Zhiy Yin, Ting Zheng, Chi-Tng Ho, Qingrong Hung, Qingli Wu,c,d, Mn Zhng,*

a Department of Food Science, Rutgers University, NJ 08901, USA

b New Use Agriculture and Natural Plant Products Program, Department of Plant Biology, Rutgers University, NJ 08901, USA

c Department of Medicinal Chemistry, Rutgers University, NJ 08854, USA

d Center for Agricultural Food Ecosystems, The New Jersey Institute for Food, Nutrition and Health, Rutgers University, NJ 08901, USA

Keywords:

Tea polyphenols

Encapsulation

Stability

Bioavailability

Bioefficacy

A B S T R A C T

Tea polyphenols (TPPs) have attracted significant research interest due to their health benefits.However, TPPs are sensitive to certain environmental and gastrointestinal conditions and their oral bioavailability was found to be very low.Delivery systems made of food-grade materials have been reported to improve the shelf-life, bioavailability and bioefficacy of TPPs.This review discusses the chemistry of TPPs; the setbacks of TPPs for application; and the strategies to counteract application limitations by rationally designing delivery systems.An overview of different formulations used to encapsulate TPPs is provided in this study, such as emulsion-based systems (liposome, nanoemulsion, double emulsion, and Pickering emulsion)and nano/microparticles-based systems (protein-based, carbohydrate-based, and bi-polymer based).In addition, the stability, bioavailability and bioactivities of encapsulated TPPs are evaluated by various in vitro and in vivo models.The current findings provide scientific insights in encapsulation approaches for the delivery of TPPs, which can be of great value to TPPs-fortified food products.Further explorations are needed for the encapsulated TPPs in terms of their applications in the real food industry as well as their biological fate and functional pathways in vivo.

1.Introduction

Tea is a widely consumed beverage obtained from the leaves ofCamellia sinensis, an evergreen shrub native to China and East Asia [1].All types of tea contain a wide variety of bioactive compounds such as tea polyphenols (TPPs), caffeine and so on.Earlier researches have revealed that the major components responsible for the health benefits of tea consumption are polyphenols [2].TPPs are known and credited with their antioxidant, anti-inflammatory, anti-cancer and other therapeutic properties [3,4], but epidemiological studies reported that large volumes of tea have to be consumed in order to obtain these health promoting effects [5].Therefore, extracting TPPs from tea leaves and fortifying them in dietary supplementations and food products have gained a lot of interest.

However, it has been brought to light that the application of TPPs in food is limited by several factors.On the one hand, the poor stability of TPPs against temperature, light, pH and oxygens largely accelerates their degradation during long-time storage [6].On the other hand, due to the harsh environment in gastrointestinal tract(GI) and low permeability across intestinal membranes, only a small proportion of TPPs remains available for absorption in human body after ingestion, leading to a low bioavailability of TPPs [7].

Encapsulation strategies, which have been successfully applied in many nutraceutical-contained supplements, represent a solution to the challenge of TPPs application.The encapsulation materials for TPPs are required to be food grade or generally regarded as safe (GRAS)for their application in food industry.The encapsulation materials can be divided into two categories based on their molecular weight:small molecules (e.g., lecithin) and large molecules (e.g., proteins and carbohydrate polymers).Besides, different types of formulations have been investigated to encapsulate TPPs in food products, including the emulsion-based systems and particle-based systems.So far,remarkable delivery systems have been developed to incorporate TPPs, and showed significant improvements on their stability during storage and their oral bioavailability [8,9].

This review covers the characteristics of TPPs and the rationale for their encapsulations.It provides a comprehensive overview of the fabrications and characterizations of TPPs-loaded delivery systems and appraises the performance of these systems regarding the enhancement of TPPs stability and bioavailability through multiplein vitroandin vivomodels.Furthermore, the challenges of current researches and future perspectives are also discussed.This review can be of great value on TPPs applications in the food industries.

2.Chemistry of tea polyphenols

Green tea and fermented tea (e.g., oolong tea and black tea)are two generally consumed forms of tea and both are rich in polyphenols.With different processing methods, green and fermented tea own different phenolic compositions.Compared to other types of tea, green tea possesses the highest phenolic content [10], in which flavonoids and phenolic acids account for up to 30% of fresh leaves dry weight.Flavan-3-ols, commonly known as catechins, are the most representative flavonoids in green tea and possess a 2-phenyl-3,4-dihydro-2H-chromen-3-ol skeleton.The primary catechins in green tea include: (+)-catechin (C), (–)-epicatechin (EC), (–)-gallocatechin(GC), (–)-epigallocatechin (EGC), (–)-epicatechin gallate (ECG),(–)-epigallocatechin gallate (EGCG) [11]as shown in Fig.1.

Fig.1 Chemical structures of major tea polyphenols in green tea and fermented tea.

Unlike green tea, which is produced by gradually drying tea leaves [12],fermented tea such as black tea is manufactured by either of two ways: the orthodox and the ‘crush-tear-curl’ (CTC) process.The orthodox process includes withering, rolling, sorting, oxidation, and drying of tea leaves, while the CTC process consists of pre-cutting the withered leaves and passing them through the CTC machine [13].The formation of TPPs in fermented tea involves oxidation and polymerization processes [14], in which the green tea catechins are partially oxidized to quinones under enzymatic catalysis, followed by nucleophilic addition and rearrangements [15].Upon termination of the fermentation process, the major polyphenols in fermented tea are catechins, theaflavins and thearubigins.The group of theaflavins is characterized by a benzotropolone structure and includes thea flavin(TF1), thea flavin-3-gallate (TF2A), thea flavin-3’-gallate (TF2B), and thea flavin-3,3’-digallate (TF3) (Fig.1) [16].

After ingestion, only a small fraction of TPPs in the small intestine can be absorbed with or without metabolism (sulfation,glucuronidation andO-methylation), while the remaining TPPs will enter the colon and subject to catabolism by gut microbiota.After TPPs and their metabolites in the small intestine and colon are absorbed, they are then transported to the liver via portal vein for a more extensively metabolism.In the liver, TPPs and their metabolites either go through an enterohepatic recirculation process and return to small intestine following the biliary secretion, or continue entering the systemic circulation and being delivered to organs and tissues.For the excretion of TPPs, generally more TPPs are excreted through feces than through urine.It has been reported that relatively high level of TPPs was absorbed in small intestinal mucosa, while high level of TPPs metabolites was detected in colon mucosa [17-19].The level of TPPs in organs and tissues after oral administration is in the order of kidney > brain > lung > heart > spleen > liver.Thet1/2(Ka)of TPPs is generally ranged 1-3 h, and EGCG has the longestt1/2(Ka)among TPPs in all tissues and organs [20-22].

Numerous animals and clinical studies have documented the biological activities of TPPs.The hydroxyl groups on TPPs can scavenge reactive oxygen species (ROS), which cause cellular oxidative stress and damaging proteins and DNA in their high levels.Thus, TPPs are reported to demonstrate good antioxidant effect by breaking down the chain reactions of free radicals and preventing the hazardous consequence [2,23].Studies also suggested that owing to the binding capacity with proteins, TPPs can modulate redox-sensitive transcription factors, inhibit mitogen activated protein kinases and proteases, and decrease the aberrant arachidonic acid metabolism [7].These activities of TPPs further lead to cell apoptosis and proliferation inhibition [24], resulting in reduced risks of cancers [25].In addition, TPPs also have been reported to exhibit anti-cardiovascular activity [16], anti-obesity [26]and anti-diabetic effects [27], and neuroprotective properties [28].Moreover, plenty of research have provided evidence of the gut health benefits of TPPs.TPPs are found to modulate gut microbiota by binding on bacterial cell membrane, altering membrane permeability, and suppressing the growth of pathogenic bacterial species [29-31].

