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Preventive Efficiency of Green Tea and Its Components on Non-alcoholic Fatty Liver Disease Jie Zhou, Chi-Tang Ho, Piaopiao Long, Qilu Meng, Liang Zhang, and Xiaochun Wan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05032 • Publication Date (Web): 20 Mar 2019 Downloaded from http://pubs.acs.org on March 20, 2019

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Journal of Agricultural and Food Chemistry

Preventive Efficiency of Green Tea and Its Components on Non-alcoholic Fatty Liver Disease

Jie Zhou†,‡, Chi-Tang Ho‡,§, Piaopiao Long†,‡, Qilu Meng†,‡, Liang Zhang

†,‡*

and

Xiaochun Wan †,‡* †State

Key Laboratory of Tea Plant Biology and Utilization and ‡International Joint

Laboratory on Tea Chemistry and Health Effects of Ministry of Education, Anhui Agricultural University, 130 West Changjiang Road, Hefei 230036, China. §Department

of Food Science, Rutgers University, New Brunswick, NJ, USA.

*Corresponding

author:

Phone/Fax: +86-551-65786765. Email: [email protected] (L.Z.); [email protected] (X.W.).

ACS Paragon Plus Environment


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ABSTRACT

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Non-alcoholic fatty liver disease (NAFLD) is a typical chronic liver disease highly

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correlated with metabolic syndrome. Growing prevalence of NAFLD is supposed to

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be linked with the unhealthy lifestyle, especially high-calorie diet and lacking enough

5

exercise. Currently, there is no validated pharmacological therapy for NAFLD except

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for weight reduction. However, many dietary strategies had preventive effects on the

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development of liver steatosis or its progression. As one of the most common

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beverages, green tea contains abundant bioactive compounds possessing antioxidant,

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lipid-lowering and anti-inflammatory effects, as well as improving insulin resistance

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and gut dysbiosis that can alleviate the risk of NAFLD. Hence, in this review, we

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summarized the studies of green tea and its components on NAFLD from animal

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experiments and human interventions, and discussed the potential mechanisms.

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Available evidences suggested that tea consumption is promising to prevent NAFLD,

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and further mechanism and clinic study need investigate.

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KEYWORDS: NAFLD; NASH; green tea; (-)-epigallocatechin-3-gallate; preventive

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efficiency

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INTRODUCTION

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NAFLD is commonly caused by the significant deposition of triglycerides (TG) in the

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liver without history of alcohol addiction and/or other inducements such as

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steatogenic medication and genetic disorders.1, 2 Pathologically, NAFLD can be

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classified into two distinct conditions: fatty liver (non-inflammatory steatosis) and

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nonalcoholic steatohepatitis (NASH). The latter contains a wide range from fibrosis to

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cirrhosis, and finally liver failure and carcinoma.3

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NAFLD is thought to be a manifestation of metabolic syndrome (MetS) in the

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liver, frequently associated with metabolic comorbidities, such as obesity (51.34%),

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type Ⅱ diabetes (22.51%), hyperlipidemia (69.16%) and hypertension (39.34%).4,

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Clear evidence has demonstrated that cardiovascular disease is the main cause of

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death among NAFLD patients.6 With the changing of dietary patterns in recent years,

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the NAFLD has been raised speedily worldwide, and accompanied the increasing of

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MetS. It was reported that the incidence of NAFLD is about 17-46% of Western

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country adults, and 11.8-43.91% of Chinese adults.3, 7, 8

5

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Presently, there is lack of first-line effective pharmacological therapy for

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NAFLD, the alternative medications, including vitamin E, cysteamine, metformin,

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pioglitazone and pentoxifylline produce borderline efficacy in improving liver

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symptoms because of potential side effects and toxicities. The most useful treatment

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strategy for NAFLD is 10% of weight reduction by dietary restriction and regular

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exercise, which is effective to reverse NAFLD for most patients, but it is hard to

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maintain a long-term success rate in practice.1, 9



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The pathogenesis of NAFLD has not yet been fully elucidated, and the treatment

41

of NAFLD also has not been completely established, except for changing the lifestyle

42

by diet and exercise.10 Currently, NAFLD therapies are limited, clinical practice

43

guidelines for managing of NAFLD suggest that antioxidants are beneficial for the

44

improvement of steatosis, inflammation, ballooning and other symptoms of NAFLD.

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Thus, much attention has been focused on foods and beverages that are naturally rich

46

in antioxidant compounds, to provide an alternative way for NAFLD prevention and

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treatment.11 Tea is rich in bioactive components, and its regular intake is associated

48

with reducing the morbidity of a constellation of metabolic disorders.

49

According to the manufacture process, tea (Camellia sinensis (L.) O. Kuntze)

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products can be classified into six types: green, black, yellow, white, dark and oolong

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tea. The processing technology endows different taste, aroma, color and

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health-promoting effects of tea infusion, which might be due to different chemical

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profiles. Green tea is accounts for 20% of world tea consumption. Green tea is made

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of fresh tea leaves by fixation and drying to deactivate the endogenous enzymes,

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which preserves the maximum of original secondary metabolites. Tea contains high

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content of polyphenols, which account for 18-36% of the weight of the dried leaves.

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As the richest catechins in green tea, (-)-epigallocatechin-3-gallate (EGCG) accounts

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about 50-75% of the total catechins. Tea also possesses minor levels of flavones and

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their glycosides, as well as purine alkaloids. In addition, it possesses about 2%

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L-theanine, a tea-specific amino acid, of the dried weight of leaves. Other five types


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of tea are prepared by partially or fully enzyme-catalyzed oxidation and

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polymerization of catechins, dark tea even involves in microbial fermentation.12-15

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The biological activities of these teas and their components have been widely

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studied in the past 30 years with respect to prevent MetS related diseases and cancer,

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as well as other health benefits. Most of the health-promotion activities were

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established through green tea as raw materials, and to its major tea polyphenol, EGCG.

