**1. Introduction**

Tea has been known to be the world most renowned non-alcoholic beverage since ancient times and it is currently being utilized by two-thirds population of the world, owing to its taste, stimulating effects, unique aroma, and associated health perspectives [1,2]. Various types of tea like green, black and oolong tea are derived from the *Camellia sinensis* (L.) plant containing variety of components among which polyphenols are most significant ones. Basic differences among all of these teas depends upon the stage of fermentation processes from which they are produced. Purposely, green tea is not fermented while black tea and oolong tea are completely and partially fermented, respectively. Amongst all of the investigated teas around the globe, green tea is well studied, owing to its health promoting benefits. In general, tea based phenolics possess protective action against numerous metabolic syndromes.

Several nutraceutical aspects of green tea extracts depends upon the concentration of phenolics and their associated derivatives. The major biologically active moiety present in leaves of green tea are classified as catechins that nearly account for 25–35% on dry weight basis. This catechin group comprises eight phenolic flavonoid constituents, specifically, catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, and epigallocatechin gallate (EGCG) [3,4]. Among above mentioned polyphenols, EGCG is the most vital tea based catechin which is considered to be the main reason for bioactivity of green tea [5–8].

Catechins are plant secondary metabolites [9,10]. They possess numerous functions in plant survival, growth, and metabolism, but they can also interact with other living organisms if ingested or came into direct contact. Catechins or flavanols probably constitute the most abundant subclass of flavonoids and they are essentially represented by (–)-epigallocatechin-3-gallate (EGCG), (–)-epigallocatechin (EGC), (–)-epicatechin-3-gallate (ECG), and (–)-epicatechin (EC) [11]. These last four bioactive compounds are found in large amount in green tea (leaves of *Camellia sinensis*) and cocoa/chocolate, but they are also present in many fruits or seeds, such apples, blueberries (*Vaccinium myrtillus*) and grapes (red wine and beer) and peanuts [12]. This review focuses on catechins that are derived from green tea and in particular on EGCG, which represents 50% to 80% of all *Camellia sinensis* catechins (200–300 mg/brewed cup of green tea) [13]. Moreover, we analyzed preclinical works examining its pharmacological and biomolecular mechanism of action.

Epigallocatechin gallate is the most bioactive catechin that is predominantly present in tea. Among all of the tea types, it is found at maximum comncentration in green tea leaves. EGCG molecule (Figure 1) comprises two aromatic structures that are co-joined by three carbon bridge structure (C6-C3-C6) along with hydroxyl group (OH) at simultaneous carbons i.e., 3', 4', 5' of ring-B. Carbon-3 of ring-C is esterified with a gallate molecule (features of catechins originated from tea are responsible for bioactivity due to position and number of OH-group on the rings. They regulate their capability to interact with biological matter via hydrogen bonding, a hydrogen and electron transferring process contained by their antioxidative potentials. The presence and absence of a galloyl molecule differentiates EGCG from remaining three catechins [14].

**Figure 1.** Chemical structure of epigallocatechin gallate.

Pure epigallocatechin gallate is classified as an odourless crystal and/or powder available in white, pink, or cream colour. It is considered to be soluble in water as a colorless and clear solution (5 mg mL−1). It is also soluble in methanol, tetrahydrofuran, acetone, pyridine, and ethanol. EGCG have a melting point at 218 ◦C [15].

Even though EGCG is found to be the most predominant and bioactive constituent present in tea, it is considered to be poorly stable in aqueous solutions and poorly soluble in oils and fats [16–19]. This

poor stability and solubility restricts its direct addition in food products. Numerous delivery systems are, in practice, to preserve its structural integrity and shield EGCG from degradation [18,20–24]. Additionally, structural modification in EGCG has also aided in elevating its lipophilicity [25,26].

Numerous physical aspects like pH, light, oxidants contents, oxygen, temperature, and concentration of EGCG, influence the stability of epigallocatechin gallate [17,26–29]. Epimerization and auto-oxidation are two most important reactions that result in instability of EGCG and loss of its health promoting properties [30]. EGCG is oxidized at low concentration and temperature i.e., 20 to 100 μM & < 44 ◦C, respectively. On the other hand, it is epimerized to gallocatechin gallate at elevated temperature (greater than 44 ◦C) and acidic conditions (pH 2 to 5.5) [30–33]. EGCG at a concentration of 300 mg per litre has been found to be stable at 25 ◦C (24 h) in a pH range of 3 to 6, nonetheless if kept at 4 ◦C & 25 ◦C for 24 h (pH 7), a loss of 25 & 83%, respectively, was observed [29].

