: Nagendra P. Shah.

Tel: +852 2299 0836. Fax: +852 2299 9914. Email: [email protected].

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ABSTRACT

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In viewing of the potential of lactic acid bacterial (LAB) in enhancing antioxidant

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activity of food products, this work explored the effectiveness of LAB fermented black

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tea samples in alleviating H2O2–induced oxidative stress in human colonocytes. The

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antioxidant capacity of tea samples was evaluated in terms of cyto-protectiveness,

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mitochondria membrane potential (Δψm)-stabilizing activity, ROS-inhibitory effect

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and antioxidant enzyme-modulating activity. The effect on oxidative DNA damage and

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repair was studied in CCD 841 by comet assay. Results showed that the protective

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effect of tea pre-treatment was more pronounced in normal cells (CCD 841) than in

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carcinomas (Caco-2), and fermented samples were invariably more effective. Higher

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cell viability and Δψm were maintained and ROS production was markedly inhibited

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with tea pre-treatment. The fermented tea samples also remarkably stimulated DNA

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repair, resulting in less strand breaks and oxidative lesions. Our study implied that LAB

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fermentation may be an efficient way to enhance the antioxidative effectiveness of

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black tea flavonoid-enriched foods.

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Key words: Black tea; Flavonoids; LAB fermentation; Oxidative stress; Colonocytes

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INTRODUCTION

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Mounting scientific evidence implicates that the etiology of many human degenerative

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diseases and the progression of aging are highly associated with an imbalanced redox

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status induced by extra reactive oxygen species (ROS).1-3 Although a moderate amount

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of ROS is essential for maintaining cellular biological activities, excess ROS lead to

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oxidative damage to key functional elements including cellular lipids, DNA and

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proteins.4 The innate antioxidant defense systems, consisting of enzymatic and

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non-enzymatic components, play an essential part in combating ROS-induced oxidative

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stress in cells.3 In face of excessive ROS, however, extraneous antioxidants should be

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consumed to maintain a balanced oxidative state in cells.

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Oxidatively-stressed cells display several signs preceding the advancement of DNA

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damage, such as elevation in ROS level, perturbation in the mitochondria and

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accumulation of lipid peroxidation products.5,

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ROS-mediated oxidative attack and even a moderate level of cellular oxidative stress

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can cause subtle structural variations or mutations in the double helix, affecting

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protein bindings and the subsequent gene replication and transcription process.

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Therefore, searching for natural xenobiotics that are able to prevent oxidative DNA

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damage via multiple pathways is of great importance to human body.

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Much attention has been placed on natural phytochemicals, especially flavonoids, due

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to their potent antioxidant capacity and ubiquity in dietary food products, with a total

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intake estimated to be up to ca. 200 mg/day.7, 8 Black tea accounts for approximately

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80% of worldwide tea consumption, exhibiting a wide spectrum of biological activities,

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DNA substances are vulnerable to

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mainly attributed to its flavonoid composition.9 The flavonoid profile of BLACK TEA is

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much altered, compared with that of green tea, as a result of oxidation and

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polymerization during fermentation process. The modes of action of flavonoids against

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oxidative stress have been postulated to be free radical scavenging activity, transition

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metal chelating ability, antioxidant system and gene expression modulating activity.1, 10

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Tea flavonoids (TFLs) have also been shown to regulate phase II detoxifying and

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antioxidant enzymes via Nrf2 signaling pathway in response to oxidative stress.11

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Fermentation with selected lactic acid bacteria (LAB) has been found to enhance the

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overall functional properties of foods beyond original nutritional values, particularly

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on antioxidant effectiveness.12, 13 A comprehensive study on the biotransformation of

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phenolics by LAB was conducted in our lab and higher absorptivity of TFLs from

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fermented tea samples was detected in Caco-2 cell monolayers.14 This led us to

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investigate whether LAB-fermented black tea extracts (BTs) are more efficient in

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relieving cellular oxidative stress compared with the non-fermented ones. In addition,

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accumulating evidence indicates that TFLs may induce different oxidative

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environments in tumors versus normal cells.15 Thus, we sought to explore the

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influence of LAB-fermented BT or tea phenolic mixture (TPM) on H2O2–induced

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oxidative stress in both the normal CCD 841 and the cancerous Caco-2 human colon

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epithelial cells. Considering the fact that TFLs can act as antioxidants and pro-oxidants

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depending on experimental conditions,16 catalase was supplemented to some

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incubations in order to clarify the pro-oxidant potential of LAB fermented tea samples

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in modulating redox status and the differential results obtained between the normal 4



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and cancer colonocytes.