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It has been regarded as an exemplary antioxidant in vitro and in vivo, especially under

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conditions of increased oxidative stress caused by smoking, chemical carcinogens and

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aging.16 Cell culture studies often use high dose of EGCG, for instance, the significant

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effects of anticancer cell growth require 10-100 μM EGCG for different cancer cells.

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However, the dose used for cell culture study is hard to be extrapolated in human and

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animal trails. For example, even if orally treated with high pharmacological dosage,

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peak plasma concentration of EGCG only attained below 10 μM in mice and

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humans.17, 18

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So, we emphasize on the published research about the benefits of green tea and

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its components based on in vivo studies. Emerging evidence from animal and clinic

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studies support the concept that tea and tea polyphenols may have the potential to

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prevent NAFLD. The aims of this review are to summary the preventive efficacy of

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green tea and its components on NAFLD, and discuss the proposed mechanisms.

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MECHANISMS AND PATHOGENESIS OF NAFLD

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Lifestyle: Dietary and Exercise



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Numerous epidemiological evidence suggests that unhealthy lifestyle acts a vital role

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in the progress of NAFLD. The key elements of healthy lifestyle are rational nutrition

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and regular exercise.19 High-calorie diet, excessive saturated fats and high-fructose

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corn syrup (HFCS) intake may cause nutrition imbalance and induce the NAFLD.20

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Therefore, rational dietary pattern is indispensable for NAFLD treatment by energy

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restriction, optimizing macronutrient and micronutrient composition and decreasing

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alcohol intake.3,19 Deficiencies in some micronutrients such as choline and methionine

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may boost NAFLD development.21 In addition, gradually increasing in aerobic

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activity and resistance training, are important part of NAFLD lifestyle

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modification.22,23 Based on the previously published practice guidelines and literature,

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lifestyle intervention can decrease body weight, hepatic lipids accumulation and

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histological severity, improve hepatic function and aminotransferases along with

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steatosis, plasma lipid and glucose, as well as NAFLD activity score (NAS).2, 24-27

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Genetic Predisposition

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Several genes of predisposing to NAFLD have been identified, but only few of them

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have been robustly validated. Through genome-wide association studies, the

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best-characterized genetic association is PNPLA3, which has been confirmed by

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multiple cohorts and ethnicities.7 Furthermore, it was also reported that TM6SF2 gene

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was another disease modifier.28 PNPLA3 and TM6SF2 are both associated with the

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output of very low-density lipoprotein (VLDL). Higher content of liver fat and NASH

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risk were found in people carrying of the PNPLA3 I148M and TM6SF2 E167K

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variants.3, 9


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Pathogenesis of NAFLD

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A better understanding the etiology of NAFLD is very vital for early intervention,

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drug developments, and mitigation of NAFLD by lifestyle modification. In 1998, Day

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and James proposed a “two-hit” theory, which has become the extensively accepted

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pathogenesis of NAFLD.29 It considered that the first hit causes hepatic steatosis by

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lipid deposition in hepatocytes, and the second hit causes NASH and even worse by

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progressed inflammation and fibrosis.1

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The progression from normal healthy liver to hepatic steatosis is tightly

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associated with the occurrence of obesity and insulin resistance (IR). Hepatic steatosis

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occurs when the TG accumulation exceeds elimination ability in the liver. Hence, this

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can be led by various metabolic disturbances. First, excessive dietary fat intake or

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lipolysis from adipose tissue increase the delivery and uptake of fatty acids into

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hepatocytes. Second, enhanced hepatic synthesis of TG and de novo lipogenesis

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(DNL) also produce more TG in liver. Third, impaired hepatic mitochondrial

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β-oxidation reduces the lipid clearance. At last, inadequate production and secretion

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of VLDL impede the transportation of TG from liver to blood circulation.30, 31 The

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above-mentioned factors may induce the hepatic steatosis, but which could be

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potentially reversible without permanent hepatic injury.

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In addition, there is infrequent but lethal ‘hit’, which is hepatic steatosis

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accompanied by inflammation in 5% of individuals. Under this progression, oxidative

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stress, lipid peroxidation, cell death, pro-inflammatory cytokine-mediated hepatocytes

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injury, ischaemia-reperfusion injury, and hyperinsulinemia are pivotal to the



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incidence of NASH. Once chronic inflammation has started, there would be rapidly

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developed to liver fibrosis and cirrhosis.31-33

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NAFLD diagnosis based on two types of assessment, the first is non-invasive

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assessment by medical imaging, and the other one is serum biochemical

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measurements, including alanine and aspartate aminotransferase (ALT and AST),

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γ-GT, hyaluronic acid, laminin, collagen-Ⅳ and procollagen-Ⅲ-peptide, to obtain a

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comprehensive profile and make a preliminary evaluation. So far, no non-invasive

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indexes or markers were proposed for NAFLD diagnosis. The second one is hepatic

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histological biopsy, which is recognized as a golden standard for diagnosing the

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presence and severity of NAFLD by NAS.34-36

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POTENTIAL PREVENTIVE MECHANISMS OF Green TEA AND ITS

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COMPONENTS ON NAFLD

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Studies in Animal models

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Dozens of studies have revealed that tea and its components ameliorated the

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symptoms of NAFLD in different rodent models. Among these studies, green tea

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extracts and their components were commonly used, other tea types also had some

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reports, such as fermented green tea extract,37 kombucha tea,38 pu-erh tea and its

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extract,39-42 Fuzhuan brick tea extract,43,

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theaflavins.46 Tea and its components protect against the most commonly used

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dietary-based animal models, namely, high fat diet (HFD), methionine and choline

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deficient diet (MCD), and a leptin-deficient (ob/ob) mouse model. Sucrose and HFCS

44

large-leaf yellow tea45 as well as


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are important ingredients in modern Western diet. Excessive consumption of these

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sweeteners have been closely linked to increased risk of NAFLD.47 Hence, sucrose or

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HFCS were used singly or combined with high fat in the diet to mimic human dietary

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style. Although the treatments of dosage, routing and intervention time varied among

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these studies, green tea and its components could significantly improve blood

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biochemical profiles, liver histological parameters and alleviate liver lipids

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accumulation in different degrees when compared with control group.

