Zhiling Li, Chngchun Feng, Hongjin Dong, Weiin Jin,Wenying Zhng, Jinfeng Zhn,*, Shuzhen Wng,*

a College of Life Science, Huanggang Normal University; Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources Comprehensive Utilization; Hubei Collaborative Innovation Center for the Characteristic Resources Exploitation of Dabie Mountains; Hubei Zhongke Research Institute of Industrial Technology, Huanggang 438000, China

b College of Agriculture, Yangtze University, Jingzhou 434000, China

Keywords:

(–)-Epicatechin-3-gallate

Polyphenolic compounds

Pharmacological properties

Anticarcinogenic agent

Natural antioxidants

Antimicrobial properties

A B S T R A C T

(–)-Epicatechin-3-gallate (ECG), a bioactive polyphenolic compound, has contributed a lot to the health benefits of green tea.Great attention has been focused on (-)-epigallocatechin-3-gallate (EGCG), but limited research has been performed towards ECG.Like EGCG, ECG also possesses various pharmacological and physiological properties, such as mediation of antioxidant activities, anti-inflammation response, regulation of cell proliferation and apoptosis, as well as anticancer properties during angiogenesis, invasion and metastasis stages.Nontoxic ECG has various molecular targets within the cells, including CYP enzymes, phase II detoxification and antioxidant enzymes, as well as pro-inflammatory mediators.The antineoplastic mechanism contains inhibition of phase 1 CYP enzymes, induction of phase II detoxification and antioxidant enzymes,high anti-inflammatory efficacy, arrest of cell cycle progression, regulation of apoptosis, as well as mediation of metastasis processes.In particular, the gallate moiety of ECG is critical for mediating inhibitory effects towards cancer cells.Besides regulation of intracellular signaling pathways, ECG also inhibits RNase A and matrix metalloproteinase enzymatic activity via chelating metals (copper and zinc) in cancer cells.This review has summarized recent studies on pharmacological properties of ECG, and discussed corresponding mechanism on modulation of cellular signaling events by ECG, hoping to broaden its multiple usage.

1.Introduction

Tea beverages, brewed fromCamellia sinensisleaves, have been consumed around the world for nearly 5 000 years, especially in China.Tea catechins, known as natural antioxidants, account for 60%-80%of total tea polyphenols.The main catechins are (–)-epicatechin (EC),(–)-epicatechin-3-gallate (ECG), (–)-epigallocatechin (EGC), and(–)-epigallocatechin-3-gallate (EGCG) (Fig.1), which account for about 6%, 14%, 20%, and 60%, respectively [1,2].Catechins have been well reported for health-promoting multifunctionality, especially anti-inflammatory, antioxidant, and anticancer effects with multitargets [3,4].Tea catechins, especially ECG and EGCG, show great potentials in maintaining cellular redox homeostasis through free radical scavenging, and the antioxidant capacity is even stronger than glutathione, vitamins, and flavonoids [1,2].Once in the body,catechins undergo metabolic processing in liver, small intestine, and colon, resulting in the formation of glucuronide, sulfate conjugates,and even methyl epicatechins [1].As stabilizers, human serum albumins bind to catechins and then transport them [5].In particular,EGCG has attracted lots of attention due to abundance and activities,especially anticancer, anti-inflammatory, antioxidant, antibacterial and antiviral activity [2,6].However, poor bioavailability has been reported towards EGCG, as only 0.1% of EGCG is bioavailable [6].

Fig.1 Chemical structure of ECG, EGCG, EGC, and EC.

As another major antioxidative polyphenolic compound in green tea, ECG also exerts growth-inhibitory potential of cancer cells with even more potential than EGCG, especially in genderbased carcinomas [7].The values of ECG in tea powders are in the range of 18.969 to 39.469 mg/g in samples ofCamellia sinensis(L.)O Kuntze cultivar collected during four different flush seasons [8].After intragastric administration of green tea, ECG is absorbed by monocarboxylate transporter and accumulated in lumen with concentrations of 75–300 mmol/L (higher than EGCG and other catechins) [9].Moreover, ECG is less cytotoxic, higher hydrophobic,more susceptible to degradation during storage of tea leaves, and more stable at intracellular level than EGCG [10-12].Furthermore,ECG might be conjugated with glucuronic acid or sulfate in plasma,and accumulate in various cells in body [10].However, the precise mechanisms for health benefits of ECG remain largely unknown.Therefore, deeply understanding of cell signaling pathways, and clarifying exact molecular targets responsible for health promotion of ECG are greatly needed.In this review, the recent activity research on ECG has been summarized, especially anticarcinogenic, regulating of energy balance, and antimicrobial activities, aiming to provide references for the development of ECG as efficient agents.

