Nanoencapsulation of Green Tea Polyphenols

NANOENCAPSULATION OF GREEN TEA POLYPHENOLS

8

Sayantani Dutta, S.K. Sivakamasundari, Jeyan A. Moses, C. Anandharamakrishnan Computational Modeling and Nano Scale Processing Unit, Indian Institute of Food Processing Technology (IIFPT), Ministry of Food Processing Industries, Government of India, Thanjavur, India

8.1 Introduction Tea is the most consumed beverage globally, next only to ­water. Tea is mainly prepared from a infusion of leaves and bud of the plant Camellia sinensis, member of the Theaceae family (Pasrija and Anandharamakrishnan, 2015). Tea was principally an oriental beverage, popular especially in southeast Asia. Countries such as China, Japan, Lower Asia, and those in the Indian subcontinent have been the primary producers and consumers of tea at the outset. According to the legends, in 2737 BC, Shen Nung, the then emperor of China, unexpectedly discovered tea as a beverage. The first book on tea, “Ch’a Ching” was written by Lu Yu, a Chinese scholar and author, during the Tang dynasty (618–906 AD). Thereafter, the popularity of this beverage traveled through the monks to Japan and it sequentially spread to the rest of Asia. During the 16th century, tea was introduced to Europe by traders who traded in this part of the globe. Eventually, Tea gained popularity there too (UK Tea and Infusions Association, 2017). Today, tea has gained substantial popularity and has become a household name for a warm refreshing beverage throughout the world. It has become a savory both for the rich and the poor, transcending the economies of the world. The demand of tea is segregated according to its type. Depending on the manufacturing processes and chemical composition, there are three major types of tea, namely green (nonfermented), oolong (partially fermented), and black (fermented) tea. Different ethnicities have different preferences for these types of tealeaves. Among the three, green tea is the least processed and therefore, it retains most of the nutritional ingredients in natural form (Table 8.1) (Mak, 2012; Pasrija and Anandharamakrishnan, 2015). The bioactive constituents in green tea Nanoengineering in the Beverage Industry. https://doi.org/10.1016/B978-0-12-816677-2.00008-9 © 2020 Elsevier Inc. All rights reserved.

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230  Chapter 8  Nanoencapsulation of Green Tea Polyphenols

Table 8.1  Composition of Fresh Green Tea Leaf Components

% Weight (Dry Basis)

Amino acids Ash Carbohydrates Carotenoids Chlorophyll Lignin Lipids Methyl xanthines Organic acids Polyphenols Proteins Volatiles

4 5 25 <0.1 0.5 6.5 2 3.5 1.5 36 15 <0.1

(Source: Graham, H.N. 1992. Green tea composition, consumption, and polyphenol chemistry. Prev. Med. 21, 334–350.)

are chiefly polyphenols (catechins) and flavonols, which contribute to antioxidant activity (Table 8.2). The characteristic bitterness and astringency in green tea is also owing to the concentration of the ­polyphenols (Shi et  al., 2005). Green tea also possesses l-­theanine and caffeine, both of them works on neurotransmitters in the central nervous system (CNS) and minimizes the sensation of fatigue in human health (Furuse et  al., 2009; Cabrera et  al., 2006). Owing to the comparatively higher health benefits, this chapter elaborates first, on various nutraceutical benefits of the green tea per se, different methods for extraction of polyphenols from green tea and then the methods employed for encapsulating the bioactives therein. The disquisition presented herein would embrace the benefits of green tea, directly or indirectly, for nutritional and therapeutic applications, relevant to the present day.

8.2  Health Benefits of Green Tea Green tea is an important dietary source of polyphenols which are reportedly known to have antimutagenic, antidiabetic, antibacterial, antiinflammatory, and hypocholesterolemic properties (Cabrera et al., 2006). (−)-Epigallocatechin-3-gallate (EGCG) is the most significant flavonoid in green tea among other catechins such as (−)-epigallocatechin (EGC), (−)-epicatechin-3-gallate (ECG), and (−)-epicatechin (EC) (McKay and Blumberg, 2002) (Fig. 8.1). The pyrogallol-type structure on the B-ring contributes strong antioxidant activity to the EGCG

Table 8.2  Composition of Green Tea Beverage Components

% Weight (Dry Basis)

Ascorbic acid Carbohydrates Catechins Flavonols Gallic acid Methylxanthines Minerals Other amino acids Other depsides Other flavanoids Other organic acid Quinic acid Theanine Theogallin Volatiles

1–2 10–15 30–42 5–10 0.5 7–9 6–8 4–6 1 2–4 4–5 2 4–6 2–3 0.02

(Source: Graham, H.N., 1992. Green tea composition, consumption, and polyphenol chemistry. Prev. Med. 21, 334–350.)

OH

OH OH

HO

(A)

OH HO

O

OH

O

OH O

HO

OH

OH

OH OH

(B)

OH

OH

(C)

OH

OH OH

O

HO

OH O

HO

OH

O OH

O

O OH

C

OH

O

OH

C

OH

(D)

OH

OH

OH

(E)

OH

Fig. 8.1  Structures of different green tea polyphenols. (A) (+)-Catechin, (B) (−)-epicatechin, (C) (−)-epigallocatechin, (D) (−)-epicatechin-gallate, and (E) (−)-epigallocatechin-gallate. (Source: Weiss, D.J., et al., 2006b. Analysis of green tea extract dietary supplements by micellar electrokinetic chromatography. J. Chromatogr. A 1117, 103–108.)

232  Chapter 8  Nanoencapsulation of Green Tea Polyphenols

molecule and its galloyl moiety (D-ring, gallate group) is responsible for the inhibition of fatty-acid synthase leading to cytotoxicity in human cancer cells (Mereles and Hunstein, 2011) (Fig. 8.1E). Several researchers have worked on the beneficial effects of green tea on bone density, cognitive function, dental caries, and kidney stones (Cabrera et al., 2006). Recent studies indicated that consumption of green tea can decrease the severity of coronary atherosclerosis and incidence of myocardial infarction (Geleijnse et  al., 2002; Sasazuki et  al., 2000). Yang et  al. (2004) reported that habitual consumption of green tea (minimum 120 mL/day) for one year significantly reduces the risk of developing hypertension in the Chinese population. Potenza et al. (2007) investigated the effects of EGCG to simultaneously improve metabolic and cardiovascular pathophysiology in spontaneously hypertensive rats. Long-term EGCG treatment can reduce the development of obesity and symptoms associated with fatty liver (Bose et al., 2008). Several researchers have attested the chemopreventive potential of green tea catechins against a wide range of cell culture and preclinical studies, for example, cancers of the skin (UV radiation and chemically induced), lung, breast, colon, liver, oral cavity, esophagus, stomach, and prostate (Khan et  al., 2006; Pasrija and Anandharamakrishnan, 2015). Green tea polyphenols block the natural process of angiogenesis thereby inhibiting the development of new blood vessels, also, it impair the interaction of carcinogens with DNA leading to mutations, thus preventing carcinogenesis (Pasrija and Anandharamakrishnan, 2015). The liposome system showed higher potential than its free form to serve as effective carriers of EGCG and to increase its stability inside the vesicles and activate higher accumulation within basal cell carcinoma (BCC) and breast cancer cells (Fang et al., 2006b; de Pace et al., 2013). However, poor biopharmaceutical properties and pharmacokinetics including reduced stability in gastrointestinal tract, low intestinal permeability and short plasma half-life, have hindered the therapeutic application of EGCG (Rodrigues et  al., 2013). Therefore, researchers are exploring to enhance the bioavailability by different approaches, such as, coadministration with piperine, vitamin C or sucrose; enzymatic or chemical transformation of catechins and several encapsulation techniques (Bhushani et  al., 2016). The finds of researchers described above emphatically establish the nutritive and therapeutic gains accrued from green tea being a part of the daily diet. This brief collation elucidates the usefulness of the green tea bioactives and their numerous applications and therapeutic possibilities it offers at a granular level. It is essential to further investigate the findings which pave the path for application of the said bioactives, that is, the methods of extraction of the green tea bioactives. Essentially and desirably, the methods ought to be benign, nondestructive toward the

Chapter 8  Nanoencapsulation of Green Tea Polyphenols    233

bioactives as well as have the potential to extract the bioactives from the natural matrix in a feasible way. The discussion on the said objective, based on the findings of previous researches, has been next discussed.

8.3  Extraction of Polyphenols From Green Tea Leaves The concentration of polyphenols in tea depends on many factors, such as tea to water ratio and method of infusion, to name a few. Recent nutritional research indicates that consumption of at least five to ten cups of tea per day is required to achieve the recommended concentration of the said bioactives in the blood (Puligundla et al., 2017). Since consumption of tea above five cups a day would be a difficulty in a daily routine, efficient extraction of tea polyphenols is required. It should be emphasized here that although green tea is a beverage, the approach in this chapter focuses on maximizing the availability of nutraceuticals from tea, green tea in particular; rather than on the methods of tea preparation for best obtaining the said polyphenols. Extraction of catechins from green tea leaves depends on various factors including solubility, pH, extraction time, and temperature (of boiling). Hot water extraction of polyphenols have been reported by few researchers where they found that the technique was time consuming (0.5–6.0 h) and it required high temperature (80–100°C) (Shrikhande et  al., 2003; Vuong et  al., 2011). Liang et  al. (2007) observed that the bioactives EGCG and ECG of tea leaves were partially epimerized into gallocatechingallate (GCG) and catechingallate (CG), respectively, after heating at 100°C for 2 h. Alternatively, a mixture of ethanol and water (1:1) was employed for extraction of polyphenols by Row and Jin (2006) and Liang et  al. (2007). On the other hand, Goodarznia and Govar (2009) and Bharadwaz and Bhattacharjee (2012) have worked with water extraction followed by decaffeination to obtain tea catechins. Adopting another approach, microwave-assisted extraction of polyphenols was employed by Pan et al. (2003) using preleached tea leaves (ethanol: water = 1:1, room temperature for 90 min) as sample matrix. Comparative study of efficiencies of different solvents (water, 80% ethanol, 80% methanol, and 80% acetone) for extraction of green tea polyphenols was conducted by Drużyńska et  al. (2007). In that investigation, authors found highest yield of polyphenols (9.8%, w/w) for acetone solution. Comparison of different extraction techniques (ultrahigh-pressure extraction, microwave extraction, ultrasonic ­extraction, and heat reflux extraction) revealed maximum recovery of polyphenols along with the highest antioxidant potency

234  Chapter 8  Nanoencapsulation of Green Tea Polyphenols

for ­ultrahigh-pressure extraction (using 50%, v/v ethanol) (Jun et al., 2011). However, purification and drying procedures of polyphenol post recovery, limit the solvent extraction methods. Therefore, supercritical fluid (SCF) extraction technique has been utilized by few researchers to circumvent the cumbersome downstream processing (Pasrija and Anandharamakrishnan, 2015). Carbon dioxide (CO2) is the most preferred SCF for extraction since it is natural, clean, generally regarded as safe (GRAS), noninflammable, nontoxic, nonpolluting, and an inexpensive solvent (Ghosh, 2016). Another advantage of supercritical CO2 (SC-CO2) is that, it facilitates the extraction at near ambient temperature (31.1°C), thus minimizing thermal degradation of bioactives. SC-CO2 extraction of green tea polyphenols have been investigated by Chang et al. (2000) and Kim et al. (2008) using cosolvents (water and ethanol). Change in the polarity of SC-CO2 using cosolvent or modifier has been revealed in these finds, which therefore, facilitates improved solubility of catechins in SC-CO2; thereby enhancing the extract yield.