3.Setbacks of tea polyphenols

3.1 In vitro: poor stability

To date, despite plenty of evidences have shown promising therapeutic effects of TPPs, their application to the food industry was limited by their poor stability [32].TPPs are very sensitive to environmental factors such as high temperature and neutral to alkaline pH conditions [33], which may cause the brown and darker appearance and unwanted odors.Li et al.[15]has performed a storage stability study of black TPPs in harsh environment.They found that even though catechins and thea flavins in solid black tea extract can tolerate high temperatures (up to 75 °C) with prolonged storage,these polyphenols are unstable and susceptible to fast degradation in aqueous solution or in a highly humid environment [15].Thea flavins were reported to be more vulnerable to degradation than catechins, in conditions with alkaline pH and elevated temperature [6].Moreover,EGCG and EGC are less stable than EC and ECG: the former two were completely degraded after 6 h incubation at pH 7.4 aqueous condition, while the latter two were only degraded by less than 35%.An analysis performed by colorimeter showed that TPPs solution remained stable at a pH of 3-6 and temperature of 4 °C or 25 °C, but it became less green and even deeper yellow in color with increased temperature and alkalinity [34].In addition, the TPPs became unstable when subjected to light or oxidants exposure [35,36].The scavenging properties of TPPs make them readily react with free radicals such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) [37], hydroxyl radicals,superoxide anions [38], and lipid free radicals [39].Sang et al.[40]confirmed the structures of eight products derived from epicatechin oxidation and reported that B-ring is the initial reacted site of tea catechins for their oxidation products yielding from reactions with peroxyl radicals.It also has been noted in a photostability study that the blue light illumination (BLI) can trigger the polymerization of catechins and generate the superoxide anion radical via photosensitive oxidation [41].The degradation triggered by pH, temperature, light,and oxidants usually weaken the health promoting properties of TPPs such as antioxidant activity.Together, given the instability of TPPs, it is desirable to incorporate them into the formulations which could provide protection from environmental challenges during food processing and storage.

3.2 In vivo: low bioavailability

Bioavailability is defined as the fraction (%) of an administered drug or an ingested nutraceutical that reaches the systemic circulation and specific sites where it exerts biological functions [42].In previous literature, it is considered that the bio-efficacy of TPPs is limited due to their low oral bioavailability [43].Several factors that hinder the oral bioavailability of TPPs have been proposed, including poor GI stability, low intestinal absorption rate, and scarce biodistribution [44].

The environment of GI tract (GIT) is quite harsh for TPPs due to the elevated pH, residual dissolved oxygen, and metabolic enzymes [45].It has been shown that after passing through the salivary, gastric fluid and reaching upper small intestinal phases in anin vitrodigestion model, C, EGC, and EGCG show significant degradation and low recovery of 5.3%, 4.6%, and 6.1%, respectively [46].In addition to degradation, TPPs also suffer from the auto-oxidation and epimerization induced by residual dissolved oxygen in GIT [47].Moreover, a fraction of TPPs undergoes metabolic transformation by enzymes such as sulphotransferases (SULTs), UDP-glucuronosyltransferases (UGTs), and catechol-O-methyltransferase(COMT) in the small intestine and liver.Researchers have found that the liver microsomes have the greatest catalytic efficiency for the glucuronidation of EGC compared to other organs, and EGC is mainly presented as the glucuronidated conjugate (57%-71%) or sulfated conjugate (23%-36%) in human bodies [48,49].Methylation of TPPs can also occur and subsequent metabolites such as 3′- and 4’-O-methyl-EC, 4’-O-methyl EGC, 4’’-O-methyl ECG and EGCG,and 4’,4’’-di-O-methyl-EGCG were found in plasma [50].

The poor intestinal permeability of TPPs also plays a vital role in limiting their availabilityin vivo.Intestinal absorption for nutraceuticals is influenced by several factors, such as the active efflux, compound’s polarity and molecular weight.The mechanism for TPPs being transported across the small intestine epithelium is mainly via passive diffusion [51,52].Cell studies have showed that TPPs have low apparent permeability coefficients (Papp) because they underwent active efflux after being absorbed, during which a large amount of them are pumped back to lumen by ATP-binding cassette (ABC) transporters in epithelial cells and leading to low biotransportation rate [51,53-55].In addition, the low bioavailability of TPPs is also related to their molecular features.According to Lipinski’s Rule of Five [56], a compound is likely to have low bioavailability, if it has a molecular weight over 500, or contains at least five hydrogen bond donors, or holds at least 10 hydrogen bond acceptors.The multiple hydrogen bond donors and acceptors on the TPPs’ structures are expected to form a large hydration shell and increase the apparent size of these hydrophilic molecules, increasing the difficulty in passing through the lipophilic intestinal membrane.Accordingly, the EGC and EC, with fewer hydroxyl groups, are predicted to be more bioavailable than EGCG, while thea flavins and thearubigins, are expected to have extremely low bioavailability [7,57].

Moreover, a large fraction of TPPs pass into the colon after oral administration.They would be transformed to hydroxyphenyl-γvalerolactones through ring fission metabolism by gut microbiota,and further metabolized to small-molecular phenolic acids.These metabolites will be either re-absorbed into system circulation or eliminated from body via feces [58].

4.Encapsulation strategies for TPPs

The encapsulation of TPPs is desired for two reasons:1) improving their stability against harsh environment for shelflife extension; 2) improving their bioavailability for bio-efficacy enhancement.It is necessary to mention that only food grade or GRAS status materials could be used for the formulation designed for delivering TPPs in food applications [59].In general, the reported delivery vehicles can be classified into two categories, including emulsion-based systems and nano/microparticle-based systems.Parameters such as loading capacity (LC) (calculated by Equ (1)),encapsulation efficiency (EE) (calculated by Equ (2)), particle size, and zeta potential have been used for the characterization of each delivery system.The important delivery systems for TPPs are summarized in Fig.2.

Fig.2 Important delivery systems for TPPs.(a) liposomes, (b) double emulsions (W/O/W), (c) Pickering emulsions (left: Pickering emulsion with TPPs encapsulated inside, right: TPPs-particles stabilized Pickering emulsion), (d) nanoemulsions (left: O/W emulsion, right: W/O emulsion), (e) carbohydrate-based particles (left: cyclodextrin inclusion, right: other carbohydrate-based particles), (f) protein-based particles, (g) biopolymers-based particles.

4.1 Emulsion-based systems

Emulsion-based delivery systems are commonly used strategies for encapsulating TPPs.So far, the reported emulsionbased formulation can be generally classified into four categories,including liposomes, nanoemulsions, Pickering emulsions, and double emulsions (Table 1).With the aids of high speed/pressure homogenization, ultrasonic emulsification, and other preparation technologies, stable emulsions can be produced [60].

Table 1Emulsion-baseddelivery systems for TPPsa.

4.1.1 Liposome

A liposome is a spherical vesicle composed of phospholipids and compatible with a lipid bilayer structure [61].Liposomes offer several advantages as a drug/nutraceutical delivery system, including biocompatibility, the capacity of self-assembly, and modification acceptability [62].

Several technologies were involved in facilitating the preparation of TPPs-loaded liposomes.For example, Zou et al.[63]developed the EGCG loaded nanoliposome (EN) through an ethanol injection method combined with dynamic high-pressure microfluidization.The obtained EN possessed EE of 92.1%, droplet size of 71.7 nm and a low polydispersity index of 0.286.Besides, the stability of EGCG in simulated intestinal fluid (SIF) was improved significantly by encapsulation, which led to a higher antioxidant activity after digestion as compared to free EGCG.Similarly, green tea polyphenols were encapsulated into liposome by the thin film ultrasonic dispersion method with a mean droplet size of 160.4 nm and the ζ-potential of –67.2 mV.The formulation was optimized by tuning several parameters, including the ratio of TPPs to lecithin (0.125:1); the ratio of lecithin to cholesterol (4:1); the pH of phosphate buffered saline (PBS) (6.62) and ultrasonic time (3.5 min) [64].Through microfluidization and ultrasonication techniques, Dag et al.[65]encapsulated green tea extract into liposomes, and the developed liposomes had unchanged size (about 40 nm) and uniform shape during 1 month storage period as shown by TEM images.