2.Health benefits of ECG

As natural antioxidant compound, ECG shows obvious antioxidant, anti-inflammation, anticancer, and metabolic regulation properties with multi-targets bothin vitroandin vivo.

2.1 Anticancer potential of ECG

Cancer is uncontrolled cell division and the aggregation of cells to form tumors [13].Chemicals from dietary and environmental sources could be converted by phase I enzymes (members of CYP450 monooxygenases superfamily) to water-soluble metabolites, and then the polar metabolites are excreted after conjugation reactions catalyzed by phase II detoxifying enzymes, including glutathione-S-transferase (GST), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1) [13].Therefore, negative effects of these chemicals on cellular DNA are effectively avoided.Without phases II enzymes, the metabolically active carcinogens would form a covalent adduct with DNA, and lead to mutations in critical genes involved in cell growth, proliferation, apoptosis, and even the initiation and development of cancers [7].In particular, carcinogenesis could be influenced by surrounding non-malignant cells and tumor microenvironment, including hypoxia, reactive oxygen species(ROS), nitrogen species (RNS), growth factors, cytokines, matrix metalloproteinase (MMPs), and vascular endothelial growth factor(VEGF) [14].Particularly, copper level is considerably elevated in nearly all types of cancers [15].ECG could inhibit arachidonic acid metabolism and further prevent inflammation-induced carcinogenesis in both human colon mucosa and colon tumors [7].During the multistage carcinogenesis process, cellular alterations go through three stages: initiation, promotion, and progression [7].Therefore,effective chemopreventive agents contain inhibitors, blocking agents,and suppressing agents.ECG shows to be an ideal chemopreventive agent through interacting with various molecules (enzymes,transcription factors, etc.), blocking or modulating molecular expression during carcinogenesis processes, and reversing various intracellular signal transduction pathways [7,16].ECG treatment could effectively inhibit cancer cell growth in dose- and timedependent manner with an equal or even more potency than EGCG, including colon, breast, prostate, lung, pancreas, liver,melanoma, and tongue cancer cells (Table 1) [3,17-21].

Fig.2 Schematic illustrating the proposed anticancer mechanisms of ECG during.

Table 1Main anticancer mechanisms of ECG during different carcinogenesis stages.

2.1.1 Anticancer properties of ECG during initiation stage

During initiation stage, certain carcinogenic agents are absorbed and transported to organs and tissues through metabolic activation,interact with target cell DNA covalently, lead to stable genotoxic damage, and finally result in transformed cells [7].ECG could inhibit phase I enzyme CYP450 isoforms through blocking their activation in both rat cells (CYP1A1/2) and human cells (CYP3A4, CYP2A6,CYP2C19, and CYP2E1) [22].Moreover, ECG could induce internucleosomal DNA fragmentation in both human cancer cells(epidermoid and prostate cancer) and mouse lymphoma cells, but showed no effects towards normal epidermal keratinocytes [15].Tea extracts, mainly ECG and EGCG, could induce phase II enzymes (GST and quinone reductase), and inhibit ornithine decarboxylase activity in mouse mammary organ cultures, rat tracheal epithelial cells, and human lung tumor epithelial cells [23].In passage-2 NHNE cells pretreated with exogenous H2O2, ECG could decrease phosphorylation and internalization of EGFR at cell surface, reduce intracellular ROS generation, and restrain MUC5AC overexpression [24].