8.4  Bioavailability and Stability of Polyphenols Systemic delivery and bioavailability of a bioactive are the key factors which indicate the in vivo efficiency of the drug (Puligundla et al., 2017). These two issues are more challenging especially for oral administration of the nutraceuticals, since after consumption, the active ingredients undergo different stages in vivo: liberation, absorption, distribution, metabolism, and elimination (LADME) phases (Rein et al., 2012). Several factors limit the bioavailability of green tea polyphenols, such as bioaccessibility, delivery matrix effect, transporters, molecular structures, and metabolizing enzymes (Puligundla et al., 2017).

8.4.1 The Need for Encapsulation Encapsulation of tea bioactives is a necessity and research finds corroborate that. It has been reported that, tea catechins are stable under acidic conditions and they rapidly degrade in body fluids at pH 7.4 (Proniuk et al., 2002). Post oral absorption, tea catechins undergo extensive transformation by methylation, glucuronidation, and sulfation in the enterocytes of the small intestine, and further metabolism in liver decomposes these bioactives, unfortunately even before reaching the target tissue (Kadoma et  al., 2006; Conte et  al., 2016). A study by Dube et al. (2010b) observed that an 80% EGCG degradation took place after 1 h incubation in simulated intestinal fluid. Poor intestinal transport and lack of specific receptors on the surface of

Chapter 8  Nanoencapsulation of Green Tea Polyphenols    235

small intestinal epithelial cells also stimulate the poor bioavailability of tea polyphenols (Puligundla et al., 2017). Several other factors, such as, low serum albumin, hard water comprising of high concentrations of Ca2+ and Mg2+ or consumption of milk together with EGCG are reported to inactivate the bioactive (Ishii et al., 2011; Lorenz et al., 2007). Furthermore, these polyphenols are susceptible to light, heat, and the presence of oxidants which again reduces their activity (Shpigelman et al., 2010). For improved bioavailability and chemical stability of polyphenols several approaches are reported. Researchers had utilized different chemical additives for this purpose, such as reducing agents, dissolving agents, inhibitors of phase I and/or II enzymes, lipids or protein, and conjugation with pro-moiety groups (Conte et al., 2016). Few researches are reported for protection of EGCG by these conventional methods. Peracetate protection groups were used by Lam et al. (2004) under slightly alkaline conditions which provided six times more stability to EGCG compared to its natural form. During oral administration to mice, a 2.4-fold increase in the plasma exposure was observed for peracetylated EGCG compared to EGCG solution (Lambert et al., 2006). Encapsulation is a popular method to enhance the bioavailability of green tea polyphenols as well. Polyphenol extracted from China green tea has been reportedly encapsulated in sodium-caseinate (Na-caseinate) and calcium-caseinate (Ca-caseinate) beads to preserve the antioxidant potency of the extracts (Dehkharghanian et al., 2009). Elabbadi et al. (2010) have encapsulated green tea extract (GTE) using a different wall material. They have used calcium carbonate and dibasic sodium phosphate for production of green tea microparticles, with success. In another study, microencapsulated GTE in maltodextrin was administrated orally (50 mg/kg body weight/day) to rats having high fructose diet, for treatment of different metabolic syndromes (Jung et  al., 2013). ι-carrageenan and β-lactoglobulin were utilized to prepare oil-in-water (O/W) submicrometer emulsions to encapsulate EGCG. This encapsulate exhibited enhanced in  vitro anticancer activity compared with free EGCG (Ru et  al., 2010). Semicontinuous supercritical antisolvent process was employed by Sosa et  al. (2011) involving carbon dioxide as antisolvent and dispersing agent to coprecipitate green tea polyphenols with poly-ε-caprolactone (MW 25,000). Small particles (3–5 μm) produced in this study showed high retention of green tea antioxidants in the crystalline domains of the polymer matrix. Nano-encapsulation is an emerging technique for encapsulation of green tea polyphenols; for delivering target compounds to human systems and to increase the stability and bioavailability of the bioactives (Krishnaswamy et al., 2012).

236  Chapter 8  Nanoencapsulation of Green Tea Polyphenols

8.5  Significance of Nanotechnology in Encapsulation Nanotechnology is the most popular and emerging field in encapsulation of bioactives to protect and improve the bioavailability and therapeutic property of the important compounds. The concept of nanostructure was first proposed by Richard Feynman in the year 1959; later Nario Taniguchi coined the term nanotechnology in 1974. According to the British Standards Institution, the term, “nanotechnology” signifies production, processing, and application of particles with sizes less than 1000 nm, with special properties associated with them (Dungan, 2015; Ezhilarasi et al., 2013). Reduction in particle size of bioactives to nanoscale increases their surface-to-volume ratio, which sequentially increases the bioactivity of the compounds by many folds. This supports in various unique and novel applications of the bioactives. Several evidences have indicated that drugs encapsulated in nanoparticles exhibit distinct pharmacokinetic and pharmacodynamic profiles (Man et  al., 2016). In food applications, nanoparticles act as tiny system which delivers the nutraceuticals with aided protection from the environmental factors with simultaneous improvement in the importance of the compound through enhanced stability, reactivity, loading, and delivery of the ingredient (Dungan, 2015). Application of nanotechnology is reportedly appreciated in enhancing water solubility, thermal stability, and oral bioavailability of bioactive compounds (Ezhilarasi et al., 2013). “Nanoparticles” are colloidal systems including nanospheres and nanocapsules. Nanospheres are matrix structure in which the bioactives are uniformly dispersed; whereas nanocapsules are vesicular systems where a unique polymer membrane encloses the inner cavity containing the bioactives (Fig. 8.2) (Kamil et al., 2015). Since incorporation of bioactive in various parts of the body directly depends on the size of the particle and bioactive loaded into nanoparticle can easily

Fig. 8.2  Schematic structure of (A) nanocapsule and (B) nanosphere. (Source: Ezhilarasi, P.N., et al., 2013. Nanoencapsulation techniques for food bioactive components: a review. Food Bioprocess Technol. 6 (3), 628–647.)

(A)

Nanocapsules Bioactive compound

(B)

Nanospheres

Polymer or lipid or other wall material

Chapter 8  Nanoencapsulation of Green Tea Polyphenols    237

enter the bloodstream from gut, therefore, encapsulation of bioactive in nano system is able to enhance the targeted delivery along with bioavailability of the active compound (Ezhilarasi et al., 2013).

8.6  Nanoencapsulation of Green Tea Polyphenols According to the Biopharmaceutical Classification System (BCS), catechins are categorized under class III drugs or bioactive compounds with high solubility in gastrointestinal fluid and low intestinal permeability; bioavailability of these bioactives can be improved by encapsulation in engineered nanoparticles (Oehlke et  al., 2014) (Table 8.3). It is reported that, nanoencapsulation of bioactives helps significantly to increase blood circulation times and reduce liver accumulation in mice, as compared with the nonencapsulated free drugs (Man et al., 2016). Along with improved bioavailability, nanoencapsulation also allows sustained release and localized or targeted delivery of polyphenols. Several methods have been used by researchers to prepare nanoencapsulated green tea polyphenols. These methods are either top-down or bottom-up approaches. In top-down approach, size reduction is carried out generally by applying mechanical energy, such as homogenization and ultrasonication. In bottom-up approach, nanoparticles are constructed by self-assembly and self-organization of molecules, such as inclusion complexation and nanoprecipitation. Various factors, such as pH, temperature, concentration, and ionic strength of the system influence the formation of nanoencapsulate in this approach (Ezhilarasi et al., 2013; Zhong et al., 2015). In the following section, we have discussed different methods used by researchers to encapsulate green tea polyphenols.

8.6.1 Ionic Gelation Ionic gelation is a widely used method for encapsulation, employing polysaccharides, for example, chitosan (CS), alginate, gellan, and pectin (Racoviţǎ et al., 2009). This is a simple and popular approach for nanoencapsulation employing CS for few reasons, such as absence of organic solvent and harsh reaction conditions and higher stability of synthesized particles (Anandhakumar et al., 2017). In this method, solution of the polysaccharide is added drop wise to the solution containing other counter-ions under constant stirring and due to ionic cross-linking between these two oppositely charged compounds, polysaccharides form spherical particles and eventually precipitate (Racoviţǎ et al., 2009).

Table 8.3  Nanoncapsulation of Green Tea Polyphenols Sl. No.

Wall Materials Used

Method of Synthesis

Outcomes

References

1

Zein

Electrospraying technique

Bhushani et al. (2017)

2

Soy lecithin

3

Sunflower oil

Improved bioaccessibility and intestinal permeability of catechins

Bhushani et al. (2016)

4 5

Higher encapsulation efficiency of catechins Effective method for the encapsulation of bioactive component

Park et al. (2016) Liu et al. (2016)

6

Sodium alginate and Chitosan Sulfobutyl ether-β-cyclodextrin sodium and chitosan hydrochloride Phospholipid and Cholesterol Chitosan and Polyaspartic acid

8

Paraffin oil

Emulsification

9 10

Chitosan Bovine serum albumin

Continuous stirring Desolvation method

11

Chitosan

12 13

Chitosan and Poly (γ-glutamic acid) Chitosan

14

Cholesterol and Soy lecithin coated with chitosan β-cyclodextrin

Stirring, sonication and dialysis Ionic gelation method Ultrasonication, dialysis and freeze drying Sonication method

Better encapsulation efficiency with sustained release of catechins under simulated gastrointestinal conditions Enhanced stability of catechins under gastric and intestinal conditions Particle size of nanoencapsulates remained constant with enriched bioactive stability Superior emulsion stability Increased stability and antioxidant activity of encapsulates with sustained in vitro release Improved anticancer potential of the encapsulated green tea polyphenol Effective antioxidant activity upon nanoencapsulation of catechins Improved anticancer activity of catechins than unencapsulated and control samples Improved stability along with anticancer potential of catechins even at lower concentrations Efficient encapsulation method for delivering bioactive components Enhanced stability of catechins Better anticancer potential

Zou et al. (2014b)

7

Microfluidization and ultrasonication High speed and high-pressure homogenization High-pressure homogenization Inclusion complexation and ionic gelation Ethanol injection and microfluidization Ionic gelation

Improved in vitro gastrointestinal stability and Caco-2 cell permeability of catechins Enhanced storage stability of catechins

Effective method to encapsulate catechins thus protecting it from various processing parameters Enhanced transdermal delivery of catechins in the presence of ethanol Improved intratumor effect with controlled release of core material due to the lipid bilayer

Hu et al. (2008)