Recently, several modified liposome delivery systems have been developed by incorporating functional molecules on the liposome surface.The reported modification significantly improved the stability of liposomes and provided controlled release property and higher EE of TPPs.For examples, Minnelli et al.[66]encapsulated EGCG into the Poloxamer-407 modified anionic liposomes, which were consisted of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and cholesteryl hemisuccinate.The magnesium salt was added in the preparation at 5:1 Mg2+/EGCG molar ratio to maximize EGCG internalization [67,68].The EE of EGCG in the final emulsion was 100% and a multilamellar structure of this EGCG-loaded liposome was confirmed by X-ray diffraction.Thein vitrostudy showed that this formulation contributed to the sustained release of encapsulated EGCG.Zou et al.[69]prepared amphiphilic chitosan (CS) derivative cholesterol nanoliposome with surface modification by dextran sulfate, and the encapsulated EGCG exhibited a high EE (90.8%) and a good sustaining release property.CS and dextran sulfate coatings increased the zeta potential in magnitude of the liposome droplets, which might contribute to stronger electric repulsion force between each liposome and result in higher stability against aggregation.In addition to CS, which is positively charged, applying the negatively charged biopolymers such as gum Arabic (GA) and whey protein in the formation of liposome layers have been also elucidated as alternative ways to improve liposomes stability against coalescence and decrease internal lipid oxidation [70].For example, Dag et al.[70]found that by incorporating anionic and cationic biopolymers (GA, whey protein,lysozyme, and CS) into the green tea extract loaded liposomes, the whole system gained stability against changes of particle sizes and zeta potential during storage.

TPPs-loaded liposomes have been used as delivery vehicles applied into functional food products.For example, Rashidinejad et al.[71]developed the soy lecithin liposomes for the delivery of catechin and EGCG.The EE were greater than 70% with LC of 80% approximately.Electron microscopy revealed the lamellae and central core of the liposomes and the additions of TPPs increased the liposome phase transition temperature.The retention study in a lowfat hard cheese system showed that encapsulation by liposomes is a promising method to protect catechin and EGCG in a cheese matrix against interactions with other food components and degradation during food processing.

With suitable preparation methods, liposomes can be used as the co-delivery systems for TPPs and other nutraceuticals.It was reported that aqueous soluble TPPs and insoluble vitamin E can be loaded simultaneously into the nanoscale complex liposome by reverse-phase evaporation method, and the EE of TPPs and vitamin E were (50.81 ±1.91)% and (94.05 ± 3.45)%, respectively [72].

4.1.2 Nanoemulsion

Nanoemulsion is widely investigated as the delivery carrier for TPPs.These systems typically consist of aqueous phase, oil phase and stabilizers, where chemical surfactants and food-grade emulsifiers are commonly used.The mean droplet size of nanoemulsion is usually< 500 nm which gives the emulsion a clear or hazy appearance.Ultra-low interfacial tension and large interfacial area of provide nanoemulsions higher thermodynamic stability compared to other types of emulsions [73].In order to produce nanoscale-emulsion,different emulsification technologies such as homogenization and sonification are usually applied in the preparation processes [74].

Tian et al.[75]combined the hydrogel and the water-in-oil (W/O)emulsion to sustain the release of TPPs.The TPPs were firstly loaded into xanthan gum (XG)/locust bean gum (LBG) hydrogel(XG:LBG = 6:4), and the rheological properties of this hydrogel network was determined to be dense and highly viscous.Then, the whole mixture was used as the dispersed phase to prepare W/O emulsion by using PGPR as the emulsifier.It was found that the emulsion stability increased with increasing concentration of either PGPR or the XG/LBG.The application of binary gum hydrogel into the W/O emulsion significantly improved the retention rate of TPPs during long-time storage [75].Besides of W/O emulsion, oil-inwater (O/W) emulsions were also developed to encapsulate TPPs.As TPPs are polar in nature, their solubility in oil is very limited.Incorporation of TPPs in O/W emulsion is challenging but could be realized in several studies.For example, previous research showed that catechins could be readily dissolved in oil rich in C12fatty acids [76].Other solubility enhancers such as 1-dodecanol was also applied to enhance loading of TPPs in oil combining with lecithin.Bhushani et al.[77]used soy protein stabilized O/W nanoemulsion to encapsulate green tea extract (containing 0.38% catechin, 9.5% EGC,35.64% EGCG, 11.3% ECG, and 6.56% EC) with the aid of highspeed and high-pressure homogenizations.During the preparation of nanoemulsion, green tea extract was dissolved in sunflower oil(SFO) at the heating condition of 55 °C.The mean droplet diameter of the optimized nanoemulsion was (268.33 ± 1.15) nm and the total system was stable against the phase separation and pH change for a period of 15 days.Gadkari et al.[78]developed nanoemulsions using Tween 20, Tween 80 and lecithin as the emulsifiers and investigated the influence of oil phase (SFO and palm oil (PO)) on nanoemulsion formation.The TPPs extract (containing 4.86% EGC, 12.37% EGCG,2.12% EC and 1.25% ECG) was successfully encapsulated in both SFO and PO at a concentration of 0.1% (m/m) of the whole system.In another study, they further encapsulated isolated-EGCG into the O/W nanoemulsion and the solubility of EGCG in SFO was improved to(85.98 ± 2.31) mg/g in SFO by using 1-dodecanol as carrier material.The optimum formulation possessed a droplet diameter of (280 ± 10) nm and EE of (83.16 ± 1.12)% [79].Peng et al.[9]encapsulated TPPs(containing 54.91% EGCG, 20.04% EGC, 11.17% ECG, 3.37% EC,and 0.51% C) in the O/W emulsion consisting of corn oil and Tween 80 through high-pressure homogenization.The formed nanoemulsion had droplet sizes of (99.42 ± 1.25) nm and this system achieved the controlled release of EGCG.

4.1.3 Pickering emulsion

The Pickering emulsions have been demonstrated as good carriers of TPPs in current studies.Different from classical emulsions stabilized by surfactants, Pickering emulsions are stabilized by the food-grade solid particles synthesized from proteins or starch.The detachment energy for particles on the interface is very large.Once these solid particles absorb on the Pickering emulsion interface, it is very difficult for them to detach.Therefore, Pickering emulsions have good stability against coalescence, as compared to conventional emulsion [80,81].On the one hand, Pickering emulsion is able to contain high internal phase, which advantages for improving the loading of TPPs.On the other hand, TPPs-protein/carbohydrate solid particles can also act as stabilizer to produce surfactant-free emulsions.

The droplet size of Pickering emulsions is often in the micron size range, which is much greater than that of liposomes and nanoemulsions.Zhang et al.[80]prepared an EGCG-loaded Pickering emulsion and demonstrated its application in improving the bioaccessibility of EGCG.The EGCG-loaded zein nanoparticles were first developed, and then these particles were further used to form Pickering emulsion.The whole system showed high stability against the change of pH and ionic strength with droplet size ranging from 15.4 μm to 58.3 μm.Similarly, Shao et al.[82]prepared taro starch nanoparticles with particle size of 460 nm and a contact angle of 81.5°.After fully gelatinization of taro starch nanoparticles with TPPs,these gelatinized particles were able to form Pickering emulsion with 50% oil fraction and with 67% EE of TPPs.Ye et al.[83]developed the TPPs loaded W/O Pickering emulsion using zein as a stabilizer.In this formulation, zein nanoparticles, Span 80 and Tween 80 were applied as stabilizers at the interface of grapeseed oil and aqueous phase as stabilizers.The water phase containing TPPs was added dropwise into the oil phase at the ratio of 8:10 under magnetic stirring following by high-speed homogenization.The optimum emulsion had an average size of 3.43 μm and the LC of 0.053%.

4.1.4 Double emulsion

Double emulsions such as W/O/W and O/W/O emulsions are complex polydisperse systems which are stabilized by multiple amphiphilic molecules with different hydrophilic-lipophilic balance(HLB) values.They can serve as efficient carriers for lipid-soluble and water-soluble bioactives simultaneously.

W/O/W emulsions are usually applied in TPPs encapsulations [84-87].Tian et al.[84]applied the XG/LBG hydrogel system into the inner water phase of W/O/W emulsion.The formed double emulsion used lecithin and XG as the outer phase emulsifiers,and results showed that the double emulsion with a gelled inner phase not only provided protection for TPPs but also maintained more than 50% of the antioxidant capacity of TPPs.Guzmán-Díaz et al.[85]developed and characterized the green tea extract loaded gelleddouble emulsions.Different biopolymers such as chia mucilage,carrageenan, LBG, thixogum, and whey protein concentrate were used as gelators.Their work demonstrated that the gelled double emulsions are the alternative ways to preserve green tea extract.By applying the two-step emulsification method, Aditya et al.[86]developed the W/O/W double emulsion for co-delivering hydrophobic and hydrophilic nutraceuticals.The curcumin and tea catechin loaded double emulsion prepared showed high EE of 88%-97% for the two phytochemicals, and the droplet size was around 2.8-3.0 μm.Evageliou et al.[87]prepared the W/O/W emulsion containing 0.8% PGPR, 0.25% bacterial cellulose, 1% whey protein isolate and NaCl (1.6%–8%).Both EGCG and the stearic acid esterified EGCG were successfully incorporated in the double emulsion, and the EE decreased in the order: ‘EGCG in the inner water phase’ > ‘EGCG in the oil phase’ > ‘esterified EGCG in the inner water phase’.