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As the published preventive effect of tea types and bioactive compounds on

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NAFLD, GT, GTE and EGCG were researched predominantly. EGCG is the major

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compound of GTE (approximately 50%) and a common compound of all the tea types.

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Therefore, the underlying molecular mechanisms of tea and its components on

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NAFLD were mainly based on the studies of EGCG, GTE and GT on NAFLD in vivo

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(Figure 1).

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Balance Lipid and Glucose Metabolism

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The homeostasis of hepatic lipids is involved in a complicated system of

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signaling/transcriptional pathways regulated by hormones, transcription factors and

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nuclear receptors. According to the “two hits” theory, lipids accumulation is the initial

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step in the pathophysiology of NAFLD and derived from the unbalance of lipids

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absorption, synthesis and utilization.48 To decrease serum and hepatic lipid levels, one

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strategy is to inhibit the lipid absorption and biosynthesis, the other one is to enhance

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the lipid β-oxidation and excretion. The suppression of gluconeogenesis, lipogenesis

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and cholesterol synthesis and the enhancement of lipolysis by green tea and catechins



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are mainly mediated by AMPK, which is an energy-sensing molecule. In response to

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excessive intake of high-calorie foods, AMPK is activated to reduce energy storage

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by lipids and glucose, and promote catabolism to produce ATP.

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Dietary absorption and de novo lipogenesis (DNL) are two main approaches for

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liver lipid accumulation. Hepatic lipid synthesis is regulated by several key enzymes,

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such as FAS, ACC, SCD1, DGAT, which are regulated by the activation of two

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critical transcription factors, SREBP-1c and ChREBP. As shown in the Table 1, mice

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and rats fed with HFD, and gavaged EGCG (50 mg/kg.bw/day) or GTP (200

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mg/kg.bw/day), the phosphorylation of AMPK and its upstream kinase and governor,

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LKB1 and sirtuin 1 were increased significantly in the liver.49, 50 Activation of hepatic

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AMPK can down-regulate expression of SREBP-1c and lead to decrease in the

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expression or activities of enzymes involved in hepatic DNL, gluconeogenesis,

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triglycerides, glycerolipid and cholesterol synthesis, thereby reduce liver fat

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deposition.51 GTE and EGCG supplementation down-regulated the expression of

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hepatic DNL genes, such as FAS, ACC and SCD1, and their master regulator

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SREBP-1c.49,

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suppressed the other transcription factor ChREBP that activated the enzymes

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responsible for DNL.49 Recently, it was reported that 12-weeks’ oral gavage of GTE

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to HFD-fed mice dose-dependently decreased the expressions of GPAT and HMGR,

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which were responsible for glycerolipids synthesis and cholesterol synthesis,

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respectively.53 In addition, GT or its components treatment also decreased the

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expression of other hepatic lipogenesis related enzymes, such as PPARγ (for lipid

52, 53

A 16-weeks’ oral gavage of EGCG (50 mg/kg.bw/day) also


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synthesis and storage), Elovl6 (for fatty acid elongases) and G-6-P (for

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gluconeogenesis), DGAT1 and DGAT2 (for triacylglycerol synthesis).52-54

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Hepatic fatty acids oxidation is the primary pathway of lipids expenditure, which

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mainly occurs in mitochondria and peroxisomes to provide energy. Dysfunction of

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mitochondria and peroxisomes induces the accumulation of hepatic lipids, and causes

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IR. PPARα is highly expressed in liver and upregulates enzymes involving

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mitochondrial and peroxisomal fatty acid β-oxidation. The activation of PPARα

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facilitates hepatic lipid β-oxidation mainly through carnitine palmitoyl transferase 1

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(CPT-1). Dietary supplemented with 0.32% EGCG increased the hepatic expression

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of PPARα.55 Meanwhile, another study found that daily gavaged 50 mg/kg.bw of

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EGCG to HFD fed Swiss mice could enhance the activity of mitochondrial complex

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chain, complex Ⅰ and Ⅳ, which contributed to the oxidative phosphorylation.56

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CD36 is the best-characterized transporter for the transportation of long-chain

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fatty acids through hepatic cell membrane. High levels of CD36 expression can lead

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to lipid accumulation, which positively related to liver fat content.57, 58 It is important

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for improving uptake and intracellular trafficking of free fatty acids (FFAs) as well as

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esterification into TG. GTE and EGCG not only reduced the fatty acid synthesis, but

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also inhibited the expression of CD36, so that inhibited the accumulation of FFAs in

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hepatic cells.52, 54, 55

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Furthermore, green tea and its components have ability to increase insulin

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sensitivity to obtain lipid-lowering effect. IR is also a key feature of NAFLD. In IR

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state, the input of FFAs to the liver is increased and β-oxidation of FFAs is impaired,



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and further causes lipid accumulation and lipotoxicity of the liver. IR in adipose tissue

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can lead to failure in inhibition of HsL and release of FFAs from adipose tissue into

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circulation. Park et al. found that leptin-deficient (ob/ob) mice supplemented with

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GTE (0.5-1%) in diet dose-dependently suppressed the expression of HsL in adipose

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tissue, thereby decreasing lipid accumulation in liver.59 Insulin receptor substrates

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(IRS) genes deficient mice can lead to serious IR in liver and impaired glucose

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intolerance, with β -cell hyperplasia.60,

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polyphenols can improve IR by facilitating the functional recovery of insulin receptor

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substrates with up-regulation of IRS-1 and IRS-2 tyrosine-phosphorylation.56, 62

61

However, supplementation of tea and tea

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Liver is the main organ of insulin action, clearance, and degradation, where

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majority (~80%) of endogenously secreted insulin decomposed by insulin-degrading

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enzyme (IDE, insulysin). IDE is the key enzyme responsible for insulin degradation,

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regulates insulin levels and sensitivity. The liver of IDE deficient mice has less insulin

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degradation, leading to hyperinsulinemia and glucose intolerance.63 Clinical studies

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have also observed similar results in NAFLD patients and subjects with high liver fat

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content. Gan et al. reported that daily treatment with EGCG (10, 20, 40 mg/kg.bw, i.p.)

