Bioactivity of Epigallocatechin Gallate Nanoemulsions Evaluated in Mice Model

Abstract

The hypothesis that incorporation of epigallocatechin gallate (EGCG) into nanoemulsions may increase its bioactivity compared with EGCG aqueous solutions was examined in mice. After an in vitro study in a model system with stimulated gastrointestinal conditions, the following EGCG nanoemulsions were used in a mice experiment: Emulsion I: emulsion water in oil (W/O), which contained 0.23 mg/mL EGCG in aqueous phase; Emulsion II: emulsion oil in water (O/W), which contained 10% olive oil and 0.23 mg/mL esterified EGCG in fatty phase; and Emulsion III: emulsion O/W in water (W1/O/W2; 8:32:60), which contained 32% olive oil and 0.23 mg/mL EGCG in aqueous phase. After 2 h of mice administration by gavage with 0.1 mL of EGCG nanoemulsions, total antioxidant capacity (TAC) of plasma and some tissues (especially colon, jejunum, heart, spleen) was measured with Ferric-Reducing Antioxidant Power (FRAP) and Oxygen Radical Absorbance Capacity (ORAC) assays. No toxic effects were observed after administration of 0.23 mg/mL esterified EGCG in CD1 mouse strain. The study concluded that administration of mice with the three EGCG nanoemulsions did not increase their TAC in specific tissues, compared with an aqueous EGCG solution at the same concentration. Nevertheless, the esterified EGCG emulsion (Emulsion II) exerted an increase in mice plasma compared with aqueous EGCG and showed higher values of TAC in several tissues, compared with Emulsions I and III. EGCG nanoemulsions could be considered a useful method in plethora functional food applications, but further research is required for safer results.

Introduction

Bioactivity, referring to various end points in health and disease, may be expressed through different biochemical pathways or effects, such as total antioxidant capacity (TAC).^1

–4 It has been shown by clinical trials and mouse studies that flavonoids ingestion increases the antioxidant capacity of plasma and organs.^5

–10 Flavonoids and their metabolites are transferred after absorption through blood circulation to various organs where they exert bioactivity. Absorption, metabolism, pharmacokinetics, tissue distribution, and rate of excretion in urine or feces reflect the bioavailability of flavonoids and, thus, their biological role in the organism.^11,12 Interactions between flavonoids and meal constituents (metal ions, proteins, fat, and carbohydrates) occur mainly in the small intestine and are considered important factors that affect the extent of its absorption and, thus, its bioavailability and bioactivity.^1,13

Catechins have been extensively studied as a basic flavonoid group with possible beneficial effects and a putative role in disease prevention.^1,2 Epigallocatechin gallate (EGCG) displays the highest bioactivity in comparison with other catechins, due to the presence of a gallic group in its molecule.^1,13 EGCG has been studied widely for its potential beneficial effects, especially antioxidant properties by scavenging free radicals,¹⁴ anti-bacterial,¹⁵ anti-inflammatory,¹⁶ cardioprotective,¹⁷ anti-cancer,¹⁸ neuroprotective,¹⁹ and other biological activities in human organisms. Due to its specific structure and hydrophilicity, there is evidence that EGCG interacts with other constituents in the lumen, is extensively metabolized, and is easily excreted from the human body; thus, it exerts relatively low bioavailability.²⁰ For these reasons, it is necessary to find innovative technologies, such as nanotechnology, for increasing EGCG stability and bioavailability and, consequently, bioactivity.

Nanotechnology takes advantage of properties of structures that are sized 100 nm or lower; in recent years, nanotechnology has been studied and applied in various fields, including the development of novel foods with enhanced nutritional properties.²¹ Nanoemulsions, one of the approaches of nanotechnology, consist of two immiscible liquids; one is in the form of droplets of 10 to a few hundred nanometers in diameter. Lipid nanocapsules constitute one form of nanoemulsions that may allow the delivery of bioactive compounds, particularly when hydrophobic or sensitive to acidic conditions. The nanoparticles have the ability to encapsulate the desired compounds, and therefore to stabilize and transfer them, to specific points in the gastrointestinal track that can impart their beneficial properties.^22
–24

In this study, we tested in mice the hypothesis that encapsulation of EGCG into nanoemulsions may increase its bioactivity, particularly its TAC. Previously, several EGCG water solutions and nanoemulsions were evaluated both in vitro and in a model system with stimulated gastrointestinal conditions to carry out an initial comparison.

Materials and Methods

Sample preparation

Green tea

The green tea used was rich in catechins (Green Linea Label' Unilevers). The tea beverage was prepared by pouring 250 mL boiling water over a tea bag (2.3 g) and brewing for 3 min. The concentration of catechins in green tea was 9 mg/mL according to the manufacturer.

EGCG in aqueous solution

EGCG was used at two levels of concentration: 1.2 mg/mL EGCG corresponds to the EGCG concentration that can be found in brewed green tea, and 22.5 mg/mL EGCG corresponds to a saturated solution.

EGCG used in the study was of tea origin and was provided by Biochemical Reagent (Ausmausco Pharma, Co., Ltd.).