2.1.2 Anticancer properties of ECG during promotion stage

During relatively slow tumor promotion process, the transformed cells undergo many changes, and finally form preneoplastic cells,as chronic inflammatory is critical during this stage [14,25,26].Therefore, blocking inflammation signaling is recognized as effective mode for chemoprevention.In human colonic tumor tissues, ECG could effectively suppress activities of cyclooxygenase-2 (COX-2)and lipoxygenase (LOX), production of PGE2, as well as formation of TBX and HHT [27].In keratinocytes (HaCaT) cells, ECG could inhibit H2O2production, extracellular signal-regulated kinase (ERK)phosphorylation, and further prevent UVA-induced damage [28].In human hepatoma cell line HepG2, ECG significantly reversed IL-6,and reduced synthesis of negative acute-phase protein transthyretin and retinol binding protein (RBP) in different inflammatory states [29].In related to hepatocyte growth factors (HGFs), ECG showed antiinflammatory effects through preventing IL-17A-mediated CC chemokine ligand 20 production, inhibiting p38 MAPK and ERK,and attenuating IL-17 receptor expression [30].In mouse ears, ECG effectively inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation (edema) [31].ECG also showed inhibitory effect towards inflammation through attenuating lipopolysaccharide(LPS)-induced inflammatory mediator expression and intracellular ROS generation via induction of Nrf2/antioxidant response element(ARE)-driven GSH and HO-1 levels, interference with NF-κB and Nfr2/ARE transcriptional activities, as well as suppressing MAPKs (JNK1/2 and p38) and PI3K/Akt signaling pathways [32].In particular, ECG could inhibit the production of IL-6 and IL-8 in dental pulp cells pre-treated with LPS or PG [33].In HDPC cells,ECG might mitigate pulpal inflammation through significantly reducing LPS- or PG-mediated VEGF production, and inhibiting COX-2 expression in a dose-dependent manner [34].

Cell cycle is regulated by a list of proteins, including cyclindependent kinase (CDK), cyclins, CDK inhibitors (CDKIs), and check point kinases, as these molecules may drive cells from one phase to the next [35].In transformed and malignant cells, cell proliferation is often uncontrolled.In B[a]P-induced lung carcinogenesis, ECG suppressed abnormal cell growth through blocking growth factor,as well as inducing p53 and downstream target genes (Bcl-2, Bax,H-ras, cmyc, cyclin D1, CDKI p21 and p27) [36].In SCC7 cells,ECG caused arrest in G1 phase of the cell cycle progression through down-regulatingβ-catenin/T-cell factor (TCF)-mediated cyclin D1 gene transcription [37].In HCT11664 and A549 cancerous cells, ECG inhibited proliferation and cell growth by down-regulatingNUDT6andhnRNP B1mRNA expression, respectively [38].In colon cancer cell line SW480, ECG could induce cell cycle arrest at the G0/G1-S phase border, stimulate p21 and p53, as well as suppress cyclins D1 and B1 [39].

Tumor could be induced by ER cross-talks with growth factors through mediating intracellular kinase cascades, activating downstream transcription factors, and regulating target gene expression involved in carcinogenesis processes [38].In breast cancer cell line MCF-7 and immature female C57BL/6 mice, ECG could inhibit tumor growth through blocking ER activity [40].In rastransformed 30.7b cells, ECG could effectively decrease activator protein 1 (AP-1) activity and inhibit cell proliferation, as AP-1 transcription factor might regulate various genes coding products involving in inflammation, survival, proliferation, transformation,and cell death [41].In A172 human glioblastoma cells, ECG might inhibit platelet derived growth factor (PDGF)-BB induced tyrosine phosphorylation of PDGF b receptor (PDGFRb) [42].In multidrugresistant cell lines CH(R) C5, BEL-7404/DOX, and KBC2, ECG exerted anticancer effects through inhibiting P-glycoprotein (P-gp)efflux pump activities, and enhancing intracellular accumulation of P-gp substrates (daunorubicin and rhodamine-123) [43-45].In B16F10 melanoma cells, tea catechins could significantly suppress tyrosinase activity and melanin synthesis through cAMP/CREB/MITF pathway, while the inhibitory effect of ECG is stronger than that of EGCG, and even the well-known depigmenting agent arbutin [2].Like EGCG, ECG also exerted strong inhibitory effects towards mushroom tyrosinase activityin vitro[2].