15 16 17

Molecular inclusion method Thin film hydration method Desolvation method

18

Cholesterol Gelatin surrounded with polyelectrolytes Chitosan

19

Egg phosphatidylcholine

Thin film hydration method

20

Egg phosphatidylcholine

Thin film hydration method

Ionic Gelation

Dag and Oztop (2017)

Hong et al. (2014) Mahmood et al. (2014) Kailaku et al. (2014) Yadav et al. (2014) Siddiqui et al. (2014) Tang et al. (2013) Khan et al. (2014) de Pace et al. (2013) Krishnaswamy et al. (2012) Lu et al. (2011) Shutava et al. (2009)

Fang et al. (2006b) Fang et al. (2004)

Chapter 8  Nanoencapsulation of Green Tea Polyphenols    239

CS is known to have several favorable properties for oral drug delivery. These include enhanced drug permeability through the paracellular route through a reversible opening of epithelial tight junctions and adherence of CS to intestinal mucosal surfaces (negatively charged) through ionic interactions (Dube et al., 2010b). Thus it increases the intestinal residence time of drugs after oral consumption (Smart, 2005). Several researchers have used ionic gelation method using CS for nanoencapsulation of green tea polyphenols, especially EGCG, as discussed in this section. Catechins are highly unstable in alkaline solution, therefore, encapsulation of (+)-catechin and EGCG using CS-tripolyphosphate nanoparticles (CS-TPP-NPs) has been investigated by Dube et  al. (2010b) to protect degradation of these bioactives in potassium hydrogen phosphate buffer (pH 7.4). CS-TPP-NPs have exhibited three times higher stability for (+)-catechin and four times higher stability for EGCG compared to the nonencapsulated bioactives in their study. Stabilization of these bioactives in encapsulated form was found further enhanced at the intestinal absorption site, in mouse jejunum (Dube et  al., 2010a). These researchers have also reported 1.5 times enhancement of plasma exposure of total EGCG and also enhancement in concentrations of the same in the stomach and jejunum of mice for encapsulated EGCG compared with EGCG solution (Dube et al., 2011). CS-TPP-NPs were used by another group of researchers also to optimize fabrication conditions for encapsulation of tea catechins (Hu et al., 2008). They observed effects of molecular mass and concentration of CS, initial pH value of CS solution, concentration of tea catechins, contact time between tea catechins and CS and CS-TPP mass ratio on encapsulation efficiency, and in vitro release profile of tea catechins from the nanoparticles were estimated. In a separate appraisal, Hong et al. (2014) have investigated encapsulation of EGCG by CS (3–5, 14–22, or 50–150 kDa) and polyaspartic acid (10–30, 30–50, or 50–100 kDa) and have optimized the composition (3–5 kDa CS and 30–50 kDa polyaspartic acid) to obtain EGCG nanoparticle with average particle size 102 nm. EGCG nanoparticles obtained in this condition have exhibited improved stability in gastric and intestinal conditions (simulated gastric and intestinal fluids, respectively) and enhanced effectiveness against rabbit atherosclerosis compared with EGCG alone (P < .01) (Hong et al., 2014). β-chitosan (β-CS) nanoparticles were utilized for nanoencapsulation of catechins and Zn-catechin complex by ionic gelation technology (Zhang et al., 2016aa). These bioactive loaded nanoparticles having particle size 208–591 nm, polydispersity index (PDI) 0.377–0.395, and Zeta-potential 39.17–45.62 mV showed improved antibacterial activity against Escherichia coli and Listeria innocua. Hence, catechins or Zn-catechin loaded β-CS nanoparticles can be used as natural food preservative.

240  Chapter 8  Nanoencapsulation of Green Tea Polyphenols

In another investigation, EGCG loaded in fluorescein isothiocyanate (FITC) labeled nano-complex (150  ± 4 nm particle size; 32.2 ± 3 mV surface charge) composed of CS and bioactive peptide caseinophosphopeptides (CPPs) have demonstrated a dose and time dependent uptake of nanoparticles by Caco-2 cells (Hu et al., 2012). Findings on enhancement in intestinal permeability and absorption of EGCG confirmed the utility of this nano-complex as a biocompatible and efficient system for increasing bioavailability of green tea polyphenols. Therefore, this system was further examined in human hepatocellular caricinoma (HepG2) cells and studied for cellular antioxidant activity. These researchers observed a significant (P < .01) improved activity of EGCG after delivery with CS-CPP nano-complex (Hu et  al., 2013). They suggested that during intracellular transport, the CS-CPP nanoparticles might have protected EGCG by inhibiting contact between EGCG and glycosylase and methylase. Thus, CS-CPP nano-complex could be a possible approach to enhance its antioxidant activity in biological systems. Another edible polypeptide, poly (γ-glutamic acid) (γ-PGA) has been worked upon for nanoencapsulation of tea catechin. As reported, the resulted nanoparticles showed retention of antioxidant activity and enhanced paracellular transport of tea catechins (Tang et al., 2013).

8.6.2  Molecular Inclusion Molecular inclusion is an encapsulation method where a ligand (encapsulated bioactive) associates with a cavity-bearing substrate by hydrogen bonding, van der Waals force or an entropy-driven hydrophobic effect (Ezhilarasi et al., 2013). Though this technique exhibits high encapsulation efficiency, only a few compounds, such as, cyclodextrin (CDs) and lactogloglobulin are suitable as shell (wall) materials in this method. CDs are a group of naturally occurring cyclic oligosaccharides originated from starch, with six, seven, or eight glucose residues linked by α-(1–4) glycosidic bonds (Fang and Bhandari, 2010). The outer part of CD is a hydrophilic, while the inner part of the same is hydrophobic in nature. In this structure, the glucose chains form a cone-linked cavity into which hydrophobic compounds can be attached, thereby changing their hydrophobicity (Connors, 1997) (Fig. 8.3). The size of the cavity of CD depends on the number of glucose units in the structure: α-cyclodextrin (six glucose unit, 5.2 Å), β-cyclodextrin (β-CD) (seven glucose unit, 6.6 Å) and γ-cyclodextrin (eight glucose unit, 8.4 Å) (Lira et al., 2009). Among them, β-CD has been investigated and employed for modification of hydrophobicity of several bioactives. Usnic acid and curcumin were encapsulated in β-CD by inclusion complex before formation of liposome to enhance the s­ olubility of these compounds in the aqueous core of the liposomes (Lira et al.,

Chapter 8  Nanoencapsulation of Green Tea Polyphenols    241

OH O

OH O OHO O HO

(A)

O OH HO HO

OH

O HO HO

OH O

b-Cyclodextrin O OH HO O OH HO O O OH OH OH O O HO O HO

OH

Hydrophilic exterior E

Hydrophobic interior

(B)

b-Cyclodextrin

Inclusion complex

2009; Aadinath and Anandharamakrishnan, 2016). Similarly, findings show that, complexation of resveratrol with β-CD and that of kaempferol, quercetin, and myricetin with hydroxypropyl-β-cyclodextrins (HP-β-CD) increased their aqueous solubility (Lucas-Abellán et  al., 2007; Mercader-Ros et al., 2010). Several scientists have employed inclusion complex method for encapsulation of tea catechins by β-CD. Analysis of the structure of the inclusion complex of β-CD and EGCG (1:1 stoichiometric complex) from 1H NMR and 13C NMR spectra (at 500 and 125 MHz) revealed that A ring and a portion of the C ring of EGCG molecule were included at the head of the phenolic hydroxyl group, which was attached to C7 of EGCG molecule in the β-CD cavity, from the wide secondary hydroxyl group side (Ishizu et al., 2006). In another study, tea polyphenol such as catechin, EGCG and gallocatechin gallate (GCG) were incorporated in β-CD, HP-β-CD and Heptakis-2,6-O-di methyl-β-cyclodextrin (DM-β-CD) (Folch-Cano et al., 2010). Study of stability constants by steady-state fluorescence measurements inferred that, terminal groups of the derivatized βCDs showed more interactions with tea catechins compared with the native β-CD. Further, ORAC-fluorescein and ORAC-pyrogallol red assays exhibited preservation of antioxidant potency of GCG+βCD, GCG+HPβCD, and EGCG+HPβCD complexes with respect to their free forms. EGCG incorporated in β-CD, HP-β-CD, and DM-β-CD in

Fig. 8.3  (A) Chemical structure of β-cyclodextrin, (B) schematic structure of nanoencapsulation of green tea polyphenols by molecular inclusion; E represents EGCG molecule.

242  Chapter 8  Nanoencapsulation of Green Tea Polyphenols

aqueous solution were further subjected to 2D-ROESY and selective 1D-ROESY studies which revealed that, although the complexes had similar inclusion geometries, the exposition degree of the antioxidant rings of EGCG (pyrogallol and galloyl groups) were different. This study also indicated that the inclusion process was entropy-driven for β-CD complexes and enthalpy-driven for derivatized CDs complexes (Folch-Cano et al., 2013). Nanoencapsulation of tea polyphenols by complexation method has been studied by Liu et  al. (2016) considering EGCG as the most abundant compound in the same, using sulfobutyl ether-β-­ cyclodextrin sodium (SBE-β-CD) as the cross-linking agent. Formation of inclusion complex was verified by fluorescence spectroscopy (from 320 to 480 nm) and one-dimensional 1H NMR spectroscopy (at 400 MHz). This inclusion complex was further cross-linked with chitosan hydrochloride (CSH) by ionic gelation method for formation of tea polyphenol nanoparticles. This system was further studied as an effective approach for preparation of edible film/packaging. Tea polyphenol nanoparticles (TPNs composed of CSH and SBE-β-CD) incorporated in gelatin film were investigated as packaging material for sunflower oil. This composite film showed oxidation inhibitory effect and a homogeneous dispersion of nanoparticles in the film was also achieved (Liu et al., 2015). The release of polyphenols from films was dependent on fatty food simulant (50% ethanol at 4°C and 95% ethanol at 25°C) and encapsulation efficiency (51.3%, 83.3%, and 96.9%) of the bioactive in the nanoparticle (Liu et al., 2017). The release increased for lower concentration of ethanol and with lower encapsulation efficiency. TPNs incorporated in films increased the compactness and isotherm hysteresis of films and decreased moisture diffusion. Results indicated that this composite film could preserve long-term antioxidant property of the films and thereby provide better protection to the food product.

8.6.3 Microfluidization One of the traditional methods of preparation of liposomes and emulsions in pharmaceutical industries is the “microfluidization technique.” This method uses “microfluidizer” equipment that is responsible for production of very small electron droplet size which resulted in enhancing the encapsulation efficiency of the bioactives (Jafari et al., 2007). To enumerate the process in brief, microfluidization technique applies high pressure to force the streams of two crude emulsions from two opposite microchannels to collide with each other in an impingement area. This high-pressure-induced collision creates cavitation and a tremendous shearing action that creates an exceptionally fine emulsion (Mozafari et al., 2008; Jafari et al., 2007).