Nanoemulsion preparation

The nanoemulsion composition is presented in Table 1.

Table 1.

Description of Emulsion Composition

	Emulsion type	Emulsion name	EGCG	WPI (%)	BC (%)	Olive oil (%)	PGPR (%)	NaCl (%)
Emulsion I	W/O	Aqueous EGCG	0.23 mg/mL EGCG in aqueous phase	5	1	10	—	—
Emulsion II	O/W	Control 1	—	5	1	10	—	—
Emulsion III	O/W	Esterified EGCG	0.23 mg/mL Esterified EGCG in fatty phase	5	1	10	—	—
Emulsion IV	W1/O/W2 (8:32:60)	Control 2	—	1	0.25	32	0.8	4.3
Emulsion V	W1/O/W2 (8:32:60)	Double emulsion of EGCG	0.23 mg/mL EGCG in aqueous phase	1	0.25	32	0.8	4.3

BC, bacterial cellulose; EGCG, epigallocatechin gallate; O/W, emulsion oil in water; PGPR, polyglycerol polyricinoleate; W1/O/W2, emulsion O/W in water; W/O, emulsion water in oil; WPI (Lacprodan DI-9224), whey protein isolate.

Preparation of emulsions I, II, and III

The nanoemulsions I, II, and III were prepared according to Paximada et al. ²⁵ Briefly, extra virgin olive oil was added to bacterial cellulose (BC) and whey protein isolate (WPI; Lacprodan DI-9224) solutions and subsequently mixed in a high-shear mixer at 1200 g for 2 min (Ultra Turrax T25; IKA). Emulsions were further treated with an ultrasonic homogenizer model Sonopuls 2200 (Bandelin Electronic Gmbh & Co.) at a frequency of 20 kHz for 4 min. The resulting emulsion achieves a final concentration of 1% wt. BC, 5% wt. WPI, and 10% wt. olive oil (Emulsion II). In the case of Emulsion I, pure EGCG was incorporated into the aqueous phase of the emulsions under continuous stirring before the emulsification.

In the case of Emulsion III, esterified EGCG was incorporated into the oily phase; it was first dissolved in ethanol, then the appropriate amount of oil was added, and finally the solution was placed in a rotary evaporator until the ethanol was evaporated. EGCG esterification was achieved via a reaction of EGCG dissolved in ethyl acetate with an acylating agent, namely the stearoyl chloride (SA) at a molar ratio of 1:1 in the presence of pyridine at 40°C for 3 h under constant stirring. The procedure was followed by cooling to ambient temperature and filtration of the mixture, washing of the filtrate with distilled water (60°C), collection of the organic layer, removal of the washing water, evaporation of organic solvent, and storage of the final product at −20°C until further use. The result of the esterification was a light yellow dry powder, a mixture of EGCG-stearic acid polyesters.

Preparation of emulsions IV and V

The “double emulsions” IV and V were prepared according to Mun et al. at ambient temperature (25°C) and at pH 3.6.²⁶ Initially, water in oil emulsions were produced, where the dispersed W1 phase (20% wt.) consisted of the citrate buffer solution and the continuous oil O phase (80% wt.) was extra virgin olive oil containing varying polyglycerol polyricinoleate (PGPR) concentrations (4%, 6% or 8% wt.) as lipophilic surfactant (Grindsted PGPR 90 Kosher was from Danisco, Copenhagen, Denmark). Mixing was performed at ambient temperature (20°C) by Ultra Turrax T25, (IKA) at 1120 g for 3 min. Afterward, double emulsions were formed by dispersing 40% wt. W1/O emulsion into 60% wt. W2 external aqueous phase by using the same homogenizer at 20°C (first run 1120 g for 3 min and second run 1680 g for 2 min). The W2 phase consisted of varying the BC concentration (0.125% or 0.25% wt). The overall composition of the final emulsion O/W in water (W1/O/W2; 8:32:60) emulsion was 0.23 mg/mL EGCG, 4.3% wt. NaCl, 0.8% wt. PGPR, 0.25% wt. BC, and 1% wt. WPI (Emulsion V). A blank W1/O/W2 emulsion was also fabricated without adding EGCG in the W1 phase (Emulsion VI).

Antioxidant capacity in vitro

Test material was tested in vitro and in a model system after stimulated gastrointestinal conditions. Specifically, TAC and total phenolic content of water, green tea, and aqueous EGCG in two concentrations (1.2 and 22.5 mg/mL) were measured by using Ferric-Reducing Antioxidant Power (FRAP) and Folin assays (described below). Green tea, EGCG 1.2 mg/mL, and EGCG 22.5 mg/mL solutions were further stimulated in an in vitro model that mimicked the gastrointestinal process in vivo. For this purpose, 2 mL of water or green tea or one of the two aquatic solutions of EGCG prepared as described earlier was subjected to the in vitro digestion procedure described by Argyri et al. ²⁷ TAC using the FRAP method (described in Analytical techniques section)²⁸ and total polyphenol content using the Folin–Ciocalteau method (described in Analytical techniques section)²⁹ were measured in the soluble, low-molecular-weight fraction of the digest that simulates the fraction absorbed in vivo. All samples were run in duplicate, and each experiment was repeated two times. The pH of all samples was adjusted to 2.5 with HCl 6 or 1 M.