The highly ordered multi-step process of apoptosis (programmed cell death) serves as critical protective mechanism against cancer, which is initiated by genotoxic compounds and various environmental stresses through extrinsic pathway or intrinsic mitochondria pathway [46].Unfortunately, some cancerous cells could escape from apoptosis through over-expression of growth-promoting oncogenes and antiapoptotic proteins (like Ras and Bcl-2), or by down-regulation or mutation of pro-apoptotic proteins such as p53 [47].In KATO III cells, ECG initiated apoptosis by blocking okadaic acid-induced TNF-α release and causing DNA fragmentation [48].In NCI-H460 cells, ECG could induce apoptosis through increasing p53 expression and reducing Bcl-2 expression [49].In HCT116 cells, ECG also exerted apoptosis induction and antitumorigenic activity in p53-independent pathway, as ECG could enhance pro-apoptotic NAG-1(non-steroidal anti-inflammatory drug activated gene 1) expression via mediating transcription factor 3 transcriptional activity and inducing TSP1 expression [50].In DU145 cells, ECG could increase ROS formation and mitochondrial depolarization, finally induce apoptosis [51].Moreover, ECG effectively induced caspase-3 activity, DNA fragmentation, as well as H2O2generation in carcinoma HSC-2 cells [52].The exposure of SW480 cells to ECG also resulted in apoptosis through regulating activities of caspase-3 and MAPKs,inhibiting AKT and RNA degradation, as well as activating p53-p38/JNK cascade [39].In H9C2 cardiomyocytes pretreated with ECG(20 and 30 μmol/L), significant increase of DOX-induced apoptosis(16%–18%) was observed, as Bax/Bcl-2 ratio increased significantly after 1 h pretreatment with ECG, and phosphorylation of AMP-activated protein kinase (AMPK) was also enhanced [53].

2.1.3 Anticancer properties of ECG during progression stage

Tumor cells gradually convert to invasive cells with increased metastatic potential during progression stage, which could be divided into angiogenesis, invasion, and metastasis [14].For cancer patients,the migration and proliferation of cancer cells from primary tumor to distant organs will promote cancer progression and even result in death.However, tumor cells could be modulated by various microenvironmental factors, including MMPs, growth factors, as well as cell-cell and cell-substrate adhesion molecules at each metastatic step [14].In particular, MMPs (collagenases and gelatinases) server as important mediators of ECM degradation involved in restraining tumor invasion [54].Compared with EGCG, ECG has stronger inhibitory effect on both eukaryotic and prokaryotic cell-derived collagenase and MMP-7 activities [55].Moreover, ECG inhibited HT1080 cells invasion through blocking MMP-9 and MMP-2 activities [56].ECG could impair the adhering and spreading of mouse lung carcinoma 3LL and melanoma B16F10 cells to fibronetin and endothelial cells, respectively [57,58].ECG also inhibited invasion of vascular smooth muscle cells (VSMCs) through direct inhibiting membrane-type MMP activity and reducing thrombin-induced activation of MMP-2 [59].In addition, ECG could inhibit cell motility and scattering through blocking HGF-stimulated phosphorylation of Met, ERK and AKT [60].ECG could suppress invasion of highly metastatic A549 cells through reducing the activities of MMP-2 and urokinasetype plasminogen activator, reversing TGF-β1-induced epithelial-to-mesenchymal transition, upregulating epithelial markers like E-cadherin, and inhibiting mesenchymal markers (fibronectin and p-FAK) [61].Similarly, subcutaneous inoculation of ECG also inhibited growth of lung cancer cells A549in vivo, showing effective anti-cancer and anti-invasion potentials [61].

2.2 Antioxidant potential of ECG

ECG protects cells from oxidative damage through directly scavenging ROS, and enhancing expression and synthesis of body’s detoxifying/antioxidant enzymes [7].In HepG2-ARE-C8 cells,ECG showed anti-oxidant and free radical-scavenging activities by activating MAPK proteins (ERK, JNK, and p38) [62].In HepG2 cells treated with tert-butylated hydroperoxide, ECG could suppress cellular lipid peroxidation mainly by decreasing TBARS (thiobarbituric acid reactive substances) accumulation, GSH-Px activity and GSSG content, increasing GSH level, as well as reducing cytotoxicity [63].In human TCCSUP and T24 BlCa cells (H2O2-stimulated bladder urothelial cells), ECG could effectively protect against oxidative stress/damage, and even cell death through reducing ROS production [64].ECG could protect Caco2 cells against induced oxidative stress and subsequent cellular death through inducing glutathione peroxidase (GPx),reducing ROS production, and preventing caspase-3 activation [65].In addition, ECG was capable of catalyzing oxidative DNA degradation in lymphocyte cell nuclei via mobilization of nuclear copper (reduction of Cu2+to Cu+) and induction of ROS production [15].