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The advantages of microfluidization technique are absence of any toxic solvent for preparation of liposome, flexible control of emulsion droplet size distributions, and ability to produce fine emulsions from many materials (Mozafari et  al., 2008; Jafari et  al., 2007). However, use of microfluidization is limited in food applications due to the type of surfactant used. The high pressure used in this technique can damage the structure and/or function of the bioactives to be encapsulated causing material loss and contamination of bioactives (Jafari et al., 2007). Zou et  al. (2014b) have formulated tea polyphenol nanoliposomes (TPN) utilizing dynamic high-pressure microfluidization technique along with ethanol injection method. Ethanol injection method is one of the simplest methods of preparation of liposomes in which the lipid solution in ethanol is injected rapidly into an excess of saline or other aqueous medium, through a fine needle (Pons et  al., 1993). The force of injection produces small liposomes with narrow distribution. In the aforementioned study, phospholipid, cholesterol, Tween 80, and tea polyphenols were mixed (mass ratio of 8:1.2:2:1) in ethanol and injected into phosphate-buffered saline (PBS; pH 6.0, 0.05 M). Liposomes thus prepared were subjected to microfluidizer (120 MPa for one cycle) for formation of TPN, which after assay, displayed equivalent antioxidant activities compared with the tea polyphenol solution. A good sustained release of polyphenols was observed for TPN (29.8% release of polyphenol after 24 h) and it was also confirmed that the encapsulation had improved the stability of tea polyphenol in alkaline solution (Zou et al., 2014b). The abovementioned method was also utilized to prepare EGCG nanoliposome (EN) for enhancement of its stability in near-­neutral and alkaline conditions. Similar to TPN, EN also exhibited good sustained release along with enhanced (10–12 times) stability in SIF (Zou et al., 2014a). In another study, two different homogenization techniques were examined for preparation of liposomes containing GTE in acetate buffer and water, separately. The extract along with soya lecithin (1%, w/v) was homogenized at 20,000 rpm for 2 min and the “prehomogenized solutions” were subjected to two different techniques for preparation of liposomes: high-pressure microfluidization (130 MPa for five passes) and ultrasonication using probe sonicator (75% amplitude for 5 min). Among the different combinations green tea liposomes prepared in distilled water (pH: 6.5) by microfluidization technique showed highest stability during one-month storage (at 4°C wrapped in aluminum foil) with no significant difference (P > .05) in mean particle size (40 nm), total phenolic content (111.864 mg GAE/L sample), and antioxidant activity (15.051 mg DPPH/L sample) (Dag and Oztop, 2017).

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8.6.4 Nanoemulsification Nanoemulsion or microemulsion (5–100 nm) is formed by the spontaneous self-assembly of the hydrophobic or hydrophilic parts of surfactant molecules. The system comprised of a mixture of water, hydrocarbons, and amphiphilic compounds which form homogeneous, optically isotropic solutions. They have several applications over a wide range of areas, including pharmaceutical, cosmetics, oil recovery, as models for biological membranes, and as reaction media. The major difference between a microemulsion and a nanoemulsion is, microemulsions are thermodynamically stable; whereas nanoemulsions are described as “approaching thermodynamic stability” (Flanagan and Singh, 2006). Nanoemulsification is reportedly known for enhanced gastrointestinal absorption and reduced inter- and intra-individual variability for different drugs; their large interfacial area provides excellent drug-release properties, and they can also protect drugs and improve difficult organoleptic properties of the active components (Swarbrick and Boylan, 2001). Nanoemulsion of green tea extract (NGTE) was prepared by niosome technology using high-pressure microemulsifier (at 93.08 MPa) (Kim et al., 2012). These newly formed NGTEs were investigated for in vitro antioxidant and in vivo hypolipidemic effects. For the said study, C57BL/6 mice were fed with high-fat diet along with GTE or NGTE (both contained similar total catechin concentrations) diet for 4 weeks. Investigation was also conducted for effects of green tea on factors involved in cholesterol metabolism, such as, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, cholesterol 7α-hydroxylase (CYP7A1), LDL receptor and sterol regulatory element binding protein 2 (SREBP-2). The findings indicated that antioxidant properties were comparable for both GTE and NGTE and hypocholesterolemic effects were greater (1.6 times) in NGTE compared with GTE. Down-regulation of expression of HMG-CoA reductase, increase in protein expression of LDL receptor and increase in gene expression of CYP7A1 were also observed for both GTE and NGTE (Kim et al., 2012). High-pressure homogenization is one of the methods of preparation of nanoemulsion which uses high pressure (100–2000 bar) and high shear stress to disrupt the particles to form nanoparticles. Optimization of process parameters for nanoencapsulation of EGCG in alginate-chitosan nanoparticles by high-pressure homogenization was conducted by Park et al. (2016). Effects of the number of homogenization cycles and alginate-to-chitosan ratio on the physicochemical properties (mean particle size, PDI, surface charge, encapsulation efficiency, and free radical scavenging activity) were investigated. Findings reflected the optimized condition of encapsulation to be

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three cycles of high-pressure homogenization and alginate and chitosan concentrations of 0.01% (1:1) that exhibited small size (293 nm) of nanoparticles and a zeta potential of +137.49 mV. High-pressure homogenization was also utilized by Bhushani et al. (2016) to encapsulate green tea catechins using soy protein stabilized oil-in-water based nanoemulsion system. Along with physicochemical stability, in  vitro bioaccessibility and epithelial permeability of green tea catechins were investigated. Nanoemulsion containing 10% oil and 0.5% catechins was stable at 4 ± 1°C against creaming, phase separation, sedimentation, and changes in droplet size, pH, and catechin content. Bioaccessibility of major catechins was found to be increased in encapsulated form compared with their counterpart. A significant increase in intestinal permeability of catechins in encapsulate was also observed in Caco-2 cells. Formulation of C and EGCG liposomes utilizing soy lecithin was conducted using homogenization by a high shear blender (24,000 rpm) (Rashidinejad et al., 2016a). These liposomes were further incorporated into low-fat hard cheese (ripened for 90 days at 8°C). In vitro gastrointestinal digestion of the liposome-fortified (250 ppm) cheese revealed a significant increase (P < .01) in the total phenolic content and antioxidant activity of the product without hampering its composition or pH. Findings indicated that this method can be used to increase the antioxidant potential in the human diet (Rashidinejad et  al., 2016a). Same group of authors further prepared full-fat hard cheese fortified with nanoliposomes containing C or GTE. From transmission electron microscopy (TEM) and Fourier transform infrared (FTIR) spectroscopy analyses, association of nanoliposomes with the surface of milk fat globules inside the cheese matrix was confirmed. Similar to their previous study, findings of this investigation revealed significant (P ≤ .05) increase in total phenolic content and antioxidant activity of the cheese along with retention of encapsulated catechins in the cheese curd after digestion (Rashidinejad et al., 2016b).

8.6.5  Desolvation Method Nanoparticles can be formed by desolvation of macromolecules from a wide range of polymers by desolvation by charge and pH changes, or by addition of a desolvating agent such as ethanol or concentrated inorganic salt solutions (Reis et al., 2006). This method does not require any organic solvent or surfactant. It uses two miscible solvents without any harsh operation such as involvement of high shear rate, heating, and sonication which may damage the tertiary structure of proteins (Bagheri et al., 2013). Shutava et  al. (2009) have encapsulated EGCG in gelatin-based nanoparticles (200  nm) using two-step desolvation method.

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Relatively low antigenicity and availability of gelatin make it a suitable natural substrate for nanoencapsulation (Coester et al., 2000). Although preparation of gelatin nanoparticles using desolvation method was previously reported by Marty et al. (1978), the process experienced few stability problems, such as the produced particle formed irreversible aggregates during cross-linking and they also showed tendency of aggregation more extensively after cross-­linking (Coester et  al., 2000). Therefore, Coester et  al. (2000) have developed a two-step desolvation method with a reduced tendency for aggregation. In the study of Shutava et al. (2009), authors prepared layer-by-layer gelatin-based EGCG nanoparticles using acetone as desolvating agent and glutaraldehyde for cross-linking the nanoparticles. For polyelectrolyte encapsulation (5–20 nm thick shells) of gelatin nanoparticles, solution of polyanion [polystyrene sulfonate (PSS), poly-l-glutamic acid (PGA), dextran sulfate (DexS)], polycation [polyallylamine hydrochloride (PAH), poly-l-lysine (PLL)] or protamine sulfate (PtS) was added to the gelatin nanoparticles. Findings indicated that, release of EGCG from encapsulated nanoparticles was 8 h, compared with few minutes release for nonencapsulated nanoparticles. Both encapsulated and nonencapsulated EGCG nanoparticles exhibited anticancer property by blocking HGF-induced intracellular signaling in the breast cancer cell line MBA-MD-231. Two-step desolvation method was further used for encapsulation of EGCG using bovine serum albumin (BSA) by Li et al. (2014). Nanoparticles were coated with poly-3-lysine or chitosan. All three nanoparticles, BSA-EGCG (BEN), poly-3-lysine coated BEN (PBEN) and chitosan coated BEN (CBEN) exhibited spherical morphology (186, 259, and 300 nm, respectively) and moderate loading efficiency (32.3%, 35.4%, and 32.7%, respectively). Release of EGCG from nanoparticles into SGF and SIF with or without digestive enzymes showed that, coating improved the sustained release of the bioactive. Findings also indicated improvement of stability of EGCG in coated nanoparticles during storage at 60°C. Incubation of all nanoparticles along with free EGCG in Caco-2 cells exhibited significant improvement in the absorption of EGCG by CBEN. Encapsulation of tea polyphenols by BSA using desolvation method was also performed by Yadav et al. (2014). They have encapsulated C and EC to improve their bioavailability and stability. Encapsulation efficiency of C and EC on nanoparticles was found to be 60.5% and 54.5%, respectively. Both nanoparticles showed slow and sustained in vitro release of bioactives and improved stability in solution at various temperatures of 37°C, 47°C, and 57°C. The percentage cellular inhibition indicated enhanced efficacy of nanoparticles for inhibition of A549 (human lung carcinoma cells) cell lines.

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8.6.6  Multiple Emulsion Emulsion technology is a popular method of preparation of nanoparticles in the form of nanoemulsion to protect bioactive compounds in aqueous solutions. Though the extremely small emulsion droplet sizes provide high kinetic stability to nanoemulsion, the formation of the same requires high energy input, generally from mechanical devices or from the chemical potential of the components (Ezhilarasi et al., 2013). Multiple emulsion is an excellent delivery technique that contains multiple functional components in a single system. Oil-inwater-in-oil (O/W/O) and water-in-oil-in-water (W/O/W) emulsions are the common examples of this method (Ezhilarasi et al., 2013). It is possible with this system to release an aqueous-phase component trapped within the inner water droplets to a specific site in the body, such as the mouth, stomach or small intestine (Weiss et  al., 2006a). Multiple emulsion is reportedly known as an important method of encapsulation for preparation of skin care products with prolonged action (Patravale and Mandawgade, 2008). Green tea and lotus extracts (5% extract, w/w) as functional cosmetic agents in the W/O/W multiple emulsions was investigated by Mahmood et al. (2014). In this study, hydroxypropyl methylcellulose (HPMC) performed as the thickening agent, cetyl dimethicone copolyol was used as lipophilic emulsifier and a blend of polyoxyethylene (20) cetyl ether and cetomacrogol 1000 was employed as hydrophilic emulsifiers. The emulsions were prepared by a two-step emulsification strategy. Results revealed that after 12 months of storage the multiple emulsions showed no phase separation maintaining their globule size (8–10 μm). From the findings of conductivity and rheological analyses, it was confirmed that, multiple emulsion helped in sustained release of the bioactives and use of elevated temperatures showed shifting of the emulsion toward Newtonian behavior. Thus, it can be concluded that multiple emulsion can be used to encapsulate multiple bioactives for their long term stability (Mahmood et al., 2014).