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MATERIALS AND METHODS

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Microorganisms and culture conditions. Stock of L. brevis NPS-QW 145 (L. brevis

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145, isolated from kimchi in our lab17) and L. plantarum ASCC 292 (L. plantarum 292,

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Australian Starter Culture Collection, Werribee, Australia) was stored at −80°C. For

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activation, 10 mL aliquots of sterile MRS broth were inoculated with 2% (v/v) of each

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organism and incubated at 37°C for 18 h. After the second transfer in MRS, the

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activated organisms were used for fermentation of tea samples.

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Preparation of black tea extract and tea phenolic mixture. Black tea (Dianhong,

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Yunnan Province, PRC) was extracted as per the method of Zhao and Shah.18

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Freeze-dried black tea powder was dissolved in MRS broth containing 0.2% DMSO at 9

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mg/mL to produce BT.

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Tea phenolic mixture consisted of eight phenolic compounds (HPLC-grade standards)

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most abundant in BT, namely gallic acid (GA), epigallocatechin gallate (EGCG) and

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quercetin-3-O-rutinoside (QR) (Sigma Chemicals St. Louis, MO, U.S.A.), epicatechin

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gallate (ECG), epicatechin (EC) and epigallocatechin (EGC) (Zelang Medical Technology,

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Nanjing, China), quercetin-3-O-glucoside (QG) and kaempferol-3-O-glucoside (KG)

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(Herbpurify Co., Ltd, Chengdu, China), each at the same concentration in BT as

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determined by HPLC13. Specifically, stock solutions of GA, EGCG, EGC, ECG, EC, QG, KG

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and QR, prepared in DMSO, were diluted 500 times in MRS to concentration levels at

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0.300, 0.064, 0.032, 0.080, 0.040, 0.072, 0.032 and 0.025 mg/mL, respectively.

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Individual phenolic stock was stored at -30°C in dark and used within one month. 5


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Preparation of fermented black tea samples. MRS solutions of TPM and BT were

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inoculated with L. plantarum 292 and L. brevis 145, each at 2% (v/v) and incubated at

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37°С for 48 h. Non-inoculated BT and TPM were also incubated for 48 h under the

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aforementioned conditions for comparison. Fermented TPM or fermented BT was

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referred to as fTPM or fBT, respectively. MRS and fermented MRS (fMRS) were used as

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controls in some assays. Upon completion of fermentation, samples were diluted

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two-fold with 30% ethanol and centrifuged at 10,000 × g for 10 min. The supernatants

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were alkalized to pH 6.6 by dropwise addition of 2 M NaOH prior to lyophilization. The

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lyophilized powder was re-dissolved in cell culture media without fetal bovine serum

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(FBS) (Gibco BRL, Grand Island, NY, USA) and filter-sterilized. Working solutions of

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black tea samples were diluted twice (for CCD 841) or thrice (for Caco-2) with cell

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culture media containing 10% FBS immediately before experiment. The final

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concentration of major tea phenolic compounds in cell culture media was determined

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using HPLC13 and results are presented in Table 1.

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Cell culture. The normal human epithelial colon cell line CCD 841 (ATCC CRL-1790)

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was kindly provided by Prof. Tao Qian from the Cancer Epigenetics Laboratory in the

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Chinese University of Hong Kong, and cultured in EMEM (ATCC, Rockville, MD, USA)

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supplemented with 10% FBS and 1% penicillin-streptomycin (10,000 U/mL), (Gibco

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BRL). Cells were used between 8 and 20 passages. The human colorectal

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adenocarcinoma Caco-2 (ATCC HTB-37) was cultured in DMEM (Gibco BRL)

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supplemented with 10% FBS and 1% penicillin-streptomycin (10,000 U/mL). Cells

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were used between 38 and 52 passages. All cells were incubated at 37°C in a 6



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humidified incubator (US Air Flow, NuAire, USA) containing 5%. Cell viability was

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measured using MTT assay. Dilutions of black tea samples were made based on the

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viabilities of two cell lines following 24 h incubation in various samples. The average

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survival rates for CCD 841 and Caco-2 were 96% and 84%, respectively.