4.2 Nano/microparticles-based systems

The nano/microparticles-based delivery systems are prepared from biocompatible and biodegradable polymers where TPPs are dissolved in, entrapped or attached to a particle matrix.Typical encapsulation techniques used for preparing nano/microparticles include spray drying, extrusion, coacervation, crosslinking reactions, electrospraying,electrospinning and layer-by-layer (LbL) self-assembly [88].Nanoparticles are in the size range of 10-500 nm, while microparticles are in the micron size range and can be up to 800 μm [89,90].In general, nano/microparticles-based systems designed for TPPs encapsulation can be divided into three sections based on the polymer types: protein-based, carbohydrate-based and bi-polymer-based systems (Table 2).

Table 2Particle-based delivery systems for TPPsa.

Table 2 (Continued)

4.2.1 Protein-based particles

Food grade proteins are attractive ingredients in industries because of their nutritional value.Their functional properties, such as emulsification, gelation and binding capacity, make them useful in developing TPPs-loaded delivery systems.

Gelatin is a protein obtained from partial hydrolysis of naturally occurring collagen.It can form thermo-reversible hydrogels which make it a promising material for carrying TPPs.Compared with gelatin B (pI = 4-5), gelatin A (pI = 7-9) is more widely used in TPPs encapsulation because positively charged gelatin A can interact with negatively charged TPPs at neutral pH condition.Kulandaivelu et al.[91]prepared TPPs encapsulated gelatin nanoparticles with surface modification using LbL assembly technology.Briefly, TPPs were encapsulated by six polyelectrolyte layers, including polystyrene sulphonate (PSS), poly-L-glutamic acid (PGA), polyallylamine hydrochloride (PAH), poly-L-lysine (PLL), protamine sulphate(ProtS), and dextran sulphate (DexS).The highest EE was achieved by adding six polyelectrolyte layers and incubating for 4 h, and the formed nanoparticles were spherical with the size of 50 nm [91].Shutava et al.[92,93]also applied LbL method to form EGCG-loaded gelatin A nanoparticles with polystyrene sulfonate/polyallylamine hydrochloride layers.The loaded nanoparticles have the LC of 30%(m/m) and particle size of 50-300 nm.Gomez-Mascaraque et al.[94,95]assembled the gelatin/EGCG capsulate via electrospray.The LC, EE and particle size of the nanoparticles were 10%, 96% and 470 nm,respectively, which were better than those of particles formed by spray drying.

Milk proteins have been reported as the good carriers for TPPs.The amino acid residues, hydrophilic and hydrophobic domains of milk proteins contribute to the different affinities with TPPs.Chanphai et al.[96]investigated the EE of catechin, EC, ECG and EGCG with different milk proteins.They found that the order of protein affinity toward TPPs wasβ-casein >α-casein >β-lactoglobulin(β-Lg).The EE was 30%-50% for TPPs-milk protein adducts and the polyphenols with larger molecular weight formed more stable protein complexes.The binding between heated and nativeβ-Lg with EGCG were studied in several experiments [35,97,98].Heat treatments makeβ-Lg undergo dissociation and conformational modifications, which can provide greater affinity for TPPs compared with nativeβ-Lg.The sizes of formed nanoparticles were all below 100 nm, and the EE were in the range of 54%-94%.The EGCG loaded casein also have been studied for their nano-structure and anticancer ability [99-101].

Except for milk proteins and gelatin, other proteins from different plant sources have been used to fabricate TPPs encapsulated nanoparticles.For example, ferritin [102], zein [103], and rice bran isolate [104]were reported to form nanocomplexes with green tea catechins and improve the stability and efficacy of TPPs.

4.2.2 Carbohydrate-based particles

Carbohydrates are preferred as encapsulation materials due to their good biodegradability and biocompatibility.To improve the formulations’ thermal or mechanical properties, it is necessary to choose the carbohydrates properly and chemically/enzymatically modifications on carbohydrates are usually employed.

Starch and its derivatives have been investigated for their performance to encapsulate TPPs.Goncalves et al.[105]formulated EGCG-loaded particles using modifiedn-octenyl succinate anhydride(OSA)-starch through the particles from gas saturated solution drying.This non-cytotoxic solid formulation was in the range of micrometers and the EE was up to 80.5%.Cyclodextrin (CD) is a cyclic oligosaccharide produced from starch by enzymatic conversion and it can form complexes with TPPs through hydrophobic interaction and hydrogen bonding.Ho et al.[106,107]developed catechin/β-CD inclusion complex, and found that catechin loadedβ-CD not only masked the bitterness of catechin but also provided protection against degradation in the fortified milk, cheese and yogurt.Maltodextrin(MD) is also a partially hydrolyzed product from starch and is commonly used in the industry as wall material.By combining with spray drying technique, Zokti et al.[108]fabricated green tea polyphenols loaded maltodextrin microparticles.The microparticles were on a scale of 40-226 μm and the EE was 96%.By using the same wall materials, Rocha et al.[109]produced EGCG encapsulated nanoparticles with smaller particle size (120 nm), with the aid of homogenization and spray drying.

CS has good biodegradable and non-toxic properties, and therefore is widely used in delivery systems.The formation of TPPs/CS nanoparticles is generally based on the weak and non-covalent bonds,such as hydrogen binding and weak ionic binding [110].For example,Liu et al.[111]developed EGCG loaded nanoparticles by crosslinking CS hydrochloride and sulfobutyl ether-β-CD sodium, and the particle size was around 200 nm.Liang et al.[112,113]prepared TPPs loaded nanoparticles using carboxymethyl CS and CS hydrochloride as carriers.The developed nanoparticles were non-spherical shape with an average size of (407 ± 50) nm, and the LC and EE were 8%-16% and 44%-83%, respectively.Hu et al.[114]fabricated CS-tripolyphosphate nanoparticles for delivering tea catechins with yielding EE of 24%-53%.Hong et al.[115]fabricated the EGCG loaded pH-responsive CS-aspartic acid nanoparticles with an average diameter of 102 nm.According to a rabbit study, it indicated that this nano-formulation could be a promising method to increase therapeutic effect of EGCG on atherosclerosis.

Besides of starch and CS, alginate [116], inulin [117], and GA [118]have also been used to encapsulate TPPs with the assistance of homogenization or spray drying.

4.2.3 Bi-polymer-based particles

Bi-polymeric particles are engineered from biocompatible and biodegradable polymers.By combination of proteins/peptides and polysaccharides, complexes with desired properties can be developed through the self-assembly process [119].In this process, proteins/peptides and polysaccharides generally form electrostatic complexes when they have opposite charge at specific pH condition.The interactions between proteins/peptides, polysaccharides, and TPPs are important in design of such delivery system.Due to the high affinity with proteins, TPPs are often firstly interacted with the proteins through hydrogen bonding or hydrophobic interaction.Then the loaded proteins can be crosslinked with oppositely charged polysaccharides in the solution to form bi-polymeric particles by the electrostatic interaction.The protein/peptide-polysaccharide nanoparticle is a promising way because it can achieve high EE and LC, and possibly control the release of TPPs from delivery vehicles [88].