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dose-dependently ameliorated IR in NAFLD mice through increasing insulin

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clearance by up-regulating hepatic expression and activity of IDE.64

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Tea and its components also have ability to increase lipid excretion and suppress

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lipid absorption to obtain lipid-lowering effect. Our previous study indicated that tea

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promoted lipid excretion in the feces, especially TC.43 Mice fed HFD containing

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0.32% EGCG for 16-17 weeks, fecal lipid excretion was obviously increased in


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parallel with a reduction of hepatic lipid deposition and body weight gain.55,

65, 66

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Huang et al. investigated the effect of EGCG on bile acid homeostasis and lipid

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absorption. Dietary supplemented with 0.32% EGCG for 17 weeks significantly

238

decreased bile acid reabsorption by reducing bile acid pool size, resulted in lower

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level of intestinal bile acid and higher content of fecal total bile acid, which further

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suppressed the absorption of lipid and cholesterol, while the fecal excretion

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correspondingly increased.55 Hirsch et al. treated 1% GTE to high-cholesterol diet fed

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C57BL/6J mice for 6 weeks obtained the consistent results that bile acid, feces weight

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and fecal cholesterol were increased.67

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Antioxidant Effect

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Oxidative stress is one of the major pathogenic mechanisms in the development of

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NAFLD, which aggravates the severity of insulin resistance.68 GT and its components

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supplementation for NAFLD rat or mouse models significantly elevated the level of

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endogenous enzymatic antioxidants, such as hepatic glutathione, SOD, CAT, GPx

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activity or mRNA expression, which protected liver from damage mediated by

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oxidative stress.59, 69-73

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Furthermore, GTE and EGCG are the strongest antioxidants in tea products and

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tea polyphenols, respectively. They also exerted a robust hepatic antioxidant defenses

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to scavenge free radicals and reduce the levels of ROS-mediated lipid peroxidation

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products (MDA and 4-HNE) and RNS-mediated protein nitration products (N-Tyr and

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NOx).37, 72-75 It was reported that treatment with EGCG and GTE could protect liver

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from oxidative and nitrative damage and inhibit hepatic inflammation, fibrogenesis



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and collagen deposition.74,

76

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diethyinitrosamine and CCl4, increased the expression of oxidative stress markers,

259

such as serum d-ROM, 8-OHdG (a marker of oxidative DNA damage), but EGCG

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treatment even at a low dose (0.01-0.1%) could reverse the oxidative damage to the

261

liver.71, 72

For instance, NASH rats could be induced by HFD,

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As a critical transcription factor of antioxidant defense system, Nrf2 can initiate

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the transcription of a series of cytoprotective genes, such as HO-1 and NQO-1.77 The

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GTE (2%) could attenuate the oxidative stress by lowering hepatic MDA level and

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TNF-α, MCP1 mRNA expression in Nrf2 null mice fed HFD. Meanwhile, blood

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biochemical and histological parameters were also improved to the levels of wild-type

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C57BL/6 mice fed with HFD. Most importantly, GTE increased the expression of

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hepatic Nrf2 and NQO-1 mRNA, antioxidant defense system was strengthened

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contributing an excellent effect on NASH mice induced by HFD.52

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Anti-inflammatory Effect

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Inflammation is the most important hallmark from simple steatosis to the

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development of NASH, which was caused by lipotoxicity from excessive

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accumulation of lipids in the liver.78 NASH is a sterile inflammatory disorder

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involving recruitment of lymphocytes, macrophages and neutrophils to the liver and

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activation a series of pro-inflammatory signaling pathways involving NF-κB, AP-1,

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TLR4 and NLRP3.9 Anti-inflammatory effect mainly attributed to suppression of the

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NF-κB pathway. This classical pathway is activated by phosphorylation of IκB, which

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triggers the up-regulation of inflammatory cytokines, such as TNFα and MCP-1.79


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Numerous studies have demonstrated that GTE and EGCG can alleviate hepatic

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inflammation induced by dietary components or chemicals. In the rodent NAFLD

281

models, supplementation with green tea or its components suppressed the gene or

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protein expression of TNF-α, NF-κB, MCP-1, IL-6, IL-1β, TLR4, iNOS and COX-2.

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The decreased inflammatory profile in the serum and liver could ameliorate hepatic

284

dysfunction.52, 59, 69, 71, 73-75 Furthermore, two studies also showed that GTE and EGCG

285

supplementation could mitigate liver injury during NASH by lowering COX-2

286

mediated synthesis of PGE2.73, 75 Besides, GTE and EGCG also down-regulated the

287

activity or expression of other pro-inflammatory enzymes or mediators, such as iNOS

288

and MPO.52

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NFκB-mediated inflammation is the main factor leading to NASH, which causes

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IR and oxidative stress. Long-term HFD intake will cause ROS accumulation in the

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liver, the dysbiosis of gut microbiota and increase intestinal permeability to low

292

molecular bacterial products, especially lipopolysaccharide (LPS) and endotoxins.80

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These factors contribute to NF-κB activation through inflammatory signaling

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mediated by the TNFR1 and TLR4. Recently, Li et al. successively performed three

295

studies have confirmed that 2% GTE treatment in diet protected mouse liver from

296

high-fat and genetic induced NASH by lowering hepatic TNFR1, TLR4, phospho-p65

297

and the TLR4’s adaptor protein MyD88 expression. Meanwhile, the gut-derived

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endotoxin translocation was limited to circulating via elevating the expression of

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intestinal tight junction proteins, including CLDN-1 in duodenum, CLDN-1 and OCC

300

in jejunum, OCC and ZO-1 in ileum, and to strengthen intestinal barrier function.52, 81,



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82

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dysbiosis of gut microbial, and decrease the serum LPS level.83, 84 Thus, these results

303

indicated that the anti-inflammatory activity of GTE protected liver from NF-κB

304

activation in HFD-induced NASH by attenuating endotoxin and TLR4/MyD88

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signaling along the gut-liver axis.