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Determination of cell viability following H2O2 oxidation. Cells were seeded in

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96-well microplates (Nunc, Thermo Scientific, Suzhou, China) at a density of 1 ×

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105/well and incubated for 24 h at 37°C. Cells were treated with tea samples for 2 h

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(short term) or 12 h (long term). Subsequently, cells were washed with PBS and

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treated with H2O2 (400 μM for CCD 841 and 500 μM for Caco-2 in pure culture media)

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for 90 min and cell viability was measured using the MTT assay. Under the

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experimental conditions, concentrations of H2O2 in various incubation media were

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determined by FOX-1 assay .19

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Determination of intracellular ROS level. Modulation of ROS level by tea samples in

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H2O2-stressed cells was measured by the dichlorofluorescin (DCF) assay according to

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Bellion et al. 20 with modifications. Briefly, cells were seeded in 96-well microplates at

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1 × 105/well and incubated for 24 h. Cells were incubated with tea samples for 2 h or

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12 h as aforementioned. Subsequently, cells were washed with PBS, treated with 2’,

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7’-dichlorofluorescein diacetate (DCFH-DA; final concentration at 50 μM in PBS) for 30

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min at 37°C. Cells were then washed and incubated with H2O2 (300 μM for CCD 841

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and 400 μM for Caco-2) in pure media for 90 min. In some assays, cells were treated

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with tea samples for 2 h alone, either in the presence of absence of catalase (50 U/mL)

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and then forwarded to fluorescence (FL) measurement. Control wells were not 7


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incubated with tea samples but H2O2 only. The FL, resulting from oxidation of the

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non-fluorescent probe to fluorescent DCF molecules, was recorded at 0, 30, 60 and 90

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min immediately after the addition of H2O2 using a VICTOR™ X4 Multilabel Plate

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Reader (PerkinElmer, Norwalk, CT, USA) (ex 485 nm; em 530 nm). All treatments and

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fluorimetric measurement were performed under dim light at 37°C. Results were

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expressed as relative fluorescence increase (FI%) compared with the control, using the

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following equations:

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FI = (FLt=90 − FLt=0) and FI% = 100×(FIsmpl/ FIctrl);

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where FLt=0 is the initial FL; FLt=90 is the final FL following 90 min incubation; FIsmpl/

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FIctrl is the fluorescence increase of sample/control.

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Determination of mitochondrial membrane potential (Δψm). Changes in

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mitochondrial membrane potential were measured using a potential-sensitive dye,

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JC-1

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(eBio-science Inc., San Diego, CA, USA) according to Polla et al.,21 with modifications.

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Briefly, cells were seeded in 21-cm2 culture dishes (Corning Costar, Corning, NY, USA)

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at 1 × 106/dish and incubated for 24 h. Cells were either pre-incubated with tea

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samples or with culture medium for 2 h before exposed to desired concentrations of

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H2O2 for 90 min. High concentration of H2O2 as positive control (500 μM for CCD 841;

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600 μM for Caco-2) was used to depolarize mitochondrial membrane. In some assays,

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catalase solution (50 U/mL) was added to each culture dish 5 min prior to the addition

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of treatment samples. After treatment, 1 × 106 cells were harvested and incubated with

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500 μL of JC-1 working solution (10 μg/mL) for 15 minutes at 37°C in the dark.

(5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-benzimidazolylcarbo-cyanine

iodide)

8



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Stained cells were placed on ice and analyzed within 1 hour in a BD FACSAria III flow

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cytometer (Becton Dickinson, San Jose, CA, USA) with an argon laser (488 nm for

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excitation). Peak FL emission signals were measured at 525 nm for the green

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monomers and 590 nm for the red JC-aggregates. 10,000 cells were analyzed per

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treatment.

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Preparation of cell lysate. CCD 841 and Caco-2 cells were seeded in 21-cm2 Corning

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dishes at 2 × 106/dish and 1 × 106/dish, respectively, and incubated for 24 h. Upon

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completion of tea samples and H2O2 treatment, the cells were washed twice with PBS

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and scraped off the flask/well into ice-cold 20 mM Tris-HCl buffer (pH 7.4). Following

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one cycle of freeze-thawing, cell samples were homogenized on ice for 2 min using a

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Branson Sonifier 250 (Chicago, IL, USA) at out control 4 and 40% duty cycle. The

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lysates were centrifuged at 14000 × g for 15 min at 4 °C and supernatants were stored

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at -80°C until assaying of antioxidant enzyme activities. Protein concentrations of cell

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lysates were determined by Bradford method and lysates were adjusted to the

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identical protein level of 106 cells with Tris-HCl buffer.

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Determination of antioxidant enzyme activities

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Catalase Assay. Catalase activity was determined spectrophotometrically at 25°C by

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monitoring the extinction of H2O2 at 240 nm. The H2O2 substrate (50 mM) was freshly

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prepared each assay. Cellular catalase activity was calculated as the reduction of H2O2

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(μM /μg protein) per min. Non-enzymatic H2O2 decomposition (baseline) was

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subtracted from each determination.