Mice model

Toxicity of EGCG derivatives in mice

To test the putative toxicity of the esterified form of EGCG used in tested nanoemulsions, the CD1 outbred mouse model was selected. Cohorts of five male and five female mice, aged 6–8 weeks old, were administered 0.1 mL of esterified EGCG solution 0.23 mg/mL diluted in peanut oil by oral gavage. For comparison, five mice were also administered the water-soluble form of EGCG, previously shown not to exert any toxic effect. Mouse weight was monitored daily for a duration of 2 weeks. Also, mice were inspected daily for any adverse effect or altered physical activity.³⁰

In vivo antioxidant capacity

A series of control and EGCG emulsions (emulsion oil in water [O/W], emulsion water in oil [W/O, W/O/W] and aqueous solutions were prepared as presented in Table 1 and administered in each mice group as follows. Group A: distilled water, which was used as a control; Group B: Emulsion I (W/O aqueous EGCG nanoemulsion); Group C: EGCG water solution 0.23 mg/mL (aqueous EGCG); Group D: A solution with two phases: Phase I: EGCG water solution 0.23 mg/mL (phase I was 80% of the solution) and Phase II: olive oil (phase II was 20% of the solution); Group E: Emulsion II (O/W nanoemulsion without EGCG, which was used as a control); Group F: Emulsion III (O/W esterified EGCG nanoemulsion); Group G: Emulsion VI (W1/O/W2 double nanoemulsion without EGCG, which was used as a control); and Group H: Emulsion V (W1/O/W2 double EGCG nanoemulsion).

The study protocol was in compliance with the Declaration of Helsinki principles and was approved by the authorities of the Municipality of Attica (Athens, Greece). Male mice (strain C57/BL6) were bred and housed at the Animal House Facilities of Biomedical Research Science Center “Alexander Fleming” (Vari, Greece). Cohorts of five to eight mice that were 5 weeks old, each weighing ∼25 g, were randomly assigned to the eight groups and received the eight treatments, respectively (Treatment A Group A, Treatment B Group B … Treatment H Group H).

After food withdrawal for 5 h, mice were given 0.1 mL of each treatment by oral gavage. One hundred and twenty minutes later, the animals were euthanized in carbon dioxide. Immediately after euthanasia, each animal was dissected; ∼0.4 mL of blood was drawn by cardiac puncture and transferred into heparin-containing tubes on ice; and plasma was separated by centrifugation at 3000 g for 5 min at 4°C. Aliquots of plasma were transferred into Eppendorf tubes and stored at −80°C for a maximum of 4 h, until analysis with the FRAP and Oxygen Radical Absorbance Capacity (ORAC) assays.

Different organs, namely heart, lungs, liver, spleen, kidneys, stomach, colon, jejunum, and brain, were dissected out, weighed, and immediately frozen on dry ice. After thawing, the organs were homogenized in the presence of phosphate buffer saline (pH 6.8), using a volume three times the weight of each organ. The homogenized organs were centrifuged at 13,000 g for 5 min at 4°C, according to Koutelidakis et al. ^7,10 Subsequently, the supernatants were transferred into Eppendorf tubes and stored at −80°C for a maximum of 4 h, until analysis with the FRAP and ORAC assays (described in Analytical techniques section).

Analytical techniques

All chemicals were from Sigma-Aldrich (Steinheim, Germany).

Folin–Ciocalteau assay

Total phenolic content of tea, EGCG solutions, and in vitro digestion retentates was measured according to the method of Singleton and Rossi,²⁹with some modifications (Koutelidakis et al. ⁹) by using the Folin–Ciocalteau reagent. The absorbance was measured at 765 nm, using a visible ultraviolet spectrophotometer.

FRAP assay

The FRAP of tea, EGCG solutions, and in vitro digestion retentates and of mice plasma and tissue homogenates was determined according to the method of Benzie and Strain, adapted by Koutelidakis et al.,^7,28 to employ a 96-well plate reader. Final FRAP values were expressed as micromoles of FeSO4 per tissue kg or per mL retentate, after standard curve preservation.

ORAC assay

The ORAC of mice plasma and tissues homogenates was determined according to Huang et al.,³¹ and it was adapted to employ a 96-well fluorescent plate reader. Final ORAC values were expressed as micromole Trolox equivalents per tissue gram.

Statistical analysis

The values of in vitro digestion TAC and total phenolics of retentates were expressed as mean ± standard deviation after three experiments. Differences were studied with one-way analysis of variance (ANOVA), the least significant difference test at 95% confidence interval and considered significant when P < .05. Analysis of data was carried out with the statistic program PASW Statistics 18. The values of TAC of mice plasma and organ homogenates were expressed as mean ± standard deviation after three experiments for five to six mice in each group. All differences were tested with the Kruskal–Wallis test, one-way ANOVA, and Turkey post hoc test. Differences were considered significant when P < .05. Analysis of data was carried out with the statistic program PASW Statistics 18.