8.6.7 Thin-Film Hydration Thin-film hydration method is the most simple and commonly used procedure for preparation of liposome. Generally, in this technique, a solution is prepared with membranous materials such as phospholipid and cholesterol and fat-soluble bioactives in organic solvent, eventually the solvent is removed for formation of a thin film of lipid phase. Addition of aqueous phase in the dried lipid film and dispersion using mechanical force forms liposome. Different dispersion methods are used for this procedure, such as ultrasonic dispersion method, homogenization method, and film oscillation method (Lu et al., 2011).

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Thin film ultrasonic dispersion method was employed for formation of liposomes of tea polyphenols to increase their bioavailability (Lu et  al., 2011). Polyphenols were extracted from green tea using microwave-­assisted extraction and the extract was dissolved in ­ethanol. Lecithin and cholesterol were dissolved in ether. These two solutions were mixed together and after evaporation of organic solvent, aqueous phase (phosphate buffer) was introduced to the lipid thin film and ultrasonicated for 4 min. The process conditions optimized by response surface methodology (RSM) were, tea polyphenol: lecithin = 0.125:1 and lecithin: cholesterol = 4:1; time of sonication 3.5 min. In  vitro release of polyphenols from liposomes (160.4 nm) followed first-order equation and the liposomal system showed enhanced stability of tea polyphenols (at 3–5°C) (Lu et al., 2011). Local delivery of liposomal formulations of C, EC, and EPC were investigated by Fang et  al. (2004). Egg phosphatidylcholine, cholesterol and different ionic surfactants [deoxycholic acid (DA), dicetyl phosphate (DP), and stearylamine (SA)], and thin film utrasonication method using probe sonicator were utilized for production of liposome. Green tea polyphenols are reportedly having antioxidant and anticancer activities, therefore, these researchers have studied the skin and tumor deposition of the liposomes in vivo. Skin deposition study using the aqueous solution of polyphenols revealed selective absorption of EC and faster and higher skin uptake of EGCG compared to C. Findings revealed that application of liposome reduced the skin uptake of C and EC, possibly due to the extra barrier of phospholipids or very slow release of drugs from the liposomes. On the other hand, application of liposome enhanced the uptake of EGCG for DA and SA may be because they have helped to cross the stratum corneum into deeper regions of the skin. The same study also exhibited improved deposition of polyphenols from liposomes into the tumor, revealing slow clearance of liposome from the tumor. Tumor uptake study was also conducted in another study using EGCG liposomes, prepared by probe sonication (25 W for 30 min). BCCs, melanomas, and colon tumors were treated with EGCG liposome and the results indicated EGCG liposome prepared with DA enhanced (20-folds) the deposition of EGCG compared to its free form in the presence of ethanol. Findings revealed that liposomal system enhanced the stability and cytotoxicity of EGCG against BCCs, thus reducing the effective dose of the bioactive (Fang et al., 2006b). The same group of researchers developed liposome with elastic properties to encapsulate C, EPC, and EGCG (Fang et al., 2006a). Egg phosphatidylcholine (EPC), cholesterol, anionic surfactants (DA and DP), and polyethylene glycol (PEG200 and PEG1000) in different ratio were used for preparation of liposome. The resulted liposomes were applied to various skin membranes, including intact nude mouse skin,

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SC-stripped skin, delipidized skin, and furry mouse skin. They have reported that incorporation of anionic surfactants in the presence of 15% ethanol enhanced the permeation of C by five to sevenfolds compared to the control liposome. Findings indicated that the presence of ethanol loosens the structure of the EPC bilayers, thus increasing the skin permeability of the drug. Effects of nanoliposome in enhancement of anticancer activity of EGCG and antibacterial, antioxidant, and prebiotic properties of GTEs were also investigated (de Pace et  al., 2013; Noudoost et  al., 2015). Chitosan-coated EGCG nanoliposomes exhibited improvement in the stability and sustained release of EGCG. Study with MCF7 (breast cancer) cells indicated enhanced apoptosis and inhibition of cell proliferation for EGCG liposome compared to its free form (de Pace et al., 2013). The study confirmed the use of biocompatible and biodegradable liposomal system to enhance the chemopreventive efficacy of EGCG along with reduced immunogenicity and side effects. In the investigation of Noudoost et al. (2015), researchers studied prebiotic activity of 1% liposome (containing GTE) using Lactobacillus casei and Bifidobacterium lactis. Findings confirmed that growth rate of the probiotics were significantly increased by the application of nanoliposomes. Authors suggested that enhanced solubility, stability, bioavailability, and antioxidant activity of EGCG in liposomal system facilitated the increase in the prebiotic activity of nanoliposome.

8.6.8 Use of Modified Protein Utilization of heat-modified proteins to protect bioactives with simultaneous improvement in their bioavailability is another technique of encapsulation. This method was investigated by few researchers to nanoencapsulate green tea polyphenols. Shpigelman et al. (2010) have applied thermally modified β-lactoglobulin (β-Lg) to form co-assembled nano vehicles (50 nm) of EGCG. β-Lg is the major whey protein in cow milk; which gets aggregated during heating by a chain-reaction of sulfhydryl-disulfide interchange and it is reportedly found that native β-Lg can act as a nano-vehicle. Findings from the study of Shpigelman et  al. (2010) displayed that the optimized condition of application of EGCG for nanoencapsulation was addition of the bioactive at preheated (75–85°C, 20 min) β-Lg solution during cooling and vortexing. Thermally induced protein exhibited enhanced association constant (3.5-fold higher) and considerable protection to EGCG against oxidative degradation. Further it was observed that, increase in the EGCG concentration increased the particle size and increase in the β-Lg concentration enhanced the loading efficiency of the bioactive (Shpigelman et al., 2012). Freezedried nanoparticles revealed molecular level complexation in the

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nanoparticles and in simulated gastric digestion limited release of EGCG was observed for nanoparticles. From the previous study it was found that, β-Lg can be used as an effective natural vehicle for nanoencapsulation of EGCG. In another study, similar method was followed by Li et al. (2012) using heat treated β-Lg and EGCG. In this study, they have optimized effects of pH (2.5−7.0), heating temperature of β-Lg (30–85°C), molar ratio of β-Lg and EGCG (1:2–1:32), concentration of β-Lg (1–10 mg/mL) on particle size, and ζ-potential and entrapment efficiency of EGCG. The optimum pH that provides a stable and clear solution was pH 6.4–7.0. Based on the antioxidant activity it was found that the optimum heating temperature was 85°C and molar ratio 1:2 (β-Lg: EGCG). Use of protein other than β-Lg, for encapsulation of EGCG is also reported, where authors have used ovalbumin and dextran to encapsulate the said polyphenol. Ovalbumin conjugated with dextran (Maillard reaction) (O-D) were self-assembled with EGCG (heated at 80°C for 60 min) for formation of nanoparticles at pH 5.2. EGCG-O-D nanoparticles were further cross-linked by glutaraldehyde. Stability study employing SGF and SIF revealed retention of nano size of particles in both EGCG-O-D and cross-linked EGCG-O-D nanoparticles. EGCG-O-D conjugate showed enhanced apparent permeability coefficient (Papp) on Caco-2 monolayers compared to EGCG solution indicating improved absorption of EGCG in cells (Li and Gu, 2014).

8.6.9 Phase Inversion Preparation of EGCG loaded lipid nanocapsules have also been investigated by few researchers using phase inversion process, which is an organic solvent-free method consisted of an oily liquid triglyceride core surrounded by a tensioactive cohesive interface (Barras et al., 2009). The nanocapsules thus obtained display biocompatible and biodegradable nature. In the study of Zhang et  al. (2013), both lipid (soy lecithin, glyceryl tridecanoate, glyceryl tripalmitate, and polyoxyethylated 12-hydroxystearic acid) and aqueous (EGCG and NaCl in deionized water) phases were heated to 85°C prior mixing. The mixture was then subjected to three temperature cycles of 60–85°C and 85–60°C at the rate of 4°C/min (with constant stirring) and in the last cycle the mixture was cooled to 70°C using cold deionized water (0°C). This fast cooling-dilution process produced the nanostructured lipid carriers of EGCG. The nanocapsules formed were further coated with chitosan using a magnetic stirrer for 40 min at 4°C. This study demonstrated that the phase inversion procedure of encapsulation provided improved stability of EGCG post encapsulation. Chitosan-coated nanocapsules had an increased EGCG content in THP-1 (human monocytic cell line)-derived macrophages and both

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­ anocapsules (with and without chitosan) showed significantly den creased macrophage cholesteryl ester content compared with the nonencapsulated EGCG. Chitosan-coated nanoencapsulates also revealed decreased expression of monocyte chemoattractant protein-1 (MCP-1). From these findings it can be concluded that the nanocapsules can inhibit atherosclerotic lesion development as evidenced by Zhang et al. (2013). A similar method was employed by Barras et  al. (2009) for nanoencapsulation of EGCG. They synthesized peracetate-protected EGCG (Pro-EGCG) prior to the preparation of EGCG lipid nanocapsules. Pro-EGCG mixed with caprylic/capric triglyceride was reportedly subjected to phase inversion method. In this study, the temperature cycle used was from 70°C to 90°C and then the sample was cooled to 78°C using distilled water (0°C). The nanoencapsulated EGCG exhibited 95% encapsulation rate and stable colloidal suspensions of EGCG in water for 4 weeks; whereas, degradation of free EGCG occurred within 4 h.

8.6.10 Other Encapsulation Techniques 8.6.10.1 Electrospray Electrospray is the process of atomization of liquid by electrical forces. In this technique, encapsulated particles are obtained by spraying a polymer solution using high potential electric field. The system consists of a high voltage source (1–30 kV), a blunt ended stainless steel needle or capillary, syringe pump, and a grounded collector (flat plate or rotating drum). Various factors such as system (molecular weight and microstructural characteristics of the polymer), solution (type and concentration of polymer and solvent), instrumental (electrical potential, flow rate of feed, and needle to collector distance) and ambient parameters (temperature, humidity, and air velocity in chamber) control the size of the final particle (Bhushani and Anandharamakrishnan, 2014). Encapsulation of green tea catechin by electrospraying technique was investigated by Bhushani et  al. (2017) employing zein molecule as wall material. The core-to-wall ratio of the process and effects of nanoparticles on gastrointestinal stability and permeability of green tea catechin were studied. The optimized concentration of zein solution was found to be 5% w/w that provided spherical and monodispersed nanoparticles. This study revealed improved in vitro gastrointestinal stability and enhanced cellular permeability (in Caco-2 cells) of catechins in liposomal form compared to its free form.