EGCG is an abundant source of phenolic OH-groups that leads to its health escalating properties. Various scientific studies (in vivo and in vitro) have been carried out to authenticate the potential of EGCG as an anti-oxidative, antiinflammatory, and anti-cancerous agen<sup>t</sup> [34,35].

Wei and co-authors have reported that EGCG exerts an anti-tumor effect through the inhibition of key enzymes that participate in the glycolytic pathway and the suppression of glucose metabolism [36]. Shang and coworkers proposed that tea catechins in combination with anticancer drugs are being evaluated as a new cancer treatment strategy [37]. It has a potential role as adjuvant in cancer therapy and it could enhance the effect of conventional cancer therapies through additive or synergistic effects, as well as through the amelioration of deleterious side effects [38]. EGCG interacts with catalase and highlighted as an anticancer drug [39]. Green tea polyphenols are reported as a translational perspective of chemoprevention and treatment for cancer with concrete influence on signaling pathways [2]. EGCG as a powerful antioxidant, antiangiogenic, and antitumor agent, and as a modulator of tumor cell has the potential to impact in variety of human diseases [40].

This review was prepared systematically searching for articles, papers, books from different sources, such as Embase-Elsevier, Google Scholar, Ovid, PubMed, Science Direct, Scopus, and Web of Science. The search strategy was based on the combination of different keywords, such as EGCG, cancer, tumor, mechanism of action, preclinical, and clinical data, while considering any time of publication. Only sources written in English were included.

#### **2. Occurrence of Epigallocatechin-3-Gallate in Di**ff**erent Foods**

The close relationship between food and disease is well known [41], thus EGCG consumed in food could be a natural potential source of health benefits, even in complex and intricated disorder as cancer.

Principal catechin in tea is EGCG, which is also known to be responsible for various health modulating perspectives tht are related to utilization of tea. According to Wu et al., (2012), a cup of green tea prepared by adding 2500 mg leaves in 0.2 L of water contains 90 mg of EGCG [42]. The content of EGCG in black tea is less when compared to green tea, mainly due to polymerization of catechins [43]. EGCG that are derived from tea possess various bioactive properties and, therefore, have attained attention as potent functional and nutraceutical ingredient in food and pharmaceutical industry. Table 1 lists EGCG contents in some selected foods.


**Table 1.** Occurrence of epigallocatechin-3-gallate in different food.

### **3. Signaling Pathways Involving EGCG: Proliferation, Di**ff**erentiation, Apoptosis, Inflammation, Angiogenesis and Metastasis**

A plethora of biomolecular mechanisms has been demonstrated and proposed to be involved in the use of this catechin in preclinical models. These can include, but are not limited to, the induction of apoptosis, cell cycle changes, antioxidant activity, modulation of drug metabolism enzymes, etc. EGCG possesses multiple pharmacological activities, depending on cancer cell type, thus reflecting different protein expression and epigenetic mechanisms. Every cell model can express and modulate at convenience its genetic background, and this is at the basis of the diverse biomolecular targets triggered by EGCG. More consistently, EGCG has been analyzed in different tumor cell lines and less frequently in animal models: this paragraph investigates the association of EGCG with proliferation, differentiation, apoptosis, inflammation, angiogenesis, and metastasis, i.e., the main signaling pathways and processes of living cells (Figure 2).

**Figure 2.** Epigallocatechin gallate (EGCG) involved in the signaling pathways in cancer.

#### *3.1. Cell Death Induction (Apoptosis, Necrosis, Autophagy)*

The effects of EGCG on cell death induction, including apoptosis and autophagy, have been investigated in several preclinical studies. Jian and colleagues demonstrated that EGCG induced apoptosis of human liver hepatocellular carcinoma HepG2 cells and rat pheochromocytoma PC12 cells by decreasing the Bcl-2 protein and up-regulating Bax, when used at >20 μM [51]. The proposed mechanism of action of EGCG was related to Human antigen R (HuR, also known as ELAVL1), which can control amyloid protein precursor (APP) via HuR-Erk1/2-APP pathway. APP is implicated in Alzheimer's disease and cancer, blocking ATP with EGCG might be useful in the treatment.