Results

In vitro antioxidant capacity

Table 2 presents the results of the TAC, measured with FRAP assay, of green tea and EGCG solutions before and after their submission to the in vitro digestion procedure. The highest concentration of EGCG (22.5 mg/mL) showed the highest TAC (13.32 mM FeSO4). No difference in the antioxidant capacity was observed between the samples of green tea and EGCG 1.2 mg/mL (2.64 and 2.71 mM FeSO4, respectively). The TAC of green tea and EGCG (1.2 mg/mL) was reduced after their submission to the in vitro digestion procedure (0.80 mM FeSO4 [P = .039] and 0.85 mM FeSO4 [P = .038], respectively). The TAC of EGCG (22.5 mg/mL) was also reduced (11.91 mM FeSO4), but the reduction was not statistically significant (P > .05).

Table 2.

Total Antioxidant Capacity (Ferric-Reducing Antioxidant Power Assay) and Total Phenolic Content (Folin Ciocalteau Assay) of Samples of Green Tea and Two Aqueous Solutions of Epigallocatechin Gallate Before and After Their Submission to an In Vitro Digestion Procedure

	Antioxidant capacity FRAP (mM FeSO4)	Total phenolic content (μg/mL gallic acid)
Green tea	2.65 ± 0.08b	78.95 ± 5.58b
EGCG (1.2 mg/mL)	2.71 ± 0.25b	210.53 ± 111.65b
EGCG (22.5 mg/mL)	13.32 ± 0.04c	1392.11 ± 135.84c
Water after in vitro digestion	0.10 ± 0.00a	1.45 ± 0.93a
Green tea after in vitro digestion	0.80 ± 0.08d	88.16 ± 20.47b
EGCG (1.2 mg/mL) after in vitro digestion	0.85 ± 0.02d	403.95 ± 273.54b
EGCG (22.5 mg/mL) after in vitro digestion	11.92 ± 2.08c	1231.58 ± 126.53c

Values are means ± SD. Values of means with different letters in each column are significantly different at P < .05.

FRAP, Ferric-Reducing Antioxidant Power; SD, standard deviation.

Table 2 also presents the results of the total polyphenol content of the various samples before and after their submission to the in vitro digestion procedure. EGCG (22.5 mg/mL) showed the highest total polyphenol content (1392 μg/mL gallic acid). No difference in the total polyphenol content was observed between the samples of green tea and EGCG (1.2 mg/mL; 80.1 and 210.5 μg/mL gallic acid, respectively). No significant differences in total polyphenol content were observed between the treatments before and after their submission to the in vitro digestion procedure.

Mice model

Toxicity

No toxic effects were observed after administration of 0.23 mg/mL esterified EGCG in the CD1 mouse strain. Therefore, we proceeded with analyzing the in vivo effects of selected EGCG nanoemulsions.

Plasma TAC

Plasma TAC measured by FRAP assay showed no significant differences among mice groups that received the EGCG nanoemulsions (B, F, H) and the aqueous EGCG (C) in comparison with the control groups (A, E, G; P > .05) (Table 3). However, the group that received 80% aqueous EGCG and 20% olive oil (D) had a significantly increased TAC compared with all groups (P < .05) (Table 3). Plasma TAC measured by ORAC assay was significantly increased in mice that received the esterified EGCG (group F) compared with its control group E and compared with aqueous EGCG group C (Table 4). In addition, plasma of mice that received the aqueous EGCG nanoemulsion (group B) and the EGCG with olive oil (group D) had a trend for increased values than groups A, C, E, G, and H (P < .05) (Table 4).

Table 3.

Total Antioxidant Capacity of Plasma and Organ Tissues from Mice Receiving the Eight Treatments, Measured with the Ferric-Reducing Antioxidant Power Assay