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Anti-fibrotic Effect

307

Hepatic fibrosis is the severe phase of NAFLD, and caused by the accumulation of

308

high-density extracellular matrix (ECM) proteins. As shown in the Figure 1, HSCs are

309

the principal collagen-producing cells and play an important role in the development

310

of hepatic fibrogenesis.85 HSCs keep quiescent under normal conditions, once the

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liver injured, the released TGF-β activates HSCs. The progression of hepatic fibrosis

312

in rodent models activated key members in the TGF-β/SMAD pathway and other

313

molecules (α-SMA, COL1A1, MMP and TIMP) responsible for the accumulation of

314

hepatic collagen. Recently, a study suggested that the PI3K/Akt/FoxO1 pathway

315

participated in the proliferation and trans-differentiation of HSCs.73, 86 When the rats

316

liver cells were injured by HFD, chemical reagent (CCl4) and bile duct-ligated single

317

or combined, low dose of EGCG treatment could relieve the liver injury in three

318

rodent models of liver fibrosis by lowering expression or concentrations of

319

pro-fibrogenic factors, involving TGF/SMAD and PI3K/Akt/FoxO1 pathways.72,

320

According to these studies, the expression level of TGF-β1, the phosphorylation of

321

SMAD2/4, α-SMA, procollagen-α1, PAI-1, MMP-2/9, TIMP-1/2 and collagen were

322

markedly down-regulated in the liver after EGCG treatment for 2-8 weeks.71, 73, 87, 88

Similarly, other studies demonstrated that tea or tea polyphenols could restore


73

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In addition, the hyperactivity of renin-angiotensin system (RAS), which participates

324

in blood pressure regulation, plays a pivotal role in hepatic fibrogenesis. Dietary

325

supplementation with 0.1% EGCG for 8 weeks inhibited this fibrogenesis of

326

SHRSP-ZF rats by targeting RAS activation, because EGCG significantly decreased

327

the level of angiotensin-II (AT-II) of serum and mRNA levels of ACE and AT-1R

328

(RAS components) in the liver.72

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Clinical Trials

330

NAFLD affects about 30% of population in the world-wide, with limited medical

331

treatment options and unsatisfactory results. More recently, a large-scale prospective

332

study has shown that coffee intake is inversely associated with total and cause of

333

specific mortality, including NAFLD.89 Tea is a comparable popular beverage with

334

coffee, only six human randomise controlled trials have investigated the preventive

335

effect of tea or its components on NAFLD.

336

Hepatic ALT and AST are two important biomarkers for hepatocellular injury.90

337

Additionally, there are several indexes for the NAFLD or related symptoms.

338

Liver-to-spleen attenuation ratio can judge the severity degree of NAFLD.91 While

339

urinary 8-isoprostane is a biomarker of oxidative stress.92 After catechins intervention

340

for 12 weeks, the prominent changes are the body fat ratio (%), serum ALT, urinary

341

8-isoprostane reduction, and liver-to-spleen attenuation ratio elevation.93 Fukuzawa et

342

al. extended two folds of intervention time (6 months) using a moderate dosage, and

343

obtained more significant results in body fat level and insulin resistance.94 Green tea



344

extract was inferior to catechins, only the indicators of liver function was decreased

345

that might be due to lower amount of catechins in green tea extract compared with

346

other two studies.95 It suggested that long-term intake of catechins or green tea extract

347

performed benefits on NAFLD subjects. Vitamin E and silymarin could also improve

348

the serum transaminases in NAFLD patients, which implied inflammation and

349

oxidative stress involved in the pathogenesis of NAFLD.9 Green tea extract and

350

catechins have anti-inflammatory and antioxidant activities, so that they may be also

351

alternative selection for prevention and improving the NAFLD.

352

Among these six clinic studies, half of them used tea catechins, while, as the

353

important components of GTE, theanine has hepatoprotective effect and coffee

354

caffeine intake is negatively correlated with the incidence of liver fibrosis in NAFLD

355

patients.96-99 Hence, GTE should emphatically consider in the future clinic

356

intervention study, these two components may be synergistic with polyphenols to

357

prevent NAFLD.

358

When it comes to the health benefits of tea and its components, bioavailability is

359

an inevitable issue in tissues. This problem could explain why many health benefits

360

were observed in all the tea types.14 Tea catechins are less directly absorbed into

361

circulation. Nevertheless, they may take their effects in intestinal tract. Most catechins

362

are decomposed and metabolized by the colonic microflora. The intestinal bacterial

363

metabolites of catechins might play a critical role in the bioactivities of tea catechins

364

in vivo. Because these metabolites mainly are low molecular phenolic acids and

365

valerolactones, which can be easily absorbed into blood.100 Recently, it was reported


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366

that gut microbial metabolites of catechins exerted the same biological activities as

367

original molecules.101, 102

368

Dietary supplementation of 2% GTE to rodents is equivalent to about 10

369

servings/day (120 mL per serving) for humans, which is a normal consumption level

370

with a lower incidence of liver injury in Japanese adults.103 When mice treated with

371

pure EGCG in diet, the dose of 3.2 g EGCG/kg diet equivalents to 10 cups/day (200

372

mL per cup) of green tea for an average person. These two commonly used dosages

373

may represent an attainable dose of human consumption.65 However, the potential

374

hepatotoxicity is a crucial issue. Based on toxicological and human safety data, for

375

adults with normal liver function, a safety limit has been determined to be 338 mg

376

EGCG daily intake of solid dosage, and 704 mg EGGG/day in tea beverage.104

377

Another two reviews proposed that 600 mg EGCG/day had no observed adverse

378

effect level (NOAEL) for humans, while, some European countries were cautiously

379

adjust the upper intake dose of EGCG to a half of NOAEL (300 mg/day).105, 106 Yang

380

et al. reviewed that people drinking three to four cups of tea (0.6-0.9 g catechins) per

381

day or more showed the significant beneficial effects on MetS related disease.18 Green

382

tea contains the most abundant catechins, especially EGCG, 1 g dried green tea

383

commonly contains about 120 mg catechins and 70 mg EGCG.12 Taken together, for

384

preventing against NAFLD, it suggested that it is safe and good for health,

385

consumption in solid form 600-900 mg/day tea catechins is reasonable, and drinking

386

as a beverage 5-10 g/day of green tea is more appropriate.