8.6.10.2 Nanoprecipitation Nanoprecipitation or solvent displacement technique depends on the spontaneous emulsification of the organic phase containing polymer, drug, and organic solvent into the aqueous phase. Eventually

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the polymer gets precipitated from the organic solution and the solvent diffuses in the aqueous medium. Biodegradable polymers, such as polycaprolactone (PCL), poly (lactide) (PLA), poly (lactide-co-­ glicolide) (PLGA), eudragit, and poly (alkylcyanoacrylate) (PACA) are used for nanoprecipitation. Formulation of catechin hydrate (CH) nanocapsules by nanoprecipitation method was investigated by Samanta et  al. (2016). CH is a secondary metabolite from green tea that reportedly has antioxidant, antiinflammatory, antiproliferative, anticancer, antiangiogenic, and antidiabetic activities. PLGA was used in this study as the carrier polymer and didodecyl dimethyl ammonium bromide (DMAB) was employed as stabilizing agent. Sodium meta borate (SMB) was also employed in the study as chelating agent. Results of the bioavailability of CH in serum reflected enhanced sustained release of CH from nanocapsules (size < 50 nm).

8.6.10.3  Co-Solubilization Methodology Smith et  al. (2010) investigated preparation of EGCG nanoparticles involving co-solubilization methodology. In this method anhydrous EGCG was mixed with monophasic liquid preparation and co-­solubilized by mixing at room temperature. Distilled water was mixed to the mixture for production of nanoparticles (30–80 nm). Rather than formation of micelle, this process produced lipid-EGCG complexes which improved the systemic absorption of EGCG during oral administration along with effective α-secretase activity in vitro.

8.6.10.4  Ligand-Linked Nanoparticles Zhang et al. (2016b) have formulated KOdiA-PC (CD36-targeted ligand) linked EGCG nanoparticles to study its antiatherogenic property against atherosclerosis, which is one of the major causes of cardiovascular disease (CVD). Increase in cholesterol level and inflammatory responses in intimal macrophages generally initiate atherosclerosis. Accumulation of cholesterol by macrophages form foam cells which are known as “hallmark” of atherosclerosis. Macrophage scavenger receptor CD36 is reportedly responsible for foam cell formation. Therefore, Zhang et al. (2016b) envisaged to deliver EGCG to macrophages to decrease production of inflammatory factors and/or cholesterol accumulation that could reduce the risk of atherosclerosis. In this study authors have prepared void and EGCG nanoparticles using PC, kolliphor HS15 (nonionic solubilizer and emulsifier) and (+)-α-tocopherol acetate; KOdiA-PC was associated with the nanoparticles as ligand on the surface of the nanoparticles. CD36targeted EGCG nanoparticles exhibited improved binding affinity to

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macrophages, as well as enhanced uptake by the same. The targeted nanoparticles delivered EGCG to macrophage cytosol avoiding lysosomes and significantly decreased mRNA levels and protein secretion, however, cholesterol content of macrophage did not change significantly. This novel method employed by Zhang et  al. (2016b) can be further employed for targeted delivery of bioactives to intimal macrophages for treatment or prevention of atherosclerosis.

8.6.10.5  Use of Gelatin-Dextran Conjugate A novel green method was utilized by Zhou et al. (2012) to produce nanoencapsulated tea polyphenols in the form of complex coacervation core micelles (C3Ms). In this study, gelatin-dextran conjugate was prepared using 1:1 weight ratio of dextran to gelatin; then the solution of this conjugate was mixed with tea polyphenol and C3Ms were formulated by incubation of this mix along with genipin for 24 h at 4°C. Formulated C3Ms showed high loading capacity, sustained release of polyphenol, and increased cytotoxicity against MCF-7 breast cancer cells compared to free tea polyphenols. Thus, C3Ms can be used for enhanced bioavailability and increased chemoprevention of other bioactives.

8.7  Applications of Nanoencapsulated Green Tea Polyphenols 8.7.1 Food Application Several therapeutic potencies of green tea polyphenol have been reported including high antioxidant, antiinflammatory, and chemopreventive effects which made it fourth most commonly used dietary supplement in the United States (Haratifar, 2012). One novel food application of GTE was investigated by Ghosh and Bhattacharjee (2016). In this study they blended green tea infusion with gamma-­ irradiated expeller-pressed virgin coconut oil, devoid of the characteristic rancid-acid odor of coconut oil, to design a noncarbonated antioxidant-rich ready-to-serve (RTS) beverage. Further these authors carried out microencapsulation of the said beverage by spray drying, and their findings exhibited high antioxidant activity of the dry beverage, along with a shelf-life that was 29 times higher than that of the RTS beverage per se. Both these beverages have been put forth as a health drink. Microencapsulated GTE was investigated in another study for fortification of bread which exhibited similar physicochemical properties compared to the bread prepared with GTE. Total polyphenol contents of both breads were also comparable (Pasrija et  al., 2015). These findings reflected fortification of bread with microencapsulated

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extract can provide therapeutic effects similar to that of the native extract with increased protection to the bioactive during bread baking. Nanoliposomal EGCG and catechin were incorporated into low-fat hard cheese that significantly increased (P < .01) total phenolic content and antioxidant activity of the product after in vitro gastrointestinal digestion (Rashidinejad et  al., 2016a). In another study, full-fat cheese was fortified with liposomes containing C or GTE which also enhanced the total phenolic content and antioxidant potency of the cheese (Rashidinejad et al., 2016b). In both the studies incorporation of liposome did not affect the physical properties of cheese. Therefore, application of nanoencapsulated green tea polyphenols can be investigated for other food products also.

8.7.2 Chemopreventive Application Administration of nanoliposomes prepared from green tea polyphenol has reportedly showed chemopreventive activity. EGCG nanoliposome applied to BCC cells exhibited 20-fold enhanced drug deposition and increased cellular death compared to free EGCG (Fang et  al., 2006b). Nanoliposome of EGCG also exhibited chemopreventive activity against MCF7 breast cancer cells (de Pace et  al., 2013). These nanoliposomes enhanced intracellular content of EGCG along with apoptosis of the cancer cells and showed higher antiproliferative potency compared to free EGCG. Chitosan-based oral formulation of EGCG nanoparticles were prepared by Khan et  al. (2014) for the treatment of prostate cancer in preclinical trials. Application of nanoparticles exhibited significant inhibition of tumor growth and prostate-specific antigen levels compared to free EGCG. Another study conducted with layer-by-layer gelatin coating of EGCG revealed comparable potency of the bioactive both in liposomal and free forms in preventing of HGF-induced intracellular signaling in the breast cancer cell line (MBA-MD-231) (Shutava et al., 2009). In another study, C and EC encapsulated in BSA nanoparticles exhibited improved antioxidant activity and efficacy against human lung carcinoma (A549) cells, thus established the chemopreventive potential of tea polyphenols in nanoencapsulated form (Yadav et al., 2014).

8.8 Conclusion Green tea offers many possibilities in terms of nutraceuticals, health benefits, including chemopreventive action. The multifaceted avenues of application of green tea bioactives in both curative and preventive medicine are many. Although green tea has highly prized therapeutic potencies, they are not immune to degradation under

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a­ mbient conditions and routine delivery modes. Also, targeted delivery of green tea bioactives is an everlasting challenge, which needs attention. The chapter presented herein has collated the findings of researchers’, right from method of extraction of these biomolecules from the tealeaves to their characterization and applications. The chapter combines details on the various approaches employed by researchers to encapsulate; or better, to nanoencapsulate the bioactives from green tea. The research findings revealed the greater possibilities that lie ahead in nanoencapsulation of green tea bioactives; the findings on food applications highlighted the avenues of therapy through nutrition. In aggregate, green tea has a wealth of nutraceutical benefits and should be encapsulated for better stability, bioavailability, and delivery. These encapsulated tea polyphenols should be incorporated in our daily diet for health benefits.

References Aadinath, B.,.A., Anandharamakrishnan, C., 2016. Synergistic radical scavenging potency of curcumin-in-β-cyclodextrin-in-nanomagnetoliposomes. Mater. Sci. Eng. C 64, 293–302. Anandhakumar, S., et al., 2017. Preparation of collagen peptide functionalized chitosan nanoparticles by ionic gelation method: an effective carrier system for encapsulation and release of doxorubicin for cancer drug delivery. Mater. Sci. Eng. C 70, 378–385. Bagheri, L., et al., 2013. Nanoencapsulation of date palm pit extract in whey protein particles generated via desolvation method. Food Res. Int. 51 (2), 866–871. Barras, A., et  al., 2009. Formulation and characterization of polyphenol-loaded lipid nanocapsules. Int. J. Pharm. 379 (2), 270–277. Bharadwaz, A., Bhattacharjee, C., 2012. Extraction of polyphenols from dried tea leaves. Int. J. Sci. Eng. Res. 3 (5). Bhushani, J.A., Anandharamakrishnan, C., 2014. Electrospinning and electrospraying techniques: potential food based applications. Trends Food Sci. Technol. 38 (1), 21–33. Bhushani, J.A., Karthik, P., Anandharamakrishnan, C., 2016. Nanoemulsion based delivery system for improved bioaccessibility and Caco-2 cell monolayer permeability of green tea catechins. Food Hydrocoll. 56, 372–382. Bhushani, J.A., Kurrey, N.K., Anandharamakrishnan, C., 2017. Nanoencapsulation of green tea catechins by electrospraying technique and its effect on controlled release and in-vitro permeability. J. Food Eng. 199, 82–92. Bose, M., et al., 2008. The major green tea polyphenol, (−)-epigallocatechin-3-gallate, inhibits obesity, metabolic syndrome, and fatty liver disease in high-fat-fed mice. J. Nutr. 138 (9), 1677–1683. Cabrera, C., Artacho, R., Giménez, R., 2006. Beneficial effects of green tea – a review. J. Am. Coll. Nutr. 25 (2), 79–99. Chang, C.J., et  al., 2000. Separation of catechins from green tea using carbon dioxide extraction. Food Chem. 68 (1), 109–113. Coester, C.J., et al., 2000. Gelatin nanoparticles by two step desolvation-a new preparation method, surface modifications and cell uptake. J. Microencapsul. 17 (2), 187–193. Connors, K.A., 1997. The stability of cyclodextrin complexes in solution. Chem. Rev. 97 (5), 1325–1357.