Total antioxidant capacity (μmol FeSO4/kg)	Group A	Group B	Group C	Group D	Group E	Group F	Group G	Group H
Plasma	23,606 ± 7607a	26,834 ± 6291a	22,345 ± 5850a	31,537 ± 5023b	21,200 ± 5441a	24,678 ± 1784a	23,444 ± 1656a	26,789 ± 3099a
Heart	3322 ± 1382a	9461 ± 1988a	11,869 ± 2075a	13,909 ± 4080a,b	6413 ± 4595a	20,185 ± 4838b	15,145 ± 4678a,b	22,134 ± 3727b
Lungs	6866 ± 2896a	27,342 ± 12,288b	24,600 ± 7950b	18,430 ± 5890b	14,762 ± 3294b	18,529 ± 5447b	18,976 ± 10,647b	27,876 ± 22,834b
Spleen	11,572 ± 4275a	41,234 ± 7674b	39,768 ± 15,208b	33,495 ± 18,487b	4165 ± 444a	36,792 ± 21,301b	12,345 ± 10,346a	14,567 ± 5419a
Kidneys	11,617 ± 4325a	20,675 ± 5688a	20,196 ± 4267a	13,472 ± 4268a	12,025 ± 3328a	20,005 ± 4254a	25,456 ± 5564b	27,897 ± 4472b
Liver	23,516 ± 4086a	23,987 ± 2680a	24,772 ± 3356a	26,412 ± 16,230a	37,990 ± 5723a,b	29,400 ± 12,500a	34,567 ± 17,130a	35,675 ± 6313a
Brain	12,701 ± 4684a	21,563 ± 5326a	22,147 ± 5245a	14,172 ± 3798a	21,351 ± 13,421a	22,798 ± 4446a	30,123 ± 23,037a,b	27,898 ± 7280a
Stomach	7345 ± 1829a	9876 ± 4536b	9665 ± 1852b	9422 ± 2784b	11,077 ± 4473b	12,513 ± 7338b	18,337 ± 4720b,c	21,989 ± 2113b
Colon	20,188 ± 3978a	21,345 ± 11,492a	20,020 ± 19,918a	10,395 ± 4853b	10,068 ± 12,454b	18,014 ± 5822a	14,567 ± 9395a	17,898 ± 2042a
Jejunum	16,985 ± 3599a	20,456 ± 11,427a	31,053 ± 14,054b	9617 ± 5919a	7240 ± 4216a	28,732 ± 12,999b	6765 ± 3660a	78,962 ± 2998a

Values (micromole FeSO4/kg) are means ± SD of five or six animals. Values of means within a line with different letters are significantly different at P < .05.

A, control water; B, EGCG W/O emulsion; C, EGCG solution; D, oil+EGCG solution; E, control O/W emulsion; F, esterified EGCG O/W emulsion; G, control W/O/W emulsion; H, EGCG W/O/W in water emulsion.

Table 4.

Total Antioxidant Capacity of Plasma and Organ Tissues from Mice Receiving the Eight Treatments, Measured with the Oxygen Radical Absorbance Capacity Assay

Total antioxidant capacity (μmol Trolox/g)	Group A	Group B	Group C	Group D	Group E	Group F	Group G	Group H
Plasma	245.80 ± 13.1a	255.67 ± 18.44a,b	239.21 ± 35.52a	259.36 ± 4.7a,b	248.08 ± 13.82a	295.9 ± 21.44b	235.87 ± 43.34a	238.98 ± 17.93a
Heart	175.42 ± 20.96a	194.23 ± 19.94b	201.38 ± 11.01b	218.91 ± 13.29b,c	185.56 ± 25.43a	210.98 ± 59b	185.19 ± 35.53a	180.36 ± 15.34a
Lungs	167.56 ± 15.91a	208.9 ± 3.57b	194.97 ± 14.62b	211.87 ± 16.23b	190.23 ± 23.74a	232.45 ± 68.45b	182.65 ± 31a	184.78 ± 15.26a
Spleen	165.38 ± 1.23a	195.34 ± 23.86b	196.25 ± 7.34b	215.07 ± 15.76b	180.98 ± 34.11a	178.78 ± 15.22a	169.67 ± 35.04a	200.04 ± 19.99a
Kidneys	175.82 ± 13.29a	208.98 ± 5.13b	203.95 ± 13.94b	219.7 ± 9.85b	178.67 ± 56.39a	219.54 ± 73.68b	162.54 ± 27.27a	192.14 ± 12.61a
Liver	179.01 ± 13.4a	212.34 ± 15.22b,c	220.23 ± 9.63b,c	248.83 ± 12.77c	200.78 ± 49.78a	261.78 ± 90.31c	195.87 ± 35.88a	190.11 ± 20.49a
Brain	181.07 ± 16.28a	198.67 ± 19.11a,b	202.91 ± 16.08a,b	220.65 ± 17.72b	185.78 ± 26.53a	175.67 ± 41.58a	167.12 ± 31.09a	178.63 ± 28.35a
Stomach	184.27 ± 21.04a	215.78 ± 4.62b	199.55 ± 9.34a	225.9 ± 35.43b	216.78 ± 23.81b	245.43 ± 20.78c	187.76 ± 35.46a	201.34 ± 27.38a
Colon	208.14 ± 5.75a	212.34 ± 11.03a	213.12 ± 15a	209.47 ± 10.1a	194.87 ± 33.11b	189.67 ± 8.57b	201.78 ± 23.35a	230.24 ± 19.52c
Jejunum	179.18 ± 14.82b	215.9 ± 2.24a	215.6 ± 33.63a	215.3 ± 15.05a	165.43 ± 67.87b	159.87 ± 16b	177.29 ± 28.89b	204.59 ± 10.43a

Values (micromole FeSO4/kg) are means ± SD of five or six animals. Values of means within a line with different letters are significantly different at P < .05.

A, control water; B, EGCG W/O emulsion; C, EGCG solution; D, oil+EGCG solution; E, control O/W emulsion; F, esterified EGCG O/W emulsion; G, control W/O/W emulsion; H, EGCG W/O/W in water emulsion.