2.3 Antiobesity and crucial roles in lipid metabolism

The 5α-reductases and fatty acid synthases (FAS), important enzymes involved in cellular metabolism and growth regulation,could be inhibited by ECG effectively [66,67].Moreover, ECG could affect cholesterol biosynthesis through interfering enzymes involved in mevalonate pathway, such as FPPS, MVK and MDD [68].Like EGCG but with less efficiency, ECG could induce diacylglycerol kinase-α translocation from cytoplasm to plasma membrane and subsequent activation, while the diacylglycerol kinase-α is involved in vitamin E-induced improvement of diabetic renal dysfunction [69].Methylated ECG, whose hydroxyl group at C-3 in D ring substituted by methoxy group, could effectively inhibit proliferation and differentiation of 3T3-L1 preadipocyte, and showed a relatively high antiobesity effect with low cytotoxic activity [70].Research on effects of tea polyphenols on COX-dependent and LOX-dependent arachidonic acid metabolism revealed that ECG(30 mg/mL) could inhibit LOX-dependent activity by 30%-75%,and the formation of 5-, 12-, and 15-LOX metabolites was greatly inhibited in human cancer cells [27].

Epicatechin-3-gallate-(4β→8, 2β→O→7)-epicatechin-3-gallate,named as A-type ECG dimer, could significantly reduce intracellular lipid accumulation in 3T3-L1 preadipocyte cells by targeting microRNAs (miR-27a and miR-27b) and PPARγin the early stage of differentiation, while inhibitory effects were highly structuredependent and associated with dimer-membrane interactions [71].In particular, galloyl moieties and A-type linkage were crucial for the inhibitory effects of A-type ECG dimer on adipogenesis [71].Strong disturbing effects of A-type ECG on fluidity, hydrophobicity, and permeability of 3T3-L1 preadipocyte cell membrane contributed a lot to the distinct inhibitory effects on adipocyte hyperplasia [71].Moreover, the A-type ECG dimers showed high affinity for lipid bilayer, which could form hydrogen bond with both surface oxygen acceptors and deeper inside lipid oxygen atoms simultaneously [72].High positive correlations were observed between membrane disturbing abilities of A-type ECG dimer and 3T3-L1 cells differentiation, which might be due to the strong bilayer perturbing potency of A-type ECG dimer [72].In synthetic “l(fā)ipid raft-like”liposome, A-type ECG dimer could be incorporated into the liposome with high affinity and subsequently decrease membrane fluidity [73].Similarly, the A-type ECG dimer might bind to lipid raft cholesterol via hydrogen bonding interactions, disrupt integrity of lipid rafts,and exert remarkable inhibitory effects on cell differentiation through suppressing mitotic clonal expansion process and relative mRNA levels(PPARγ, C/EBPα, and SREBP1C) in 3T3-L1 preadipocytes [73].In particular, a highly positive correlation existed between the cholesterol binding capacity and inhibitory effect on preadipocytes differentiation (R2= 0.932 8) [73].

2.4 Cardioprotective effects of ECG

In rat VSMC cells, ECG showed anti-atherosclerosis and cardioprotection through inhibition of cell adhesion on collagen and laminin, interference with integrin 1 receptor, as well as binding to ECM proteins [74].Additionally, ECG has been reported to regulate the sensitivities of intracellular Ca2+ions and type-1 ryanodine receptor(RYR1) in junctional sarcoplasmic reticulum membranes [75].In the primary culture of neonatal rat ventricular myocyte, ECG enhanced the slowly inactivating component of voltage-gated Na+currents (INa) in a concentration-dependent manner (0.1–100 mmol/L)with an EC50value of 3.8 mmol/L [75].In particular, ECG could shift the current–voltage relationship of peakINato the hyperpolarizing direction and accelerateINarecovery kinetics, as well as increase the firing rate of normal spontaneous action potential (sAP) about two-fold without waveform alteration [75].Due to great potential in clinical applications on cardiac arrhythmias as novelINaagonist,ECG has been proposed as an effective compound in improving cardiac contractility [75].In rat hearts, ECG showed cardioprotective effects and could greatly reduce cardiac mortality through triggering Na/K-ATPase/Src receptor in the cell-free system, activating CTS-like signaling pathway, and providing PKCε-dependent protection against ischemia/reperfusion injury [76].