A combination of clofarabine (640 nM and 50 nM in MCF7 and MDA-MB-231 cells, respectively) and EGCG (10 μM) also induced apoptosis in human breast cancer cells, followed by hypomethylation of the tumor suppressor gene RARβ (retinoic acid receptor β) and the upregulation of RARβ, PTEN, and CDKN1A genes [52]. Clofarfabine can inhibit DNA methylation reaction and, thus, its combination with EGCG can have a role in blocking breast cancer development.

The effects of EGCG (>80 μM) on tongue squamous cell carcinoma were recently investigated, showing that it reduced proliferation while promoting apoptosis in CAL27 and SCC 15 cells via the Hippo-TAZ signaling pathway [53]. Similar to other tumor cell models, EGCG (10–200 μM) induced apoptosis in a dose-dependent manner in human thyroid carcinoma cell lines by increasing the Bax/Bcl-2 ratio, thus pointing to an increase of mitochondrial-mediated apoptosis and the involvement of EGFR/RAS/RAF/MEK/ERK signaling pathway [54]. In addition to apoptosis, cell treated with EGCG has been associated with autophagy regulation. The treatment of human embryonic kidney cell line (HEK293T cells) with EGCG increased cell viability by promoting autophagy through mTOR pathway [55]. In hepatocellular carcinoma cells, EGCG induced autophagy via direct interaction with LC3-I protein and, consequently, led to the degradation of α-fetal protein (AFP); an effect that suggests a potential therapeutic application for the treatment of this type of tumor [56]. EGCG-induced autophagy is also involved in the regulation of ectopic lipid accumulation in vascular endothelial cells, which indicates a potential beneficial effect in preventing cardiovascular diseases [57]. Thus, EGCG is involved in autophagy, even in different tumor cell lines, but with concentration dependent on cell tissue origin. The effect of EGCG on cell death mechanisms is well documented. EGCG has been also proposed as protective agen<sup>t</sup> against necrosis induction because of its demonstrated action on reduction of oxidative stress and inflammatory responses [58].

#### *3.2. Cell Cycle Arrest (Cyclin, CDK)*

The effect of EGCG on cell proliferation and cycle has been investigated in various cancers. EGCG in a early work has been demonstrated to play an effective role as an anti-proliferative and chemopreventive agen<sup>t</sup> breast cancer, colon cancer, and skin cancers, such as melanoma in experiments while using MCF-7, HT-29, and UACC-375 cell lines [59]. Specific effects on cell cycle modulation were also investigated in a liver cancer model in which EGCG inhibited the proliferation of liver cancer cells in G2 phase and induced apoptosis after the blocking of the cell cycle progression in the G1 phase [60]. Similar effects were supported in a study on skin cancer, in which the benefits of using polyphenols, such as EGCG, in the treatment and prevention of skin cancer were extensively presented [61]. The effect of EGCG treatment has been also investigated on different cancer types in which cell cycle modulation was demonstrated through the alteration of the pattern of cell cycle proteins with specific reference to CDKs. EGCG increased the expression of CDK1 proteins and inhibited the expression of Cyclin D1 and the phosphorylation of retinoblastoma proteins. Moreover, EGCG treatment has been associated to cell cycle arrest affecting upregulation/downregulation balance in which p1/p21, Kip1/p27, p16/INK4A, cyclin D1, cyclin E, CDK2, and CDK4 proteins are involved. This effect was demonstrated to be the first step of the apoptosis cascade induction via ROS and subsequent caspase-3 and caspase-9 action [62]. These conclusions were then supported by additional in vitro and in vivo experiments in melanoma cancer cells. EGCG treatment of melanoma cells results in the modulation of cyclin-dependent kinases inhibitor network and Bcl2 family proteins with a consequent inhibition of viability and growth, cell cycle blocking, and apoptosis induction [63]. Nevertheless, it was also demonstrated that EGCG can enhance the therapeutic effect if used in synergy with IFN in both in vitro and in vivo models of melanoma, which could be an important advantage in reducing the concentration of IFN and the toxicity of the treatment [64]. The reliability of the treatment or the synergic use of EGCG is additionally supported by a large number of in vitro and in vivo scientific reports on the effects of EGCG on cell cycle modulation and apoptosis induction without affecting normal cells [65]. The studies that are presented in this paragraph point to the usefulness of EGCG in treatment against malignancies by cell cycle modulation and cell death induction, both as therapeutic agen<sup>t</sup> and synergic adjuvant [13,66].