Tang et al.[120]formed the self-assembled nanoparticles composed of TPPs, CS andγ-PGA.This nanoparticle system was suggested to be a suitable transmucosal delivery system for tea catechin in the jejunum and ileum because of their pH-responsive properties.By tuning the electrostatic interaction between CS andγ-PGA, the nanoparticles would remain stable at pH = 2.5-6.9 but disintegrate and release TPPs at the pH condition of jejunum and ileum.Another research showed that the alginate-calcium caseinate (or whey proteins) microparticles could achieve the highest EE of TPPs(up to 80%), while the alginate-bovine serum albumin (BSA) particles provided the most spherical and softer structure.Coating with CS or pectin conferred better a release profile of TPPs from the alginateprotein particles, and this phenomenon might result from the reduced porosity in the particle matrix [121].Oliveira et al.[122]developedβ-Lg-pectin/CS system to improve the catechin bioaccessibility in the simulating GI condition.Kumar et al.[123]fabricated the BSACS nanoparticles for delivery of TPPs.The LC, EE and particle size of the BSA-CS nanoparticles are (42.9 ± 2.5)%, (26.1 ± 1.3)% and 285 nm, respectively.

Apart from forming bi-polymeric complexes via non-covalent bond, developing particles via covalent bonds is another appealing option to encapsulation TPPs.Forming covalent bonds between proteins and polysaccharides have been reported to enhance the stability of conjugates against harsh environments and decelerate the burst release of the TPPs.The covalent bonding between bipolymers could be achieved through Maillard reaction or by chemical crosslinkers.

Glycosylation of protein via Maillard reaction in a mild condition may inhibit the phenols induced precipitation during encapsulation.Xia et al.[124]fabricated the casein-dextran conjugation through Maillard reaction to encapsulate EGCG.The glycosylated casein displayed high EE of EGCG, and could protect EGCG from degradation in the alkaline environment [99].Another study from this group found that BSA aggregation induced by polyphenols was suppressed by replacing native BSA by glycosylated BSA obtained through Maillard reaction [124].In their study, two dextran molecules (10 kDa and 20 kDa) were successfully linked to the BSA molecule and the conjugates were found to have a core-shell structure, which was an ideal site for carrying EGCG.Besides of Maillard reaction, chemical crosslinkers were also utilized to improve the stability of protein-polysaccharide structure through covalent bonding.Genipin can react with amino groups in CS and peptides to improve the mechanical properties of formed conjugates.Hu et al.[125]formulated the EGCG loaded caseinophosphopeptide-CS nanoparticles with particle size smaller than 300 nm.The nanoparticles crosslinked by genipin showed improved stability and a slower EGCG release profile, as compared to nanoparticles without chemical crosslinking.

Except for the polymer-based nano/microparticles, two studies have developed the lipid-based particles to encapsulate TPPs, namely solid lipid nanoparticles (SLNs) [19,126].SLNs are sub-micron colloidal carriers which consist of biocompatible lipids as the solid core and amphiphilic surfactants coated in lipid-aqueous interface.SLNs are solid at room or body temperature and they can provide several advantages compared to conventional nanocarriers, such as achieving sustaining release and preventing premature degradation of TPPs [127].Radhakrishnan et al.[126]developed EGCG loaded SLNs with the particle size of 157 nm and EE of 67.2%, and Ramesh et al.[19]developed EGCG-SLNs with higher EE of (81 ± 1.4)% and particle size of (300.2 ± 3.8) nm.Given that only few studies were reported for SLNs, more information on the fabrication, characterization and application of TPPs-SLNs need to be investigated for future studies.

5.Enhancing the stability, bioavailability and bioactivity of TPPs by encapsulations

5.1 Encapsulation systems aiming at extending shelf-life

To enhance the stability and maintain biological activities of TPPs in tea containing products before consumption, different strategies have been explored by researchers.As TPPs were susceptible to heat, the encapsulation would protect TPPs from epimerization and degradation during thermal processing [128](Fig.3).Several researchers utilized O/W nanoemulsions to encapsulate TPPs,and investigated their stability at elevating temperatures [77,79].Gadkari et al.[79]prepared nanoemulsions by solubilized TPPs in SFO with 1-dodecanol and lecithin.After 10-week storage in different temperature conditions, it was found that the degradation rate of TPPs increased with elevated temperature, with the degradation percentages being 4.25%, 15.97% and 22.78% for TPPs stored at 4, 27 and 37 °C,respectively.Similar trend was also observed in another study conducted by Bhushani et al.[77].Soy protein stabilized nanoemulsions containing 0.3%-0.5% green tea TPPs stored at 4 °C showed the highest retention of TPPs, while more than half content of EGCG and EGC in nanoemulsion were degraded at 40 °C after 15 days [77].In addition to emulsion systems, nanoparticles were also found to be effective to preserve the heat stability of TPPs.Zokti et al.[108]developed green tea extract loaded nanoparticles with CS, GA and CD via spray-drying.Among the encapsulating materials, green tea extract encapsulated in GA and CD have shown to retain total catechins up to 81.82% and 67.38%, while the tea powder without encapsulation only had a retention rate of 32.62% after 12-week storage test at 40 °C.White tea extract loaded in polycaprolactone nanoparticles were able to retain around 76% polyphenol content compared to 56.3% of unencapsulated tea extract at 40 °C after 30 days [129].

Fig.3 Enhancing stability, bioavailability and bioefficacy of TPPs by encapsulations.

Compared to temperature instability, alkaline pH condition seemed to be more detrimental to the stability of TPPs due to the proton-donating capacity of catechins, which could result in autoxidation [43].Gu?lseren et al.[130]demonstrated that EGCG encapsulated in milk phospholipid nanoliposomes showed improved stability when stored at pH 5 and 7 for 16 days without significant decrease in encapsulated content.In a nanoemulsion system, the total encapsulated polyphenols decreased by 16.28% and 35.54% of its initial content at pH of 7.0 and 9.0 after 8 weeks [79].Although the abovementioned studies did not include comparisons with unencapsulated TPPs, significant stability enhancement were provided by delivery systems considering almost complete degradation of free TPPs within 3 h at pH 7.4 as reported before [131].

In addition, the stability of the formulated TPPs have been found to be enhanced in real food products (Fig.3).Aditya et al.[132]added catechin-loaded W/O/W emulsion and free catechin to a model beverage system and compared the catechin stability after 15-day storage at (23 ± 2) °C.Catechin in emulsion showed higher stability with around 35% degradation, while free catechin underwent almost 60% degradation in the same beverage system [132].Ho et al.[106]entrapped catechins in cyclodextrin and put the complexes to several food matrices.It was noticed that the degradation of catechins was the highest (48%) in milk after storage at 4 °C for 4 weeks, following by the fortified yogurt (44%) and cheese (33%).Zokti et al.[133]found that catechin had stable shelf life for four weeks in the mango drinks with fortified catechin extract in their nanoparticle forms.

Even though various systems have been explored, only a limited number of current investigations compared the stability of TPPs in their formulated form with the unformulated form.According to the available studies, encapsulation can significantly extend shelf-life of TPPs by slowing down auto-oxidation and epimerization against unfavorable processing/storage conditions (e.g.temperature and pH).Other factors, such as formulation parameters (e.g.oil fraction and stabilizer selection) [77],interaction with other food components (e.g.unsaturated fatty acids [79]),and real food product forms still need to be taken into consideration to ensure the retention of TPPs before ingestion.

5.2 Bioavailability enhancement by encapsulation

Bioavailability enhancement by formulation can be achieved mainly in two ways, one is to improve stability of TPPs in GIT,and the other is to increase intestinal absorption (Fig.3).Intestinal stability of encapsulated TPPs has been evaluated via multiple ways and the most used method is to compare the release profile and retention rates of TPPs after incubation in simulated gastric fluid (SGF) and intestinal fluid (SIF).Several parameters in SGF/SIF, such as pH, temperature, and major enzymes, are usually adjusted to mimicin vivoconditions in GIT.Intestinal absorption enhancement can be evaluated via cell studies and animal models.Caco2 monolayer is usually used as an intestinal absorption model to evaluate permeability enhancement of TPPs in delivery systems [134].The Papp, which reflects the transport rate from apical side to basolateral side of epithelial membrane, is used as a key index for comparing absorption efficiency [135].Combing the effects of stability and absorption improvement, the overall bioavailability after orally administration of TPPs can be assessed byin vivostudy.In vivoestimation of oral bioavailability is often evaluated in rats/mice models, plasma levels of TPPs are usually determined by the area under concentration-time curve (AUC) to calculate total available amount for exerting bioactivities.Other parameters such as maximum concentration (Cmax), time to peak concentration (Tmax)and so on, are also used to study pharmacokinetic characteristics of formulated/unformulated TPPs [136].