387

CONCLUSION AND PERSPECTIVE



388

The current animal evidence indicates that tea and its components have ability to

389

prevent steatosis and its progression to NASH, which involves multiple mechanisms,

390

including lipid-lowering effect by decreasing hepatic DNL, improving insulin

391

sensitivity and increasing lipid excretion which would reduce lipid overload in the

392

liver, antioxidant effect by enhancing the antioxidant defense system to protect

393

against oxidative and nitrative damage, and anti-inflammation and anti-fibrotic effects

394

by regulate pro-inflammatory and pro-fibrotic molecular signaling pathways.

395

However, there lacks sufficient clinical trials to verify some efficacies on animal

396

models. Meanwhile, the exisited clinic studies have some limitations. First, the

397

sample size of participants is too small in trials. Second, all these clinic studies were

398

conducted in Asian countries, other European and American countries with a high

399

incidence of NAFLD have barely addressed. Third, these trails were heterogeneous in

400

trail design, such as the treatment dose and time, pathologic confirmation. Lastly,

401

lifestyle behaviors was neglected in these studies, such as dietary patterns, alcohol

402

consumption, smoking and physical exercise. Hence, the preventive efficacy of tea

403

and its components needs further research, especially in human subjects using

404

achievable dosage of tea or its components.

405

Further clinical trials should focus on examining GTE, catechins and EGCG on

406

NAFLD, including double-blinded randomized clinical trials and epidemiological

407

investigation. The treatment of dosages, duration, routes, enrollment and diagnostic

408

criteria should be considered in future human trials. It also need to enlarge sample

409

size, extend treatment duration, and combine with other therapies, such as exercise


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410

and medicines. Moreover, it is necessary that epidemiological investigation about the

411

relationship between drinking tea and the prevalence of NAFLD, which might provide

412

a dietary solution for NAFLD prevention.

413

Conflicts of interest

414

Authors declare that they do not have any conflict of interests.

415

Funding

416

This work was supported by the Young Elite Scientist Sponsorship Program by CAST

417

(2016QNRC001), the Anhui Provincial Natural Science Foundation (1708085MC73,

418

1508085MC59), Key Research and Development Projects of Anhui Province

419

(1804b06020367) and the Earmarked fund for China Agriculture Research System

420

(CARS-19).

421

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FIGURE CAPTION Figure 1 Potential preventive mechanisms of green tea and its components on NAFLD.



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Table 1. Rodent Studies Evaluated the Impact of Green Tea and Its Components on NAFLD Animal model ob/ob mice

Treatment 1–2% GTE in diet

Duration 6 weeks

Reference Bruno et al., 2008103 Park et al., 201159

8 weeks

Main outcomes a ↓Serum ALT and AST ↓Hepatic total lipid and TG ↓Serum TC, ALT, NEFA ↓Liver lipids and ALT, TNF-α, MDA ↓Adipose SREBP-1c, FAS, SCD-1, HSL, TNF-α ↑Hepatic antioxidant defenses (tGSH, Mn-SOD, Cu/Zn-SOD, CAT, GPx) ↓ROS-mediated LPO (4-HNE, NADPH oxidase activity) ↓RNS-mediated protein nitration (N-Tyr, NOx) ↓Pro-inflammatory enzymes (MPO and iNOS) expression ↓Hepatic and epididymal adipose TNF-α, MCP-1, NFκB ↑Hepatic and epididymal adipose GSH Reverse hepatic fatty acids to the normal level ↓Hepatic MDA, COX-2 and PGE2 activity, COX-2 protein ↓Hepatic MDA, α-tocopherol ↓Hepatic SREBP-1c, FAS, CD36, SCD1, DGAT1/2 and TNFα, MCP-1, iNOS and TNFR1, TLR4 mRNA ↑(C57BL/6 mice) Hepatic Nrf2, NQO1 mRNA, phospho-p65 protein ↓Serum TNFα and endotoxin level. ↓Hepatic phospho-p65, MyD88 protein ↓Hepatic TNF-α, iNOS, TNFR1, RIP1, TLR4, MD2, CD14, MyD88 mRNA ↑Duodenum OCC and ZO-1 mRNA, ileum OCC mRNA ↓Serum endotoxin and hepatic MDA level. ↓Hepatic phospho-p65, MyD88 protein ↓Hepatic TNFα, iNOS, MCP-1, MPO, TLR4 ↑Hepatic GSH, GSH/GSSG, tGSH ↑mRNA expression of CLDN-1 in duodenum, CLDN-1 and OCC in jejunum, OCC and ZO-1 in ileum ↓Hepatic CD36, PPARγ mRNA

ob/ob mice

0.5–1% GTE in diet

6 weeks

ob/ob mice

0.5–1% GTE in diet

6 weeks

Wistar rats HFD (60% kcal) Wistar rats HFD (60% kcal) Nrf2-null, C57BL/6 mice HFD (60% kcal)

1–2% GTE in diet

8 weeks

1–2% GTE in diet

8 weeks

2% GTE in diet

8 weeks

C57BL/6J mice HFD (60% kcal)

2% GTE in diet

8 weeks

TLR4-mutant C3H/HeJ mice HFD (60% kcal)

2% GTE in diet

8 weeks

C57BL/6 mice HFD (21% butter fat, 0.15% cholesterol) Balb-c mice

2, 4% GTE/CTE in diet 1% GT in diet

6-12 weeks

↓Mean diameter of hepatocytes fat globules

Sajjad et al.,


Chung et al., 201274 Park et al., 201269 Chung et al., 201575 Li et al., 201652 Li et al., 201781