2.5 Effectiveness towards neurodegenerative diseases

Alzheimer’s disease (AD), clinically characterized by progressive decline of memory and cognition due to progressive accumulation of senile plaques composed of misfolded amyloidβ-peptide (Aβ), accounts for over 60%–80% of 50 million dementia patients worldwide [77].The major pathological features of AD are extracellular aggregates of senile plaques (SPs) constituted by abnormal insoluble Aβ, high concentration of metal ions (Cu2+, Zn2+),as well as the deposits of intracellular neurofibrillary tangles [78].In mouse neuroblastoma Neuro-2a cells, ECG showed significant inhibition of Aβaggregates through mildly binding to Cu2+and Zn2+, diminishing the Cu2+- or Zn2+-induced or self-assembled Aβaggregates, modulating Cu2+/Zn2+-Aβ40 induced neurotoxicity, and reducing ROS production [79].In the brain of APP/PS1 mice, ECG could cross the blood–brain barrier to reduce Aβplaques and protect neurons from injuries [79].

2.6 Antimicrobial properties

ECG exhibited significantly antibacterial activity against pathogenic bacteriaListeria monocytogenes(MTCC-839),Pseudomonas aeruginosa(MTCC-741),Bacillus cereus(MTCC-1272),andStaphylococcus aureus(MTCC-96) (Table 2) [8].In particular,ECG possessed remarkable anti-infective potentials towardsP.aeruginosastrains through targeting both cell wall and cytoplasmic membrane [80].Then, the destructed cellular membrane ofP.aeruginosafurther resulted in membrane permeability perturbation,ascending access of hydrophobic antibiotics, release of potassium ions, and leakages of nucleotides [80].Moreover, ECG had cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory activity and showed great potential in treating CFTR-related diseases, such as secretory diarrhea [81].In ex vivostudies, ECG inhibited CFTR-mediated short-circuit currents in isolated rat colonic mucosa in a dose-dependent manner [81].Moreover, intraluminal application of ECG (10 μg) could significantly reduce cholera toxininduced intestinal fluid secretion in mice intestinal closed-loop model, while CFTR Cl-channel served as molecular target [81].Pleasantly, ECG also showed stimulatory effect on growth of eleven potential probiotics belonging to the generaEnterococcus,Bacillus,andLactobacillusspp.obtained from fermented foods of Western Himalayas [8].

Table 2Antimicrobial activity of ECG.

Green tea catechins (rich in ECG) also showed antiviral effects towards HBV, HCV, HIV, HSV, influenza, adenovirus, enterovirus,and rotavirus.In particular, the antiviral effects of catechins were exerted through inhibiting virus from binding to and entering host cells, inhibiting viral RNA or DNA synthesis, inhibiting gene transcription, and altering various viral molecules [82].Furthmore, tea catechins rich in ECG could inhibit parasite infections (Plasmodium falciparum,Babesiaspp.,Trypanosoma brucei,Trypanosoma cruzi, andLeishmania braziliensis) through decreasing parasite numbers and growth, fragmentation of parasite DNA, and reducing fatty acid synthesis [83-85].In addition, tea catechins rich in ECG inhibited fungi growth, includingAspergillus niger,Candidaspp.,Penicilliumsp.,Microsporum canis,Trichophyton mentagrophytes,andTrichophyton rubrum[86,87].Tea catechins rich in ECG also preventedβ-sheet prions from changingα-helical forms and inducing reversal ofβ-sheet forms back toα-helical forms [88].However, how much does ECG contribute to these activities is unclear.

Fig.3 Chemoprevention effects of ECG towards cancer cells.