5.2.1 Improving stability of TPPs in GIT

TPPs embedded in wall materials can partially escape digestion,avoid complete exposure to reactive oxygen species and alkaline pH in the GIT, thereby increasing intestinal stability [56,137].EGCG loaded nanoliposomes prepared with dextran sulfate modified carboxymethyl CS retained 51.1% of its original content, while EGCG solution only retained 6.3% after 1.5-h incubation in SIF [69].CS/H-2 ferritin (EHFC) nanoparticle achieved around 65% retention of EGCG in SGF and about 38% retention in SIF after 2-h digestion,which were significantly higher than unencapsulated EGCG under both conditions [137].Zagury et al.[138]also observed that EGCG either in the formulated form or free form had a significant lower retention in SIF compared to SGF.In detail, the retention rate of EGCG in theβ-Lg complexation was 37.2% and that of free EGCG system was 0.8% after 2-h incubation in SIF, while the two forms were both relatively stable during gastric digestion.Both the delivery vehicles and the molecular structure of TPPs would affect the retention rates of TPPs in the GIT.Peng et al.[9]loaded EGCG,EGC, EC, and ECG in O/W nanoemulsion consisted of corn oil and polysorbate-80.The results showed that EGC was not protected by the nanoemulsion system during small intestine digestion, while EGCG had a significantly improved retention by encapsulation.In a similar nanoemulsion system prepared with SFO and soy protein, EC possessed the highest retention rate after gastrointestinal digestion in both unencapsulated form (52.28%) and in the nanoemulsion system(109.22%), while EGCG tended to be most vulnerable with only 0.91% retention in its free form and 10.57% retention in encapsulated form [77].Although there are variations in protection efficacy,which could be due to discrepancies inin vitroevaluation methods,enhancement in stability or retention of TPPs by encapsulation was observed in the abovementioned studies.

5.2.2 Increasing intestinal absorption of TPPs

Intestinal absorption enhancement can be achieved through increasing epithelial transport, of which the major routes are transcellular and paracellular pathways.Transcellular transport of TPPs can be promoted by delivery systems by boosting active cellular uptake of nanosized structures or by inhibiting efflux pumps [110].Emulsion systems usually lead to better micellization of TPPs after digestion, resulting in facilitated transcellular transport of TPPs [139].Active uptake of nanoparticles by epithelial cell through pinocytosis was also reported to play essential role in improved transcellular shipping of TPPs [140].Paracellular pathway is mainly through opening of tight junctions between enterocytes, and this is when permeation enhancers in the wall materials such as positively charged CS play important roles.Consequently, there is a higher chance for TPPs to permeate through enterocytes, thus elevating exposure in blood stream [120].

For instance, intestinal absorption study performed on Caco2 monolayer indicated that a nanoemulsion system, which consisted of corn oil and polysorbate-80, enhanced absorption of EGCG by 28.6%and EGC by 27.1%, compared to their water solutions respectively [9].Bhushani et al.[77]found that permeation of the 4 TPPs through Caco2 cell monolayer were all enhanced by a nanoemulsion system prepared with SFO and soy protein significantly: EGC showed the highest permeability, followed by EC and EGCG/ECG.

Nanoparticles assembled with CS and poly (γ-glutamic acid)(γ-PGA) have been showed to be able to open tight junctions between adjacent cells in Caco2 monolayer [120].After encapsulating tea catechins (LC: (16.3 ± 0.7)-(74.6 ± 4.8) μg/mg) in CS/γ-PGA nanoparticles, the permeability of tea catechins across Caco2 monolayer increased from 6% to around 24%.It was also found that lower pH conditions (pH 6.6 compared to pH 7.4) could result in more positive charges on CS due to protonation on the amine groups.This would further led to stronger electrostatic interaction between positive nanoparticles and negative sites of tight junction proteins, and therefore higher permeability [120].Yang et al.[137]found that the amount of EGCG absorbed in cells increased 1.6 times when loaded in CS/H-2 ferritin composites compared to its free form.In another study, EGCG loaded in CS/caseinophosphopeptides nanoparticles also showed increased transport efficiency from apical side to basolateral side of differentiated Caco2 monolayer, with the apparent Papp elevated from 3.5 × 10-7cm/s to 1.34 × 10-6cm/s [140].

5.2.3 Evaluating bioavailability enhancement of TPPs by in vivo study

Except fromin vitrostudies, there are also severalin vivostudies confirming improved GI stability and plasma exposure of TPPs by encapsulations.EGCG was loaded in CS and tripolyphosphate nanoparticles with LC of (3.7 ± 0.1) μg/mg.With an oral dosage of 0.76 mg/kg EGCG per bodyweight, the available levels of EGCG in jejunum compartment (AUC(0?5h): ～ (12.3 ± 1.5) μmol/L·h) from Swiss Outbred mice ingested nanoparticles was found to be 2.3 folds higher than that from EGCG solution (AUC(0?5h): ～ (5.3 ± 1.1) μmol/L·h).And this enhanced bioaccessibility by encapsulation further led to 1.5 folds improvement in plasma exposure (AUC(0-5h)) after oral administration, with (116.4 ± 4.1) and (179.3 ± 10.8) nmol/L·h for the EGCG solution and nanoparticles, respectively [141].EGCG encapsulated in theβ-Lg complexation (LC = 57.3 mmol/L EGCG in 7.2 mmol/Lβ-Lg) was given to male Sprague-Dawley rats at 150 mg/kg bodyweight by gavage.The AUC(0-6h)andCmaxof EGCG in plasma reached (497.6 ± 35.4) nmol/L·h and (192.8 ± 8.0) nmol/L, while those of rats administrated with free EGCG were only (241.5 ±16.7) nmol/L·h and (134.0 ± 2.1) nmol/L, respectively [138].EGCG loaded in albumin nanoparticles was reported to increase bioavailability by 1.5 times and elongate the half time (T1/2) of EGCG to 15.6 h in rat model, compared to EGCG in its free form [142].Peng et al.[9]found that nanoemulsion largely improved the oral bioavailability of EGCG from (13.3 ± 0.2) mg·min/L in the solution to (17.1 ± 0.1) mg·min/L, and improve the bioavailability of EGC from (4.8 ± 0.0) mg·min/L in the solution to (6.1 ± 0.0) mg·min/L.The authors also noticed that the plasmaCmaxof EGCG from nanoemulsion ((166.7 ± 22.6) μg/L) was lower than that from solution((258.8 ± 135.1) μg/L), which could be explained by the prevention of burst release of EGCG by the encapsulation.EGCG-SLNs formulated with polyoxymethylene stearate and poloxamer 188 L were given to rats at a single oral dose of 10 mg/kg EGCG.EGCG-SLN showed a significantly enhanced plasma exposure of total (conjugated and unconjugated) EGCG (AUC(0-24h): (2 541 ± 278) mg·h/L) compared to that of EGCG in solution (AUC(0-24h): (798 ± 72) mg·h/L).It indicated a 3.1-fold increase in bioavailability of EGCG by formulation in rats [19].Furthermore, it was observed that the AUC(0-24h)of total EGCG in all analyzed tissues of rats receiving SLNs were significantly higher ((2 027 ± 139) mg·h/L for brain; (911 ±138) mg·h/L for kidney, (1 321 ± 61) mg·h/L for liver, and(1 882 ± 92) mg·h/L for spleen) than those in rats receiving free EGCG ((618 ± 124) mg·h/L for brain, (886 ± 101) mg·h/L for kidney, (678 ± 327) mg·h/L for liver, and (302.8 ± 52) mg·h/L for spleen).The results suggested that SLN improved EGCG overall bioavailability and its levels among tissues.The results suggested that SLN improved EGCG overall bioavailability and its levels among tissues [19].A solid double emulsion fabricated by Hu et al.[143]also improved oral bioavailability of EGCG significantly in rat model,with 1.93 times higher plasma exposure and 1.44 folds increase inCmaxcompared to free EGCG solution.It was also suggested that the bioavailability of TPPs, which were characterized with high solubility and low permeability, could be significantly enhanced when embedded in oil phase [144].Moreover, medium chain fatty acids were more suitable for enhancing systemic circulation of TPPs compared to long chain fatty acids [145].In a rabbit model,TPPs encapsulated in 2 types of gelatin nanoparticles (GNP-A and GNP-B) showed 1.63 and 1.46 folds higher AUC(0-∞)compared to free TPPs.The encapsulation also brought theCmaxof TPPs in GNP-A to 27.42 μg/mL and that in GNP-B to 24.07 μg/mL, which were both substantially higher than that of free TPPs (15.73 μg/mL) [91].