256  Chapter 8  Nanoencapsulation of Green Tea Polyphenols

Conte, R., et al., 2016. Polyphenols nanoencapsulation for therapeutic applications. J. Biomol. Res. Therap. 5 (2), 13. Dag, D., Oztop, M.H., 2017. Formation and characterization of green tea extract loaded liposomes. J. Food Sci. 82 (2), 463–470. de Pace, R.C.C., et al., 2013. Anticancer activities of (−)-epigallocatechin-3-gallate encapsulated nanoliposomes in MCF7 breast cancer cells. J. Liposome Res. 23 (3), 187–196. Dehkharghanian, M., Lacroix, M., Vijayalakshmi, M., 2009. Antioxidant properties of green tea polyphenols encapsulated in caseinate beads. Dairy Sci. Technol. 89 (5), 485–499. Drużyńska, B., Stępniewska, A., Wołosiak, R., 2007. Warsaw agricultural university SGGW. Acta Sci. Pol. Technol. Aliment. 6 (1), 27–36. Dube, A., Ng, K., et al., 2010b. Effective use of reducing agents and nanoparticle encapsulation in stabilizing catechins in alkaline solution. Food Chem. 122 (3), 662–667. Dube, A., Nicolazzo, J.A., Larson, I., 2010a. Chitosan nanoparticles enhance the intestinal absorption of the green tea catechins (+)-catechin and (−)-epigallocatechin gallate. Eur. J. Pharm. Sci. 41 (2), 219–225. Dube, A., Nicolazzo, J.A., Larson, I., 2011. Chitosan nanoparticles enhance the plasma exposure of (−)-epigallocatechin gallate in mice through an enhancement in intestinal stability. Eur. J. Pharm. Sci. 44 (3), 422–426. Dungan, S.R., 2015. Interfacial science and the creation of nanoparticles. In: Sabliov, C.M., Chen, H., Yada, R.Y. (Eds.), Nanotechnology and Functional Foods: Effective Delivery of Bioactive Ingredients. IFT Press, Wiley Blackwell, West Sussex, UK, pp. 52–68. Elabbadi, A., et al., 2010. Complexation/encapsulation of green tea polyphenols in mixed calcium carbonate and phosphate micro-particles. J. Microencapsul. 28 (1), 1–9. Ezhilarasi, P.N., et al., 2013. Nanoencapsulation techniques for food bioactive components: a review. Food Bioprocess Technol. 6 (3), 628–647. Fang, J.Y., Hwang, T.L., et al., 2006a. Enhancement of the transdermal delivery of catechins by liposomes incorporating anionic surfactants and ethanol. Int. J. Pharm. 310 (1–2), 131–138. Fang, J.Y., Lee, W.R., et al., 2006b. Effect of liposome encapsulation of tea catechins on their accumulation in basal cell carcinomas. J. Dermatol. Sci. 42 (2), 101–109. Fang, J.Y., et  al., 2004. Physicochemical characteristics and in  vivo deposition of liposome-­encapsulated tea catechins by topical and intratumor administrations. J. Drug Target. 13 (1), 19–27. Fang, Z., Bhandari, B., 2010. Encapsulation of polyphenols—a review. Trends Food Sci. Technol. 21 (10), 510–523. Flanagan, J., Singh, H., 2006. Microemulsions: a potential delivery system for bioactives in food. Crit. Rev. Food Sci. Nutr. 46 (3), 221–237. Folch-Cano, C., et al., 2010. Antioxidant activity of inclusion complexes of tea catechins with β-cyclodextrins by ORAC assays. Food Res. Int. 43 (8), 2039–2044. Folch-Cano, C., et  al., 2013. NMR and molecular fluorescence spectroscopic study of the structure and thermodynamic parameters of EGCG/β-cyclodextrin inclusion complexes with potential antioxidant activity. J. Incl. Phenom. Macrocycl. Chem. 78 (1–4), 287–298. Furuse, M., et  al., 2009. Central functions of green tea components. In: Mckinley, H., Jamieson, M. (Eds.), Handbook of Green Tea and Health Research. Nova Science Publishers, Inc., New York, pp. 1–22. Geleijnse, J.M., et al., 2002. Inverse association of tea and flavonoid intakes with incident myocardial infarction: the Rotterdam study. Am. J. Clin. Nutr. 75 (5), 880–886. Ghosh, P.K., 2016. Studies on supercritical carbon dioxide extraction, encapsulation and gamma irradiation technologies for development of novel supplements from food and horticulture products. (Ph.D. Thesis). Jadavpur University, India.

Chapter 8  Nanoencapsulation of Green Tea Polyphenols    257

Ghosh, P.K., Bhattacharjee, P., 2016. Comparative evaluation of irradiated and non-­ irradiated expeller-pressed virgin coconut oil for the design of novel functional antioxidant-­rich non-carbonated ready-to-serve and dry beverages. Nutrafoods 15, 123–133. Goodarznia, I., Govar, A.A., 2009. Superheated water extraction of catechins from green tea leaves: modeling and simulation. Trans. C: Chem. Chem. Eng. 16 (2), 99–107. Haratifar, S., 2012. Nanoencapsulation of Tea Catechins in Casein Micelles: Effects on Processing and Biological Functionalities. The University of Guelph In. Hong, Z., et al., 2014. Improving the effectiveness of (−)-epigallocatechin gallate (EGCG) against rabbit atherosclerosis by EGCG-loaded nanoparticles prepared from chitosan and polyaspartic acid. J. Agric. Food Chem. 62 (52), 12603–12609. Hu, B., et  al., 2008. Optimization of fabrication parameters to produce chitosan-­ tripolyphosphate nanoparticles for delivery of tea catechins. J. Agric. Food Chem. 56 (16), 7451–7458. Hu, B., et al., 2012. Cellular uptake and cytotoxicity of chitosan-­caseinophosphopeptides nanocomplexes loaded with epigallocatechin gallate. Carbohydr. Polym. 89 (2), 362–370. Hu, B., et al., 2013. Bioactive peptides/chitosan nanoparticles enhance cellular antioxidant activity of (−)-epigallocatechin-3-gallate. J. Agric. Food Chem. 61 (4), 875–881. Ishii, T., et  al., 2011. Human serum albumin as an antioxidant in the oxidation of (−)-epigallocatechin gallate: participation of reversible covalent binding for interaction and stabilization. Biosci. Biotechnol. Biochem. 75 (1), 100–106. Ishizu, T., et al., 2006. Structure and intramolecular flexibility of β-cyclodextrin complex with (−)-epigallocatechin gallate in aqueous solvent. Magn. Reson. Chem. 44 (8), 776–783. Jafari, S.M., He, Y., Bhandari, B., 2007. Production of sub-micron emulsions by ultrasound and microfluidization techniques. J. Food Eng. 82 (4), 478–488. Jun, X., et al., 2011. Comparison of in vitro antioxidant activities and bioactive components of green tea extracts by different extraction methods. Int. J. Pharm. 408 (1–2), 97–101. Jung, M.H., et al., 2013. Effect of green tea extract microencapsulation on hypertriglyceridemia and cardiovascular tissues in high fructose-fed rats. Nutr. Res. Pract. 7 (5), 366–372. Kadoma, Y., et al., 2006. Free radical interaction between vitamin E (alpha-, beta-, gamma- and delta-tocopherol), ascorbate and flavonoids. In  vivo 20 (6B), 823–828. Kailaku, S.I., Mulyawanti, I., Alamsyah, A.N., 2014. Formulation of nanoencapsulated catechin with chitosan as encapsulation material. Proc. Chem. 9, 235–241. Kamil, A., Chen, C.-Y.O., Blumberg, J.B., 2015. The application of nanoencapsulation to enhance the bioavailability and distribution of polyphenols. In: Sabliov, C.M., Chen, H., Yada, R.Y. (Eds.), Nanotechnology and Functional Foods: Effective Delivery of Bioactive Ingredients. IFT Press, Wiley Blackwell, West Sussex, UK, pp. 158–174. Khan, N., et  al., 2006. Targeting multiple signaling pathways by green tea polyphenol (−)-epigallocatechin-3-gallate. Cancer Res. 66 (5), 2500–2505. Khan, N., et al., 2014. Oral administration of naturally occurring chitosan-based nanoformulated green tea polyphenol EGCG effectively inhibits prostate cancer cell growth in a xenograft model. Carcinogenesis 35 (2), 415–423. Kim, W.J., et al., 2008. Selective caffeine removal from green tea using supercritical carbon dioxide extraction. J. Food Eng. 89 (3), 303–309. Kim, Y.J., et al., 2012. Nanoemulsified green tea extract shows improved hypocholesterolemic effects in C57BL/6 mice. J. Nutr. Biochem. 23 (2), 186–191. Krishnaswamy, K., Orsat, V., Thangavel, K., 2012. Synthesis and characterization of ­nano-encapsulated catechin by molecular inclusion with beta-cyclodextrin. J. Food Eng. 111 (2), 255–264.

258  Chapter 8  Nanoencapsulation of Green Tea Polyphenols

Lam, W.H., et al., 2004. A potential prodrug for a green tea polyphenol proteasome inhibitor: evaluation of the peracetate ester of (−)-epigallocatechin gallate [(−)-EGCG]. Bioorg. Med. Chem. 12 (21), 5587–5593. Lambert, J.D., et al., 2006. Peracetylation as a means of enhancing in vitro bioactivity and bioavailability of epigallocatechin-3-gallate. Drug Metab. Dispos. 34 (12), 2111–2116. Li, B., et al., 2012. Preservation of (-)-epigallocatechin-3-gallate antioxidant properties loaded in heat treated β-lactoglobulin nanoparticles. J. Agric. Food Chem. 60 (13), 3477–3484. Li, Z., Gu, L., 2014. Fabrication of self-assembled (−)-epigallocatechin gallate (EGCG) ovalbumin-dextran conjugate nanoparticies and their transport across monolayers of human intestinal epithelial caco-2 cells. J. Agric. Food Chem. 62 (6), 1301–1309. Li, Z., et al., 2014. Fabrication of coated bovine serum albumin (BSA)-epigallocatechin gallate (EGCG) nanoparticles and their transport across monolayers of human intestinal epithelial Caco-2 cells. Food Funct. 5 (6), 1278–1285. Liang, H., et al., 2007. Tea extraction methods in relation to control of epimerization of tea catechins. J. Sci. Food Agric. 87 (9), 1748–1752. Lira, M.C.B., et al., 2009. Inclusion complex of usnic acid with β-cyclodextrin: characterization and nanoencapsulation into liposomes. J. Incl. Phenom. Macrocycl. Chem. 64 (3–4), 215–224. Liu, F., et  al., 2015. Preparation of gelatin films incorporated with tea polyphenol nanoparticles for enhancing controlled-release antioxidant properties. J. Agric. Food Chem. 63 (15), 3987–3995. Liu, F., et al., 2016. Chitosan/sulfobutylether-β-cyclodextrin nanoparticles as a potential approach for tea polyphenol encapsulation. Food Hydrocoll. 57, 291–300. Liu, F., et al., 2017. Controlled-release of tea polyphenol from gelatin films incorporated with different ratios of free/nanoencapsulated tea polyphenols into fatty food simulants. Food Hydrocoll. 62, 212–221. Lorenz, M., et al., 2007. Addition of milk prevents vascular protective effects of tea. Eur. Heart J. 28 (2), 219–223. Lu, Q., Li, D.C., Jiang, J.G., 2011. Preparation of a tea polyphenol nanoliposome system and its physicochemical properties. J. Agric. Food Chem. 59 (24), 13004–13011. Lucas-Abellán, C., et al., 2007. Cyclodextrins as resveratrol carrier system. Food Chem. 104 (1), 39–44. Mahmood, T., Akhtar, N., Manickam, S., 2014. Interfacial film stabilized W/O/W nano multiple emulsions loaded with green tea and lotus extracts: systematic characterization of physicochemical properties and shelf-storage stability. J. Nanobiotechnol. 12 (1), 20. Mak, J.C.W., 2012. Potential role of green tea catechins in various disease therapies: progress and promise. Clin. Exp. Pharmacol. Physiol. 39 (3), 265–273. Man, G.C.W., Chu, K.O., Wang, C.C., 2016. Nanoencapsulation of green tea catechins and its efficacy. In: Novel Approaches of Nanotechnology in Food. Elsevier, pp. 555–586. Marty, J.J., Oppenheim, R.C., Speiser, P., 1978. Nanoparticles—a new colloidal drug delivery system. Pharm. Acta Helv. 53, 17–23. McKay, D.L., Blumberg, J.B., 2002. The role of tea in human health: an update. J. Am. Coll. Nutr. 21 (1), 1–13. Mercader-Ros, M.T., et al., 2010. Effect of HP-β-cyclodextrins complexation on the antioxidant activity of flavonols. Food Chem. 118 (3), 769–773. Mereles, D., Hunstein, W., 2011. Epigallocatechin-3-gallate (EGCG) for clinical trials: more pitfalls than promises? Int. J. Mol. Sci. 12 (9), 5592–5603. Mozafari, M.R., et  al., 2008. Encapsulation of food ingredients using nanoliposome technology. Int. J. Food Prop. 11 (4), 833–844. Noudoost, B., et  al., 2015. Encapsulation of green tea extract in nanoliposomes and evaluation of its antibacterial, antioxidant and prebiotic properties. J. Med. Plants 14 (55), 66–78.