3.Discussion

As a popular non-alcoholic beverage, tea is rich in natural polyphenols, especially catechins, which comprise around 40% of water-soluble solids in green tea [1,89].As the most concentrated catechins in green tea, EGCG and ECG might contribute great to the health benefits of green tea.Structure significantly affect the binding process of polyphenols, as binding affinity of polyphenols with other molecules could generally be affected by glycosylation and the numbers of hydroxyl groups on the second aromatic ring [90].Like EGCG, ECG also shows great anticancer potentials with multitargets and multi-functions.Nrf2 signaling has been considered to be important molecular target in cancer prevention, but whether the increase of phase II enzymes by ECG is due to modulation of Nrf2 signaling or not is unclear.In different cancer cell models, ECG shows great potentials in modulation of cell cycle progression (the arrest mainly in G1 phase) through modulating proliferative gene and growth factor, and inducing apoptosis through activation of proapoptotic protein, which are all critical strategies for chemoprevention in the multistage carcinogenesis [91].The prooxidant activity is also critical for cancer chemotherapy, as ECG might elevate ROS production over a certain threshold via lowering antioxidant defenses,subsequently selectively kill cancer cells through mobilization of endogenous copper ions (especially the copper binding to chromatin) [15,92].However, it is unclear whether the antioxidant activities of ECG directly mediates the induction of apoptosis.Although ECG could induce apoptosis of cancer cells via several mechanisms, this beneficial efficacy requires further investigation.

Chronic inflammation and infection are critical factors in the promotion and progression stages of cancers [1,26].ECG shows effective antioxidant and anti-ageing capabilities, as it may protect cells from oxidative damage through directly scavenging ROS and enhancingde novoexpression of body’s detoxifying/antioxidant enzymes [7].ECG could also enhance pro-oxidative activity in proapoptotic pathways.Furthermore, ECG could significantly reduce IL-6, which might further modulate acute-phase protein response in various inflammatory states, finally enhance host defense against inflammation and infection [29,93].Moreover, activation of Nrf2 signaling pathways is also critical in the anti-inflammatory effects of ECG [7].Additionally, anti-inflammatory effects of ECG partly require the activation of ERK1/2 signaling, which could mediate HO-1 expression and Nrf2/ARE signaling activation [32].Furthermore, ECG could interact with Kelch repeat domains of Keap1 intracellularly and bind to extra-cellular LPS, which will promote nuclear accumulation of Nrf2 protein, blockade the activation of LPS-induced downstream target signaling pathways, and attenuate pathological syndromes of LPS-induced sepsis and systemic inflammation [32].Blocking inflammation signaling is also recognized as potential mode for chemoprevention, thereby the anti-inflammatory properties of ECG make it attractive to be developed as anti-cancer agents.The anti-oxidant properties of ECG in green tea are mainly due to the phenolic hydroxyl groups in the B- and D-rings [94].

Tea extracts exert antibacterial effect on bacterial pathogens and stimulatory effect on growth of potential probiotics mainly by EGCG, ECG and EGC constituents [8].Inhibition of CFTR is a pharmaceutical approach in treating secretory diarrhea, therefore ECG could be developed as an effective pharmaceutical agent due to inhibitory effects towards CFTR [81].Green tea extracts show antimicrobial activities against most oral bacteria, and improve oral health through increasing oral peroxidase activities, preventing development and progression of periodontitis, reducing dentin erosion and tooth loss, and improving bad breath [95,96], but how much does ECG contribute to these activities need deep study.Catechins, include EGCG and ECG, can bind and damage bacterial cell membranes,increase membrane permeability, lead to cell lysis and function loss of transmembrane transporter proteins, upregulate peptidoglycan biosynthesis genes, inhibit enzyme activities (DNA gyrases, ATP synthases, protein tyrosine phosphatases, cysteine proteases, and enzymes involving in bacterial fatty acid biosynthetic pathway),inhibit dihydrofolate reductase (DHFR) activity involving in folate biosynthesis pathway, as well as alter cell wall structure [1,97-100].Through combating pathogenic microbes in various ways, ECG might work synergistically directly or indirectly with EGCG and other catechins.In particular, anti-inflammatory and antioxidant effects may also contribute to the antimicrobial effects.As natural plant products,ECG shows great potential in fighting against infectious diseases,which will save tremendous cost in healthcare.However, how much does ECG contribute to the antiviral, anti-parasite, anti-fungi, and anti-prion effects of catechins is unclear.