Tissue TAC

In both FRAP and ORAC assays, the tested EGCG nanoemulsions exerted in specific tissues increased TAC when compared with water or emulsion controls, but there were no differences compared with the EGCG solution of the same concentration. Specifically, the FRAP method showed that mice that received the esterified EGCG emulsion (group F) had a significantly increased TAC in the colon, jejunum, heart, and spleen in comparison with mice that received the control treatments (groups A and E; P < .05) (Table 3). The group of aqueous EGCG nanoemulsion (B) also exerted increased TAC in the colon, lungs, and spleen when compared with the control emulsion group E (P < .05) (Table 3).

The ORAC method showed that mice that received the esterified EGCG (group F) had a significantly increased TAC in the stomach, heart, lungs, and liver in comparison with mice that received the control treatments (groups A and E; P < .05) (Table 4). The group of double nanoemulsion (H) also exerted increased TAC in the colon and jejunum when compared with its control emulsion group G (Table 4). The group of aqueous EGCG nanoemulsion (B) exerted increased TAC in the stomach, colon, and heart, whereas the group of aqueous EGCG with olive oil (D) exerted increased TAC in the heart, lungs, stomach, and jejunum, in comparison with the water control group A (P < .05) (Table 4).

Discussion

EGCG nano-emulsification

The first important finding reported here is that mice receiving the EGCG nanoemulsions exerted increased TAC in plasma and specific tissues in comparison with mice that received the control emulsions and the water control, but no difference was observed when compared with the aqueous EGCG solution, with the exception of the esterified EGCG emulsion in plasma. Thus, the EGCG incorporated into nanoemulsions may maintain but does not seem to increase significantly its bioavailability and bioactivity, in comparison with the aqueous EGCG solution of the same concentration.

Nanoemulsions are isotropic colloidal systems and due to their nanometer-sized droplets, they have long-term physical stability.³² In addition, they are resistant to creaming because their Brownian motion is enough to overcome their low gravitational separation force and they are resistant to flocculation due to their highly efficient steric stabilization.²¹ Therefore, these tiny emulsions of nano-dimensions are sometimes referred to as approaching thermodynamic stability.³³ One of the most popular investigated applications of nanoemulsions in many different systems is that they function as bioactive ingredient delivery carriers with enhanced bioactive solubility and availability, controlled drug release, and protection of environmental stresses. Besides, innovations in material chemistry and nanotechnology have synergistically fueled the development of novel food delivery systems and nanocarriers that are biodegradable, biocompatible, targeting, and stimulus responsive.³⁴ The use of nanoemulsions with the aim of stabilizing bioactive compounds and increasing their bioavailability may be an alternative method for new functional foods production.

The second important finding in the present study is that esterified EGCG nanoemulsion in the fatty phase showed the best bioavailability in comparison with the other tested emulsions in mice in vivo, resulting in the fact of having increased TAC in several tissues with both FRAP and ORAC assays compared with its emulsion control and water, while exerting higher TAC in plasma than the EGCG solution of the same concentration; thus, EGCG nano-emulsification, as esterified in a fatty phase, could be proposed as a method for safe EGCG delivery. Nevertheless, further research is required, especially clinical trials and pharmacokinetic studies, to understand the bioavailability and metabolism of EGCG derived from nanoemulsions in the human organism.

In our study, the esterified EGCG nanoemulsion was composed in a fatty phase with olive oil by using BC. In recent years, attempts have been made to create nanoparticles of cellulose emulsions, which are isolated from natural products. Cellulose is a common biodegradable and non-toxic biopolymer that consists of glucose polymers and is used in nanoemulsions due to its high stability.³⁵ The use of BC for nanoparticle creation has been found to be more effective when it comes from olive oil-water emulsions (10% weight oil). This high stability is the result of creation of cellulose fibrils that are adsorbed on the surface of the oil droplet and form a strong matrix. Emulsions of BC seem to be unaffected by pH, ionic force, or temperature changes compared with other types of emulsifiers.²⁵

In a recent study, Fangueiro et al. reported that when EGCG was incorporated into lipid nanoparticles, its bioactivity was significantly increased due to better stability and less oxidation.³⁶ Olive oil was used in the present study, as a constituent of BC as the fatty phase in all formed emulsions at a 10–30% concentration. In addition, a mouse group (D) received 80% of the aqueous EGCG and 20% of olive oil, without emulsification, was used as a positive control. The results showed increased TAC in plasma of this group with the FRAP method and in several tissues with both FRAP and ORAC assays. These results could be explained given that olive oil is rich in antioxidant compounds, such as polyphenols, as oleuropein and hydroxyl-tyrosol, tocopherols and squalene.

Olive oil was used in this study as a stabilized factor in all the emulsions: Emulsions I (group B), II (group E, control 1), and III (group F) at a concentration of 10% and in the Emulsions IV (group G, control 2) and V (group H) at a concentration of 32%. The group D of mice received separately 80% EGCG solution and olive oil at an intermediate concentration of 20% as a positive control, to separate the basic ingredients of the emulsions to detect possible differentiation in the “behavior” of the components “out of” the emulsion.