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Glutathione peroxidase (GPx) Assay. GPx activity was measured using the cellular 9


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GPx activity assay kit (Abcam, Cambridge, UK). One unit of GPx activity is defined as

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the amount of cell lysate required to oxidize 1 μM NADPH per min.

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Determination of H2O2-induced DNA damage and repair by comet assay. The

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alkaline comet assay was performed to assess oxidative DNA damage and repair

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capacity in CCD 841 cells, with or without black tea samples treatment. Briefly, cells

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were cultured in 21-cm2 Corning dishes at 1 × 106/dish for 24 h. On the day of the

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experiment, cells in the experimental groups were treated with tea samples for 2 h as

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aforementioned before exposing to 100 μM H2O2 in pure medium on ice for 30 min.

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Cells receiving no treatment or direct H2O2 treatment were used as the negative

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control or positive control, respectively. The cells were re-suspended in ice-cold PBS at

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ca. 1 × 105 cells/mL. Cellular repair assay and oxidized purine detection was

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performed according to the modified protocol of Silva et al.22 Following H2O2 exposure,

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cells were allowed to recover in EMEM containing 10% FBS for another 30 min at 37°C

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and then harvested for the comet assay. Occurrence of oxidized lesions (OLs) was

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detected by further digesting the nucleoids with formamidopyrimidine DNA

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glycosylase (Fpg, New England Biolabs, Herts, UK), which recognizes oxidized purines

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and converts such lesions to DNA breaks.23 Cells after lysis were treated with Fpg (ca.

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8 U/mL) in enzyme buffer supplemented with 100 μg/ml purified BSA at 37°C for 30

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minutes. The content of oxidized purines (Fpg-sensitive sites) was determined by

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subtracting the amount of strand breaks (SBs, samples incubated with buffer alone) to

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the total amount of breaks obtained after digestion. All work was carried out under

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dim light to prevent artificial DNA damage. The alkaline comet assay was performed 10



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using the Trevigen's Comet Assay™ kit (Trevigen Inc., Gaithersburg MD, USA)

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following the manufacturer's instructions. The temperature during electrophoresis

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(1.0 V/cm; 300 mA; 30 min) was kept constant at 4 °C. Scores were collected from 150

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cells

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software Comet Assay IV (Perceptive Instruments, Haverhill, Suffolk, UK). DNA

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damage (DD) was expressed as %DNA in tail. Total DNA damage was counted as the

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sum of SBs and OLs. Cellular DNA repair capacity (RC) was calculated using the

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following equation:

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RC = 100×(DDt=0−DD t=30)/DD t=0;

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where DDt=0 represents DNA damage before the recovery period; DDt=30 represents

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DNA damage after 30 min’s recovery.

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Statistical analyses. For each assay, at least three independent tea samples were

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prepared and all assays were carried out in triplicate with each set of experiments.

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The data was subjected to one-way analysis of variance (ANOVA) (Tukey and

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Games-Howell tests) by SPSS 20.0 (IBM SPSS Statistics, IBM Corp, Somers, NY, USA).

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Results with p < 0.05, p < 0.01 or p < 0.001 were considered statistically significant.

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Results

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Effects of black tea samples on H2O2-induced cytotoxicity

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In general, H2O2 assault led to cell death in a concentration-dependent manner

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(Supporting Information). Caco-2 cells appeared to be less sensitive to H2O2 inflict than

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CCD 841 since they displayed at least 10% higher cell survival at respective H2O2

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concentration level except for 100 μM, where a cell viability at 100.2% or 100.4% was

per

treatment

(50

cells/slide/culture,

in

triplicate)

using

the

11


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observed in CCD or Caco-2 cells, respectively. This can be a sign of subtoxic effect that

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activated metabolic activity in the mitochondria.24 For cells subjected to 600 μM H2O2,

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12 h incubation resulted in almost 100% loss of viability for CCD, significantly higher

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(p < 0.01) than that of Caco-2.

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To assess the cyto-protective effect of tea samples, cells were pre-incubated with tea

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samples for 2 h or 12 h before oxidant stimulation. In this experiment, high

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concentrations of H2O2 (400 μM for CCD 841 and 500 μM for Caco-2) were used since

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preliminary data showed no significant improvement on cell viability (p > 0.05)

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when >80% cells were alive after direct H2O2 treatment. Results in Figure 1 clearly

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show that 2 h pre-treatment with all tea samples significantly attenuated the toxicity

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of H2O2 in both cell lines (p < 0.01). For CCD 841, 12 h incubation with all tea samples

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exhibited extended protection against oxidative damage, lowering death rates to

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Synergistic Application of Black Tea Extracts and Lactic Acid Bacteria

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