Generally, the bioavailability enhancement by encapsulation is mainly attributed to the enhanced stability of TPPs in GIT through minimizing premature degradation and sustained release from carriers during digestion [138].In addition, delivery systems can promote the absorption of TPPs through facilitating active transport and improving intestinal membrane permeability instead of passive diffusion [146,147].For the current investigations usingin vitroandin vivomodels, thein vitromodels, especially cell studies, defers from human body in environmental oxygen partial pressure, presence of antioxidants and protein concentrations [148], and animal studies usually are lack of information in tissue distribution, metabolism and excretion.Therefore, further investigation is still needed for the confirmation of bioavailability enhancement and the optimization of delivery systems.

5.3 Bioefficacy enhancement by encapsulation

Intake of TPPs including catechins, thea flavins, and thearubgins from green and black tea have been associated with various health benefits such as antioxidation [149-153], anti-inflammation [154-156],anti-obesity [149,157-160], anticancer [161-165]and so on (Fig.3).Most of the available studies evaluating the bioefficacy of TPPs are conducted on cell lines, in which high concentrations(50–200 μmol/L) were used.However such high concentration usually cannot be achieved in human bodies after oral ingestion of TPPs, due to the bioavailability issue [90].Therefore, to exert their functionality in human body, efficient delivery is necessary for achieving physiological relevant concentration at target sites.As mentioned above, encapsulation of TPPs can lead to improved bioavailability, so it is reasonable to expect that it will also result in enhanced bioactivitiesin vitroandin vivo.

5.3.1 In vitro studies

Among the availablein vitrostudies of TPPs encapsulation,anticancer assays were extensively used to evaluate bioactivity enhancement by researchers, and some of the reported data lack the comparison between TPPs with and without carriers [166,167].Even though cell models are widely applied for the bioefficacy investigation, they have limitations due to the simplified conditions.For example, the bioavailability is not considered, and solvents used in cell studies could be inappropriate for human consumption.

To evaluate anticancer activity enhancement of TPPs, various cancer cell lines, such as prostate cancer cells, cervical cancer cells,liver cancer cells etc., were used in previous research [149-151].Generally, cytotoxicity of TPPs on those cell lines was evaluated first,cell cycle arrestment and apoptosis were measured by flow cytometry to evaluate the antiproliferation effect by the formulated/unformulated TPPs.Biomarkers of cell apoptotic pathway such as caspase family,P21, P27 and cleaved PPAR, were also quantified by western blot to compare anticancer efficacy at molecular levels [168].

A SLN system was found to improve anticancer activity of EGCG on human breast cancer cells MDA-MB-231 and human prostate cancer cells DU-145 [126].Results showed that EGCG loaded in SLN demonstrated 8.1 folds higher toxicity on MDA-MB-231 cells and 3.8 folds higher toxicity on DU-145 cells compared to its free form by comparing the IC50values.Higher pro-apoptotic effect of EGCG in SLN was also observed with a substantial increase in percentage of apoptotic bodies (32.7% and 24.9%) in both cell lines, while that in cell treated with free EGCG were significantly lower (14.5% and 11.8%).In a similar study, EGCG encapsulated in polylactic acidpolyethylene glycol (PLA-PEG) nanoparticles demonstrated 10-fold dose advantage over its free form against prostate cancer (PC-3) cells by comparing the IC50values.Strengthened apoptotic effect on PC-3 cells was also observed by using 2.74 μmol/L encapsulated EGCG to achieve equivalent proapoptotic effects of 40 μmol/L EGCG in its free form.Expression of apoptotic proteins including P21, P27,Bcl-2/BAX and cleaved PARP was realized by a significant lower concentration of EGCG in PLA-PEG nanoparticles, suggesting stronger chemoprevention effect endowed by encapsulation [169].Catechin extract from green tea was also encapsulated in nanoemulsion stabilized with lecithin and Tween 80 to improve their inhibitory effect on prostate cancer using PC-3 cell line [168].After encapsulation, the IC50of catechin extract decreased to 8.5 μg/mL from 15.4 μg/mL.And catechin nanoemulsion showed a stronger phase S arresting effect, with percentage of cells in S phase raised to (23.2 ± 1.3)% from (10.9 ± 0.6)% (blank control), compared to(17.9 ± 0.7)% for cells treated with catechin solution.The percentage of early apoptosis cells in nanoemulsion treated group (18.7 ± 0.1)%was also 2 folds of that in solution treated group (9.7 ± 1.7)%.Moreover, catechin nanoemulsion enhanced pro-apoptotic protein expression such as caspase-3, caspase-8 and caspase-9 and suppressed expression of anti-apoptotic protein Bcl-2 [168].However, not all findings showed improvement in bioefficacy by formulation.For example,using carboxymethyl CS and CS hydrochloride, Liang et al.[170]assembled nanoparticles through ionic interaction to deliver TPPs with LC of 16%.It was reported that encapsulated TPPs showed a lower growth inhibitory effect on HepG2 cells compared to free TPPs at low concentrations.Cell cycle arresting effect and percentage of apoptotic cell were also both lower in cells treated with TPPs in CS nanoparticles compared to the free TPPs.

Zhang et al.[171]reported that the antiatherogenic activity of EGCG in macrophages was enhanced by nanostructured lipid carrier(NLC) and CS-coated nanostructure lipid carrier (CSNLC) (LC = 3%).It was found that cholesterol content in human monocytic cell (THP-1)derived macrophage was lessened by encapsulated EGCG compared to the equal concentration of free EGCG.Expression of TNFα at mRNA level and secretion of monocyte chemoattractant protein-1(MCP-1), were also found to be significantly lower in encapsulated EGCG treated cells.Cheng et al.[172]encapsulated EGCG in poly(lactide-co-glycolide) (PLGA)/β-CD at a LC of 16.34%, and compared the anti-inflammatory activity of TPPs in their formulated/free forms.Compared to free EGCG, nitric oxide (NO) production was significantly decreased by formulated EGCG in mouse microglial cells (BV-2) treated with lipopolysaccharides (LPS).Although the anti-inflammatory activity of encapsulated EGCG at its highest concentration was stronger than that of the free form, the authors did not clarify the EGCG concentrations used in the free form, leaving their conclusion ambiguous [172].The antioxidant activity of EGCG encapsulated in aγ-CD-based metal-organic framework (CD-MOF)decreased to 42%, while that of EGCG solution declined to 11% after 5 days as measured by DPPH assay [173].Cell toxicity of EGCG in the two forms was also evaluated on the rat glioma cell line (C6) , and the results demonstrated that encapsulated EGCG showed moderately weaker cytotoxic activity than EGCG solution [173].

Even though most of delivery systems demonstrated improved bioefficacy of TPPs from availablein vitrostudies, some of them did not bring effects in cell models as well as free compounds.Two reasons might explain this discrepancy.First, there might be a timerelease portfolio of TPPs after encapsulation in specific medium or environment.Therefore, the time point selection is crucial for bioefficacy evaluation of TPPs, considering the partial/retarded release from delivery vehicles.Another reason could be that TPPs exert functions through targeting transmembrane receptor proteins.By transducing extracellular signals into intracellular space, entry into cells is not required for modifications of cellular activities such as cell cycle arrestment and apoptosis etc.[148].Since encapsulation could prevent direct interactions between TPPs and the membrane proteins, it may result in weaker efficacy in cells as compared to their free forms.

5.3.2 In vivo studies

Bioefficacy improvement of encapsulated TPPs is consider as a result from the enhanced stability and bioavailability as discussed in the abovementioned studies.In comparison with cell studies, animal studies are more convincing for bioefficacy evaluation, since they can better mimic situations after oral consumption by incorporating processes such as digestion, intestinal absorption and metabolism as happened in human body.