Li et al., 201882

Yang et al., 201354

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HFD C57BL/6J mice HCD

1% GTE in diet

6 weeks

C57BL/6J mice HFD

30, 60, 120 mg/kg.bw GTE, gavage

12 weeks

Zucker fatty rats HFD

200 mg/kg.bw/day GTP,gavage

8 weeks

Bile duct-ligated rats

2 weeks


25 mg/kg/day EGCG, gavage 0.32% EGCG in diet


0.32% EGCG in diet

17 weeks

nSREBP-1c transgenic C57BL6 mice SD rats HFD (60% kcal) SD rats HFD (62.2% kcal) i.p. diethyinitrosamine

0.05-0.1% EGCG in diet 1 g/L EGCG

12 weeks

0.01-0.1% EGCG in diet

7 weeks

SHRSP-ZF rats HFD (56.7% kcal), i.p.CCl4

0.1% EGCG in diet

8 weeks

16 weeks

6 weeks

↓Liver TC, 4-HNE ↑Serum ALT, AST, insulin, bile acid, feces weight, fecal cholesterol ↑Hepatic TNFα, IL-6, SAA1, SAA2, iNOS, SHP,CYP27A1 mRNA ↓Liver TG, body fat percentage, adipocyte size ↓Blood glucose, serum insulin, HOMA-IR, leptin, ALT, TG, TC, LDL-C ↓Hepatic ACC, FAS, SCD-1, SREBP-1c and CD36 protein ↓Hepatic ACC, FAS, SCD-1, SREBP-1c, GPAT, HMGR and PPARγ mRNA ↑Serum adiponectin, hepatic SIRT1 and pAMPK protein ↓Serum ALT, AST, TNF-α, IL-6 level ↑Hepatic pAMPK, pACC protein ↓Hepatic pSREBP-1c protein ↓Hepatic COL1A1, MMP-2/9, TGF-β1, TIMP1, α-SMA mRNA and protein ↓Hepatic lipids accumulation ↓Plasma MCP-1 ↑Fecal lipids ↓Fatty liver incidence ↓Plasma MCP-1, CRP, IL-6, G-CSF ↑Fecal lipids ↓Liver 8-OhdG, the expression of pAkt, pIKKß, pNF-κB ↑The expressions of liver IRS-1, pIRS-1 ↓Plasma and liver MDA, CYP2E1, α-SMA expression ↑Hepatic glutathione ↓Liver GST-P-positive foci, hepatocytes proliferation ↓Hepatic TIMP-1/2 and TNF-α, IL-6, IL-1β and cyclin D1 mRNA ↓Urinary 8-OHdG and serum d-ROM ↑Hepatic CAT, GPx-1 mRNA ↓Hepatic GST-P-positive foci, α-SMA positive area, hydroxyproline content ↓Hepatic MMP-2/9, TIMP-1/2, α-SMA, TGF-β1, procollagen-1, PAI-1 mRNA ↓Serum AT-II, hepatic ACE, AT-1R mRNA


2014107 Hirsch et al., 201667 Bae et al., 201853

Tan et al., 201750 Yu et al., 201487 Bose et al., 200865 Chen et al., 201166 Ueno et al., 200962 Kuzu et al., 200888 Sumi et al., 201371 Kochi et al., 201472


SD rats HFD (30% kcal)

50 mg/kg EGCG 3 times/week, i.p.

8 weeks

C57BL/6 mice HFD Swiss mice HFD Swiss mice HFD C57BL/6J mice HFD (60% kcal)

10, 20, 40 mg/kg/day EGCG, i.p. 50 mg/kg/day EGCG, gavage 50 mg/kg/day EGCG, gavage 0.32% EGCG in diet

4 weeks

C57BL/6 mice HFD (60% kcal)

50-100 mg/kg/day GA, gavage

16 weeks

16 weeks 16 weeks 17 weeks

↓Serum d-ROM, hepatic MDA, hepatic 8-OHdG, 4-HNE, CYP2E1, JNK, p-JNK protein and GPx, CAT mRNA ↓Hepatic TNF-α, IL-6, IL-1β, MCP-1 mRNA ↓Hepatic iNOS, COX-2 mRNA and protein, TNF-α mRNA ↓Hepatic CAT, GPx mRNA, nitrotyrosine, collagen ↓Hepatic α-SMA, TGF-β1, MMP-2, TIMP-2, pSMAD2/SMAD2, pSMAD4/SMAD4 protein ↓Hepatic p27kip1 protein, TNF-α mRNA, pFoxO1/FoxO1 ↑Hepatic pPI3 K/PI3 K, pAkt/Akt ↑Insulin clearance, hepatic IDE protein and enzyme activity ↑Hepatic complex I, complex IV ↑Hepatic AKt, IRα protein ↓Hepatic SREBP-1, FAS, ACC and ChREBP protein ↑Hepatic AdipoR2, SIRT1, pLKB1 and pAMPK protein ↑Hepatic CYP7A1, CYP27A1, FXR, HMGR, LDLR, SRB1, PPARα mRNA ↑Fecal bile acids, cholesterol and total lipids ↓Hepatic CD36 mRNA ↓Intestinal bile acid content Partially normalizes HFD induced lipidomic profile ↓Liver fatty acids ↑Hepatic PUFA/MUFA ratio Disturbed lipid metabolism and ketogenesis, glycolysis, amino acids, choline metabolism and gut-microbiota metabolism partially reversed ↓Hepatic INSIG-1, SREBP-1c, SCAP protein, and FAS, SIRT, LXR-α mRNA

Page 34 of 39

Xiao et al., 201473

Gan et al., 201564 Santamarina 201556 Santamarina 201549 Huang et al., 201755

et

al.,

et

al.,

Chao et al., 201108

SD rats 10, 20, 40 mg/kg.bw 12 weeks Cheng et al., Oral administrated with EC, gavage 2017109 high-fat-cholesterol emulsion a: Arrow indicates an increase (↑) or decrease (↓) in the levels of gene expression, protein concentrations or enzyme activity. Ratio of liver weight to body weight, blood biochemical and histological assay results were not shown, all the treatment groups of green tea and its components were significantly improved in blood biochemical profile, histological parameters and alleviate liver lipid accumulation at different degrees when compared with model group.