Chapter 8  Nanoencapsulation of Green Tea Polyphenols    259

Oehlke, K., et al., 2014. Potential bioavailability enhancement of bioactive compounds using food-grade engineered nanomaterials: a review of the existing evidence. Food Funct. 5 (7), 1341–1359. Pan, X., Niu, G., Liu, H., 2003. Microwave-assisted extraction of tea polyphenols and tea caffeine from green tea leaves. Chem. Eng. Process. Process Intensif. 42 (2), 129–133. Park, S.J., et al., 2016. Fabrication and optimization of EGCG-loaded nanoparticles by high pressure homogenization. J. Appl. Polym. Sci. 133 (14), 1–7. Pasrija, D., Anandharamakrishnan, C., 2015. Techniques for extraction of green tea polyphenols: a review. Food Bioprocess Technol. 8 (5), 935–950. Pasrija, D., et al., 2015. Microencapsulation of green tea polyphenols and its effect on incorporated bread quality. LWT Food Sci. Technol. 64 (1), 289–296. Patravale, V.B., Mandawgade, S.D., 2008. Novel cosmetic delivery systems: an application update. Int. J. Cosmet. Sci. 30 (1), 19–33. Pons, M., Foradada, M., Estelrich, J., 1993. Liposomes obtained by the ethanol injection method. Int. J. Pharm. 95 (1–3), 51–56. Potenza, M., et al., 2007. EGCG, a green tea polyphenol, improves endothelial function and insulin sensitivity, reduces blood pressure, and protects against myocardial I/R injury in SHR. Am. J. Physiol. Endocrinol. Metab. 292, 1378–1387. Proniuk, S., Liederer, B.M., Blanchard, J., 2002. Preformulation study of epigallocatechin gallate, a promising antioxidant for topical skin cancer prevention. J. Pharm. Sci. 91 (1), 111–116. Puligundla, P., et al., 2017. Nanotechnological approaches to enhance the bioavailability and therapeutic efficacy of green tea polyphenols. J. Funct. Foods 34, 139–151. Racoviţǎ, Ş., et al., 2009. Polysaccharides based on micro- and nanoparticles obtained by ionic gelation and their applications as drug delivery systems. Rev. Roum. Chim. 54 (9), 709–718. Rashidinejad, A., Birch, E.J., Everett, D.W., 2016b. A novel functional full-fat hard cheese containing liposomal nanoencapsulated green tea catechins: manufacture and recovery following simulated digestion. Food Funct. 7 (7), 3283–3294. Rashidinejad, A., Birch, E.J., Sun-Waterhouse, D., et al., 2016a. Effect of liposomal encapsulation on the recovery and antioxidant properties of green tea catechins incorporated into a hard low-fat cheese following in vitro simulated gastrointestinal digestion. Food Bioprod. Process. 100, 238–245. Rein, M.J., et al., 2012. Bioavailability of bioactive food compounds: a challenging journey to bioefficacy. Br. J. Clin. Pharmacol. 75 (3), 588–602. Reis, C.P., et  al., 2006. Nanoencapsulation I. Methods for preparation of drug-loaded polymeric nanoparticles. Nanomedicine 2 (1), 8–21. Rodrigues, C.F., et al., 2013. Drug-delivery systems of green tea catechins for improved stability and bioavailability. Curr. Med. Chem. 20 (37), 4744–4757. Row, K.H., Jin, Y., 2006. Recovery of catechin compounds from Korean tea by solvent extraction. Bioresour. Technol. 97 (5), 790–793. Ru, Q., Yu, H., Huang, Q., 2010. Encapsulation of epigallocatechin-3-gallate (EGCG) using oil-in-water (O/W) submicrometer emulsions stabilized by ι-carrageenan and β-Lactoglobulin. J. Agric. Food Chem. 58 (19), 10373–10381. Samanta, A., Bandyopadhyay, B., Nirmalendu, D., 2016. Formulation of catechin hydrate nanocapsule and study of its bioavailability. Med. Chem. 6 (6), 399–404. Sasazuki, S., et  al., 2000. Relation between green tea consumption and the severity of coronary atherosclerosis among Japanese men and women. Ann. Epidemiol. 10 (6), 401–408. Shi, J., et al., 2005. Extraction of polyphenolic from plant material for functional foods— engineering and technology. Food Rev. Int. 21 (1), 139–166. Shpigelman, A., Cohen, Y., Livney, Y.D., 2012. Thermally-induced beta-lactoglobulin-­ EGCG nanovehicles: loading, stability, sensory and digestive-release study. Food Hydrocoll. 29 (1), 57–67.

260  Chapter 8  Nanoencapsulation of Green Tea Polyphenols

Shpigelman, A., Israeli, G., Livney, Y.D., 2010. Thermally-induced protein-polyphenol co-assemblies: beta lactoglobulin-based nanocomplexes as protective nanovehicles for EGCG. Food Hydrocoll. 24 (8), 735–743. Shrikhande, A.J., et al., 2003. Process for extraction, purification and enrichment of polyphenolic substances from whole grapes, grape seeds and grape pomace. Available from: http://www.google.co.in/patents/US6544581. (Accessed October 13, 2017). Shutava, T.G., et al., 2009. Layer-by-layer-coated gelatin nanoparticles as a vehicle for delivery of natural polyphenols. ACS Nano 3 (7), 1877–1885. Siddiqui, I.A., et  al., 2014. Excellent anti-proliferative and pro-apoptotic effects of (−)-epigallocatechin-3-gallate encapsulated in chitosan nanoparticles on human melanoma cell growth both in vitro and in vivo. Nanomedicine 10 (8), 1619–1626. Smart, J.D., 2005. The basics and underlying mechanisms of mucoadhesion. Adv. Drug Deliv. Rev. 57 (11), 1556–1568. Smith, A., et  al., 2010. Nanolipidic particles improve the bioavailability and alpha-­ secretase inducing ability of epigallocatechin-3-gallate (EGCG) for the treatment of Alzheimer’s disease. Int. J. Pharm. 389 (1–2), 207–212. Sosa, M.V., et al., 2011. Green tea encapsulation by means of high pressure antisolvent coprecipitation. J. Supercrit. Fluids 56 (3), 304–311. Swarbrick, J., Boylan, J.C. (Eds.), 2001. Encyclopedia of Pharmaceutical Technology. Marcel Dekker, Inc., New York. Tang, D.W., et  al., 2013. Characterization of tea catechins-loaded nanoparticles prepared from chitosan and an edible polypeptide. Food Hydrocoll. 30 (1), 33–41. UK Tea and Infusions Association, 2017. Tea—A Brief History of the Nation’s Favourite Beverage. UK Tea & Infusions Association. Available from: http://www.tea.co.uk/ tea-a-brief-history. Vuong, Q.V., et  al., 2011. Optimizing conditions for the extraction of catechins from green tea using hot water. J. Sep. Sci. 34 (21), 3099–3106. Weiss, J., Takhistov, P., McClements, D.J., 2006a. Functional materials in food nanotechnology. J. Food Sci. 71 (9), R107–R116. Yadav, R., et al., 2014. Encapsulation of catechin and epicatechin on BSA NPs improved their stability and antioxidant potential. EXCLI J. 13, 331–346. Yang, Y.-C., et al., 2004. The protective effect of habitual tea consumption on hypertension. Arch. Intern. Med. 164 (14), 1534. Zhang, H., Jung, J., Zhao, Y., 2016a. Preparation, characterization and evaluation of antibacterial activity of catechins and catechins-Zn complex loaded β-chitosan nanoparticles of different particle sizes. Carbohydr. Polym. 137, 82–91. Zhang, J., Nie, S., Wang, S., 2013. Nanoencapsulation enhances epigallocatechin-3-­ gallate stability and its antiatherogenic bioactivities in macrophages. J. Agric. Food Chem. 61 (38), 9200–9209. Zhang, J., et  al., 2016b. Formulation, characteristics and antiatherogenic bioactivities of CD36-targeted epigallocatechin gallate (EGCG)-loaded nanoparticles. J. Nutr. Biochem. 30, 14–23. Zhong, Q., et al., 2015. Delivery systems for food applications: an overview of preparation methods and encapsulation, release, and dispersion properties. In: Sabliov, C.M., Chen, H., Yada, R.Y. (Eds.), Nanotechnology and Functional Foods: Effective Delivery of Bioactive Ingredients. IFT Press, Wiley Blackwell, West Sussex, UK, pp. 91–111. Zhou, H., et al., 2012. Fabrication of biopolymeric complex coacervation core micelles for efficient tea polyphenol delivery via a green process. Langmuir 28 (41), 14553–14561. Zou, L., et al., 2014a. Improved in vitro digestion stability of (−)-epigallocatechin gallate through nanoliposome encapsulation. Food Res. Int. 64 (June), 492–499. Zou, L.Q., et al., 2014b. Characterization and bioavailability of tea polyphenol nanoliposome prepared by combining an ethanol injection method with dynamic high-­ pressure microfluidization. J. Agric. Food Chem. 62 (4), 934–941.

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Further Reading Graham, H.N., 1992. Green tea composition, consumption, and polyphenol chemistry. Prev. Med. 21, 334–350. Weiss, D.J., et al., 2006b. Analysis of green tea extract dietary supplements by micellar electrokinetic chromatography. J. Chromatogr. A 1117, 103–108.