It could be mentioned and is of high importance that the mice that received olive oil and EGCG separately (group D) showed increased antioxidant capacity in plasma and in specific tissues. This may indicate a possible synergistic action between olive oil components, such as polyphenols and other antioxidants, and the EGCG when ingested without emulsification. In addition, the groups that received emulsion with olive oil with or without EGCG exerted increased antioxidant capacity in several tissues compared with the water group. Nevertheless, these results do not downgrade the possible effect of EGCG on mice tissues' antioxidant capacity, given the fact that mice that received the emulsions with EGCG (groups B, F, G) exerted increased antioxidant capacity in plasma and some tissues, compared with those that received the control emulsions that contained olive oil. For example, ORAC showed increased antioxidant activity in plasma and lungs of mice that received the emulsion of esterified EGCG, in comparison with its control emulsion.

This study showed that the nano-emulsification to incorporate bioactive compounds into foods could be an alternative method for maintaining or increasing its bioactivity. Nevertheless, further investigation is required based on the final composition and structure of the emulsions.

In vitro digestion process

The first finding of the in vitro study was that the in vitro digestion procedure was successfully applied in samples of water, green tea, and two aqueous solutions of EGCG (1.2 and 22.5 mg/mL, respectively). The second finding of the in vitro study was that measurement of TAC and polyphenol content of the low-molecular-weight soluble fraction resulting from the submission of samples to the in vitro digestion procedure that simulates the gastrointestinal digestion is feasible and proportional to the concentration of EGCG in the original sample.

EGCG preserved its antioxidant activity in both concentration levels used after the submission of samples to the in vitro digestion procedure. Moreover, the preservation of TAC and total phenolic content after the in vitro digestion procedure depended on the initial concentration of EGCG. The TAC was reduced by 68.6% for EGCG (1.2 mg/mL) and 10.58% for EGCG (22.5 mg/mL; no statistically significant reduction, P = .098), whereas reduction was also observed in the total phenolic content. Dube et al. came to a similar conclusion after studying the stability of EGCG in alkaline solutions. The lowest concentrations of EGCG were faster degraded than the higher concentrations.³⁷ The multifactorial mechanism of polyphenol absorption and metabolism, which has been investigated in mice models and human studies, possibly explains the greater absorption of higher concentrated polyphenols than lower ones.¹¹

These findings ensure that after ingestion of EGCG solutions, especially in the concentration of 1.2 mg/mL that simulates a normal daily intake from foods, the absorbent EGCG part is significantly decreased; thus, there is a need for improving EGCG stability, bioavailability, and, consequently, bioactivity. This need could be covered by using nanotechnology, such as nano-emulsification.

Mice model, antioxidant capacity, bioavailability, and bioactivity

Another important result of this study was that TAC increased in plasma and several tissues of mice after supplementation with specific EGCG nanoemulsions, compared with control emulsions and was in most cases similar to the TAC after aqueous EGCG solution ingestion. These results suggest the possible maintaining of bioavailability and thus bioactivity of EGCG into nanoemulsions and in some specific cases, possibly increasing. The increase in plasma antioxidant capacity is an important indicator of EGCG bioavailability, since it exhibits maximum blood concentrations about 1.5–2 h after the consumption, increasing the overall antioxidant capacity of plasma.^11,38,39 Tea catechins, such as EGCG, are rapidly metabolized after ingestion. Detection of EGCG in plasma at low concentrations does not exclude the eventual bioactivity of itself and its metabolites.^11,39 According to the literature, increased TAC in plasma and tissues after the consumption of food containing EGCG may be the result of synergistic interaction between EGCG and endogenous antioxidants. Polyphenols such as catechins are metabolized in the liver after absorption and may be moved to the tissues or excreted.

EGCG or the detection and quantification of its metabolites in plasma (by high-performance liquid chromatography [HPLC] or other methods), in parallel with increased TAC, could lead to safer conclusions about EGCG bioavailability, given that TAC is a biomarker that is influenced by a lot of factors and is useful for an indirect, but not direct bioavailability and bioactivity assessment.^7,9,11,38,39 Thus, a basic limitation of this study is the lack of an analytical technique for EGCG or the determination of its metabolites in plasma and tissues of mice. This determination could explain whether the increased antioxidant capacity observed is due to EGCG specifically or is the result of the interaction between endogenous and other parameters.