Consumption of green tea could inhibit atherosclerosis, reduce total lipid levels, improve the ratio of LDL to HDL, increase insulin receptor sensitivity, inhibit digestive enzymes and absorption of fat, as well as stimulate glucose-induced insulin secretion [1,101],but whether these properties are caused by ECG or not need further investigation.Cholesterol binding might be one mechanism for ECG in modulating and controlling of metabolic dysregulation, particularly adipocyte differentiation [73].Moreover, a potent regulatory role of ECG on cardiac function may be associated with the modulations of sAP firing [75].Additionally, ECG might affect cardiovascular diseases through antiplatelet, antiproliferative, and antimigratory effects [94].As potent inducer of phase II enzymes, ECG could reduce carcinogenic formation through interacting with microsomal cytochrome P450 enzyme proteins and impairing electron transfer.The effects of tea polyphenols containing ECG on arachidonic acid metabolism in human colon mucosa and colon tumors may reduce the risk of colon cancer in humans [27].

Several limitations have been reported towards the bioactive component EGCG, such as poor stability and low absorption rate,whose bioavailability needs to be improved via modifying the structure of EGCG or delivery with nano-materials [102-104].In fact, ECG is accumulated in lumen with higher concentrations than EGCG [9].Moreover, ECG is nontoxic, which strongly protects normal cells from damage, cytotoxicity and chronic inflammatory,as well as induces apoptosis of cancer cells [7].In lead-exposed HepG2 cells, co-treatment with ECG and EGCG showed obvious synergistically protective effects through significantly increasing cell viability, decreasing lipid peroxidation, and protecting cell membrane against damage [18].In addition, the synergistically protective effects have also been observed in many studies with tea polyphenols [105-107].However, the synergistic mechanisms need to be further investigated.ECG could be separated by one-step fractional extraction from tea polyphenols with purity of 98%, yield of 94%,and output of 397 g in 24 h [108].The continuous separation and low operation cost make it a promising candidate to carry out industrial production.EGCG-modified collagen membrane and scaffold have been used to promote osteoblasts proliferation, as well as for guided bone regeneration (GBR) surgery, modulation of macrophage phenotype, and macrophage recruitment [109-111].Therefore, ECG could also be exploited as new functional agents due to multiple pharmacological properties.

4.Conclusion

As anticarcinogenic agent, nontoxic ECG has various molecular targets within the cells, including CYP enzymes, phase II detoxification and antioxidant enzymes, as well as pro-inflammatory mediators.Moreover, ECG could also induce cell cycle arrest, as well as inhibit cancer cell growth, invasion, and metastatic progression.ECG can interfere with multiple cell signaling pathways, such as Nrf2/ARE, ERK, and cAMP signaling pathways.In particular, the gallate moiety of ECG is critical for mediating inhibitory effects towards cancer cells.The antineoplastic mechanism contains inhibition of phase 1 CYP enzymes, induction of phase II detoxification and antioxidant enzymes, high anti-inflammatory efficacy, arrest of cell cycle progression, regulation of apoptosis, as well as mediation of metastasis processes.Besides regulation of intracellular signaling pathways, ECG also inhibits RNase A and MMPs enzymatic activity via chelating metals (copper and zinc) in cancer cells.ECG also serves as antioxidant, anti-obesity, cardioprotective, neuromodulatory,and antimicrobialagents.Although various pharmacological properties have been observed, mechanisms of which are still needed to be deeply clarified.The present review highlights recent significant findings about health benefits of ECG, and discusses corresponding mechanisms, hoping to develop ECG into preventive and therapeutic agents.Therefore, additional large, long-term cohort studies and clinical trials should be carried out to investigate the efficacy,pharmacology, safety and side-effects of ECG and other tea catechins.

Conflicts of interests

The authors declare that they have no competing interests.

Acknowledgement

This research reported in this paper are funded by Hubei Science and Technology Plan Key Project (G2019ABA100), Open fund of Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources Comprehensive Utilization (201932103), and fund from Assessment and Comprehensive Utilization of Characteristic Biological resources in Dabie Mountains (4022019006).