Abbrevations ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; (p-)AMPK, (phosphorylated) adenosine monophosphate-activated protein kinase; ACC, acetyl-CoA carboxylase; ACE, angiotensin-converting enzyme; AdipoR2, adiponectin receptor 2; AKT, protein kinase B; AP-1, activating protein-1; AT-II, angiotensin-II; CAT, catalase; CD36, cluster of differentiation 36; ChREBP, carbohydrate-responsive element-binding protein; COX-2, cyclooxygenase-2; CLDN-1,


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Claudin-1; CPT-1, carnitine palmitoyl transferase; CRP, C-reactive protein; CTE, cocoa tea (Camellia ptilophylla) extract; CYP2E1/7A1/27A1, cytochrome P450 2E1/7A1/27A1; d-ROM, diacron-reactive oxygen metabolites; DGAT-1/2, diglyceride acyltransferase-1, -2; EGCG, epigallocatechin-3-gallate; FAS, fatty acid synthase; FXR, farnesoid X receptor; GA, gallic acd; GTE, green tea (Camellia sinensis) extract; G-CSF, granulocyte colony-stimulating factor; GST-P-positive foci, glutathione S-transferase placental form positive foci; (t)GSH, (total) glutathione; GPx, glutathione peroxidase; HCD, high-chlosterol diet; HDL, high-density lipoprotein; HFD, high-fat diet; HsL,hormone-sensitive lipase; HOMA-IR, homeostasis model assessment for insulin resistance; HFSD, high-fat-sucrose diet; HMGR, 3-hydroxy-3-methyl glutaryl coenzyme A reductase; iNOS, inducible nitric oxide synthase; IDE, insulin-degrading enzyme; IL-6, interleukin-6; IL-1β, interleukin-1β; IRα, insulin receptor α; (p-)IRS-1/2, (phosphorylated) insulin receptor substrate-1, -2; INSIG-1, insulin-induced gene 1JNK, c-Jun N-terminal kinase; LKB1, liver kinase B1; LXRα, liver X receptor α; LDL, low-density lipoprotein; MCD, methionine and choline deficient diet; MCP-1, monocyte chemoattractant protein-1; MDA, malondialdehyde; MMP-2/9, matrix metalloproteinase-2, -9; MPO, myeloperoxidase; MUFA, monounsaturated fatty acid; NEFA, non-esterified fatty acids; NF-κB, nuclear factor kappaB; NOS, nitric oxide synthase; NOx, total nitrate and nitrite; NQO1, NADPH quinone oxidoreductase 1; Nrf2, nuclear factor-erythroid 2-related factor; N-Tyr, 3-nitro-tyrosine; OCC, occludin; p-AMPK, adenosine monophosphate-activated protein kinase; PAI-1, plasminogen activator inhibitor-1; PGE2, prostaglandin E2; PK, pyruvate kinase; PI3K, phosphoinositide-3 kinase; PPAR-α, -γ, peroxisome proliferator activated receptor-α, -γ; phospho-p65, phosphorylated-p65; PUFA, polyunsaturated fatty acid; SCD-1, stearoyl-CoA desaturase; SD, Sprague-Dawley; SIRT1, sirtuin 1; SOD, superoxide dismutase; SREBP-1c, sterol regulatory element-binding protein 1c; SCAP, SREBP cleavage-activating protein; TC, total chlosterol; TG, total triacylglycerol; TGF-β1, transforming growth factor β1; TIMP-1/2, tissue inhibitor of metalloproteinases1,2; TLR4, toll-like receptor 4; TNF-α, tumor necrosis factor α; TNFR1, tumor necrosis factor receptor-1; ZO-1, zonula occluden-1; 4-HNE, 4-hydroxynonenal; 8-OhdG, 8-hydro-2'-deoxyguanosine; α-SMA, α-smooth muscle actin.



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Table 2. Clinical Trials Carried Out with Green Tea Extract and Catechins on NAFLD Subjects

Participants

Treatment

Duration

Main outcomes a

14(NAFLD) Japan

GT with high-density catechins (catechins1080m g/day)

12 weeks

↓Serum ALT, urine 8-isoprostane ↑Liver CT attenuation improvement rate

Sakata et 2006110

17(NAFLD) Japan

200,1080 mg/day Catechins

12 weeks

↓Body fat (%), serum ALT, urinary 8-isoprostane ↑Liver-to-spleen CT attenuation ratio

Sakata et al., 201393

38 (NASH) Japan

600 mg/day Catechins

6 months

↓BMI, serum TG, TC, FBG, IRI, HOMA-IR, GA (%), AI, hs-CRP, visceral fat /subcutaneous fat ratio ↑Serum HDL-C, liver to spleen CT attenuation ratio

Fukuzawa et al., 201494

71(NAFLD) Iran

500 mg/day GTE

12 weeks

↓Body weight, BMI, serum ALT, AST, ALP

Pezeshki et al., 201595

80(NAFLD) Pakistan

500 mg/tiwce a day GTE

12 weeks

↓Body weight, BMI, HOMA-IR, lipid profile (TC, TG, LDL-C), ALT, AST, hs-CRP ↑Serum HDL-C, adiponectin, 67.5% regression of fatty liver changes on ultrasound

Hussain et al., 2017111

45(NAFLD) Iran

550 mg/day GTE

3 months

↓BMI, serum AST and FBS

Tabatabaee et al., 2017112

/Country

Reference

a: Arrow indicates an increase (↑) or decrease (↓) in the levels of protein concentrations or enzyme activity. CT, computed tomography; BMI, body mass index; hs-CRP, C-reactive protein; GA, glycoalbumin; HOMA-IR, homeostasis model assessment-insulin resistance; FPG, fasting plasma glucose; RI, immunoreactive insulin; AI, arteriosclerosis index; ALP, alkaline phosphatase; FPS, fasting plasma sugar.


al.,

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Figure 1



Table of Contents Graphic (TOC)


Page 38 of 39

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Preventive Efficiency of Green Tea and Its Components on

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