Subsequently, an important question that arises from this study is relative to the correlation between bioactivity and bioavailability and the biomarkers that determine these parameters. Food bioactivity is linked to the absorption and metabolism of its bioactive constituents such as EGCG; thus, it is linked to EGCG bioaccessibility and bioavailability. This means that catechins, as EGCG, must be absorbed into the enterocyte and moved and distributed to the organ tissues.^38,40 EGCG bioavailability may be studied directly by the measurement of individual EGCG or its metabolites into plasma, urine, and tissues of animals or humans.³⁹ Catechins, and especially EGCG, immediately after absorption across the small intestine and colon, enter blood circulation as conjugates, are metabolized in the liver, and are moved to organ tissues.^1,9,38 Measurement of EGCG absorption, metabolism, and bioactivity may be perplexed by various factors, such as the chemical structure and the biotransformation of its metabolites into lumen and the action of metabolic enzymes of enterocytes.^12,13

EGCG bioavailability may be evaluated indirectly from changes in bioactivity, which, in turn, may be measured from specific parameters in biological fluids, such as TAC.⁴¹ Koutelidakis et al. observed in mice that administration of green tea and of white tea infusions increased the TAC of plasma, heart, and lung, but it did not have an effect on the spleen, liver, brain, or kidney.⁷ Nevertheless, the concept of bioactivity in relation to tea ingestion has evolved to include other aspects or biological indices besides antioxidant properties such as gene expression, formation of oxidation products such as hydrohyperoxides, or cardiovascular and cancer biomarkers.¹ For example, nutrigenomic approaches linking nutrient supply and effects at the molecular basis of gene expression indicate that the investigation on the biological role of plant compounds may be directed on observing effects on specific genes in human or animal cells both in vitro and in vivo.^42,43 In another study, Koutelidakis et al. observed in mice that a mix of catechins administration for 1 month increased TAC in plasma and specific tissues and may be affected by the expression of several genes.¹⁰

Human studies have shown that tea administration leads to an acute increase of plasma TAC at 1.5–2 h.⁹ Nevertheless, catechin concentration in plasma does not exceed 1 mM when consumed in typical amounts (1/2 cups, 100–200 mg catechins). The total concentration of catechins (free and conjugated) is about 2–3 mM or less. Therefore, tea catechins bioavailability seems to be relatively low; only 0.2–2% of the consumed quantity of catechins reaches the plasma of healthy humans.^44,45 Thus, the use of nanoparticles for EGCG incorporation may be a useful method for its bioavailability improvement and could probably lead to the production of new functional foods, as is the concept of this study.

In this study, we used both FRAP and ORAC assays; measurement of TAC by a single assay cannot reflect the multiple reactions and mechanisms involved in oxidative stress because it represents only the chemical reactivity under the specific conditions of this assay. A relatively large standard deviation was observed in the TAC readings in the FRAP method, and this is a second limitation of this study. Previous research indicates that each organism may respond in different ways to phenolic-rich foods/beverages. The genetic differences among animals affect the extent of polyphenols absorption in lumen and thus polyphenol bioavailability and final antioxidant biomarkers.^{9,26,29,46,47}

In addition, in this study, the esterified EGCG nanoemulsion in fatty phase (F group) exhibited greater antioxidant activity in plasma compared with the control group nanoemulsion E and compared with the EGCG solution C. It also exhibited increased antioxidant activity in the colon and jejunum, tissues of great importance for catechins absorption, and in other tissues with important metabolic roles such as the liver, spleen, and kidneys. A lower effect in several tissues was observed from the double nanoemulsion (group H) and from EGCG emulsion in the aqueous phase (group B).

In previous studies, Koutelidakis et al. also reported an increase in the TAC of specific tissues (heart, liver, colon etc.) after tea infusion administration in mice.^7,10,48 Relevant to heart function, clinical trials support the possible effect of green tea EGCG on endothelial function.⁴⁹ In a meta-analysis of randomized, controlled clinical studies, Kim et al. referred to the cardioprotective effect of tea catechins due to its possible impact on lipidemic profile.⁵⁰ In addition, the esterified form of EGCG used for the preparation of the different nanoemulsions did not exert any toxicity or did not have any adverse effect on mouse physiology, similar to the aqueous EGCG, resulting in the possible incorporation of bioactive compounds into nanoemulsions being safe for functional foods production.

This study concluded that the incorporation of EGCG into nanoemulsions and its acute administration in mice did not increase its bioactivity measured as TAC in specific tissues of mice, compared with an aqueous EGCG solution at the same concentration. Nevertheless, the esterified EGCG emulsion exerted an increase in mice plasma compared with aqueous EGCG and, in general, showed high values of antioxidant activity in several tissues. Further research is needed to detect the possible effect of olive oil, which is used as a stabilized factor for the production of the nanoemulsions.

Nanotechnology use for the incorporation of bioactive ingredients, such as EGCG, could be considered an alternative method for functional food production achieving stability and bioavailability of the bioactive substances. These types of foods, as part of a balanced diet, may contribute toward maintaining and promoting human health. Nevertheless, further research, especially clinical trial conduction, is crucial as the next step with the purpose to investigate in humans the bioavailability and bioactivity of EGCG nanoemulsions.

Footnotes

Acknowledgments

The authors thank the staff of the animal house facility of BSRC Alexander Fleming for taking care of the animals. This study was conducted with funds from the research program “Novel formulations and nanostructures for enhancing the bioavailability of a bioactive compound. The case of emulsion production-NONASTRU. ESPSA 2007–2013.” The founding sponsors had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the article, and in the decision to publish the results.

Author Disclosure Statement

No competing financial interests exist.

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