Siddiqui and coworkers [169]investigated the antitumor activity of EGCG nanoparticles using a xenograft model on athymic nude mice.Results indicated that tumor volume was decreased to 707 mm3by EGCG loaded in polylactic acid-polyethylene glycol (PLA-PEG)nanoparticles (100 μg/mouse, i.p.) and 854 mm3by free EGCG(1 mg/mouse, i.p.), when that of control was 1 242 mm3at 45 days post-inoculation.Serum prostate-specific antigen (PSA) level was drastically reduced to 2.64 ng/mL by EGCG nanoparticles and 9.31 ng/mL by free EGCG, when that in control mice was 29.86 ng/mL.It suggested that EGCG loaded in PLA-PEG nanoparticles could bring 10 times dose superiority over unformulated EGCG owing to higher uptake efficiency by the tumor tissue [169].Comparably, Khan et al.[174]encapsulated EGCG in CS/pentasodium tripolyphosphate hexahydrate(CS/TPP) nanoparticles to improve its inhibitory effect on prostate cancer through oral administration.The appearance of tumor was delayed 14 days and 7 days in mice receiving EGCG CS/TPP nanoparticles and free EGCG, respectively.Average volume of tumor in mice treated with EGCG-CS/TPP nanoparticles (6 mg/kg body weight) was 216 mm3, which was half of that in EGCG(40 mg/kg body weight) treated group (514 mm3).Serum PSA level was also evaluated with 57%-72% reduction by EGCG nanoparticles,while 13%-26% reduction by free EGCG, indicating that antiprostate cancer activity of EGCG was markedly enhanced by the CS/TPP nanoparticles delivery.Even though the anticancer activity of unencapsulated TPPs has been studied extensively in humans, there are very few clinical studies investigating the activity of encapsulated TPPs.In a study, Lazzeroni et al.[8]determined the TPPs levels in the breast tumor tissues and plasma in twelve women with early breast cancer after they consumed Greenselect Phytosome (GSP),a lecithin formulation of green tea polyphenols extract.The results showed that oral administration of GSP increased the level of total(including conjugated and free forms) EGCG to 17-120 ng/mL in patients’ plasma.The total EGCG was detectable in breast tumor samples collected from all participants and the concentration was up to 8 ng/mL, which was higher than that of the adjacent normal tissues.They also found that administration of GSP decreased Ki-67 in tumor tissue, which presented a biomarker for the proliferation rate of breast cancer cells.However, the comparative analysis was not conducted for the effects of formulated and unformulated TPPs in their study [8].

The anti-atherosclerosis effect of EGCG was enhanced by nanoparticles consisting of CS and polyaspartic acid (PAA) with EGCG LC of (348.1 ± 12.4) mg/g [115].It was reported that the lipid deposition in inner artery surface decreased to 16.9% and 42.1% in rabbits fed with EGCG nanoparticles (50 mg EGCG/kg) and free EGCG (50 mg/ kg) for 5 weeks, respectively.The oral administration of EGCG nanoparticles led to improved blood lipid profiles in rabbits as compared to free EGCG, evidenced by the lower serum levels of total glycerides, total cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol [115].Metabolic syndrome ameliorating effect of EGCG/β-Lg complexation and free EGCG was evaluated in high-fat-diet (HFD)-induced obesity mouse model.It was found that EGCG/β-Lg complexation could lead to lower hepatic triglycerides in mice than free EGCG after 12 weeks treatment (both equaling EGCG 80 mg/kg body weight per day).The AUC of blood glucose during glucose tolerance test (GTT) in HFD mice treated with EGCG/β-Lg complexation was also found to be 16.2% lower than EGCG administered mice.Besides, the maximal blood glucose level after i.p.injection of insulin were lower in mice treated with encapsulated EGCG compared to free EGCG, indicating that formulated EGCG better enhanced the insulin sensitivity of mice [175].

In another study, the anti-obesity effect of encapsulated TPPs was evaluated in the human model.A recently developed TPPs phytosome in the form of coated tablets, namely MonCam, was tested in obese people (n= 100) of both genders with a hypocaloric diet.After 90 days, the group treated with MonCam showed significant weight loss (14 kg) and body mass index (BMI) reduction (12%), while the weight loss and BMI reduction for the control group receiving only a hypocaloric diet was about 5 kg and 5%, respectively.Besides,MonCam is found to improve the lipid profiles and insulin sensitivity in the plasma of participants [176].

Although the number ofin vivostudies is very limited, results all showed significant enhancement in bioefficacies of TPPs by encapsulation.It indicates that encapsulation systems are promising ways for the application of TPPs in functional foods.Considering treatments in animal models usually last for a few weeks, there is a chance that gut microbiota might play a role in influencing metabolic activities of the host and therefore alleviating some diseases.Hence, further investigation is needed to determine the relevance of bioavailability enhancement and the influence by gut microbiota on the bioactivities of TPPs.

6.Conclusion and perspective

TPPs have attracted great research attentions for years due to their prefunding health benefits.However, the application of TPPs is greatly limited by their poor stability and low bioavailability.By rational design of delivery systems, recent studies have found that the stability and oral bioavailability of TPPs could be improved by encapsulation strategies.Furthermore, several studies have confirmed that the bio-efficacy of TPPs was also enhanced by encapsulation using bothin vitroandin vivomodels.

Although the available data on the delivery of TPPs and the fortification of TPPs in functional food is encouraging, further investigations are still needed on the following issues.1) Several reported studies chose the TPPs crude extract rather than pure compounds to investigate their bioactivities before and after encapsulations.Though crude extract is more convenient to prepare in large quantity either in experimental scale or food industry applications, such studies will not reveal the actual bioactivity of active compounds.In some scenarios, the whole extract may have higher activity than pure compounds due to synergistic effects of multiple components.Thus, mechanistic studies using the pure TPPs need more research attention.2) Even though several delivery systems were evaluated in shelf-life studies, there is a lack of comparison of TPPs stability between formulated and unformulated systems.Moreover, besides of pH and temperature, other factors still need to be investigated, such as interactions between TPPs and food matrix/real food products during the processing and storage conditions.3) The toxicity and safety issues of encapsulated TPPs remain substantial challenges.The deliveries of TPPs by nanoparticles need to be applied with caution as the toxicity of TPPs should be considered when systemic bioavailability is significantly increased [177].4) Morein vivostudies toward the comparison offree and formulated TPPs need to be conducted.Currently, the enhancement of the stability and bioavailability of TPPs by encapsulations were proved mainly through thein vitrostudies, while these results cannot be directly translated into human bodies.It is notable that the conditions used inin vivoandin vitrostudies are quite different.The biological fate of encapsulated TPPsin vivoneeds further exploration.Besides,in order to investigate how encapsulated TPPs exert their functions,more molecular information (such as protein and mRNA levels)is desired in thein vivostudies.5) It is necessary to evaluate the bioefficacy of TPPs and their metabolites, especially gut microbiotamediated metabolites.The biotransformation of TPPsin vivois extensive, so thein vivobioactivity of TPPs may be more relevant to their metabolites in the systematic circulation and organs [178].For examples, some TPPs with large molecular structure such as TF3 may have extremely low systemic bioavailability, but they may still exhibit biological activity through itsin vivometabolites [59].Given that very few studies have demonstrated the influence of delivery systems on TPPs metabolism, future investigations would better elucidate TPPs’ biotransformation pathwayby the host and microbiota as well as the relationships between TPPs’ bioactivity and their metabolites.6) It is important to investigate the tissue distribution of encapsulated TPPs and its correlation with the bioactivities of TPPs.A number of cell studies have shown the improvement of TPPs bio-efficacy by encapsulation, such as anti-cancer and anti-inflammatory activities.The prerequisite condition for those cell studies is that TPPs would be contacted with or absorbed by cells in specific organs.However,there is a knowledge gap between the encapsulated TPPs and tissue distribution after oral administration.High amount of deposition in specific organs or tissues is desired for TPPs to achieve high level of bioefficacy.Therefore, morein vivostudies toward the tissue distribution and bioactivity of TPPs need to be conducted.

There are still great expectations in the fields of developing delivery systems for TPPs.Formulations with targeted release are expected to be developed for TPPs to treat specific diseases.More importantly, joint efforts from the clinical trials and epidemiological studies are critical for providing unequivocal evidence for the benefits of TPPs encapsulations.

Conflict of interest

The authors declare no conflict of interest.