Niosomes Consisting of Tween-60 and Cholesterol ... - ACS Publications

Nov 16, 2016 - Centre for Water-Soluble Polymers, North East Wales Institute, Plas Coch, Mold Road, Wrexham LL11 2AW, U.K.. •S Supporting Informatio...
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Niosomes Consisting of Tween-60 and Cholesterol Improve the Chemical Stability and Antioxidant Activity of (−)-Epigallocatechin Gallate under Intestinal Tract Conditions Rong Liang,† Ling Chen,‡ Wallace Yokoyama,§ Peter A. Williams,∥ and Fang Zhong*,‡ †

Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, P. R. China ‡ Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Food Science and Technology, Jiangnan University, Wuxi 214122, P. R. China § Western Regional Research Center, ARS, USDA, Albany, California 94710, United States ∥ Centre for Water-Soluble Polymers, North East Wales Institute, Plas Coch, Mold Road, Wrexham LL11 2AW, U.K. S Supporting Information *

ABSTRACT: In order to improve the chemical stability and antioxidant activity of (−)-epigallocatechin gallate (EGCG) in the gastrointestinal tract, niosomes composed of Tween-60 and cholesterol were developed to encapsulate EGCG in this investigation. EGCG loaded niosomes with encapsulation efficiency around 76% exhibited a small Z-average diameter about 60 nm. Compared to free EGCG, the EGCG remaining in dialysis tubes was significantly improved for niosomes at pH 2 and 7.4. Meanwhile, the residual EGCG for niosomes increased from 3% to 49% after 2 h incubation in simulated intestinal fluid (SIF). Pancreatin was found to impact the stability of niosomes in SIF mainly. Furthermore, the results from ferric reducing antioxidant power and cellular antioxidant activity tests indicated that EGCG loaded niosomes exhibited stronger antioxidant ability than free EGCG during intestinal digestion. Thus, we can infer that niosomal encapsulation might be a promising approach to improve the oral bioavailability of EGCG in the body. KEYWORDS: (−)-epigallocatechin gallate (EGCG), niosomes, digestive stability, cellular antioxidant activity (CAA)



EGCG impairs its ability to cross the cell membrane,13 and high levels are found in feces of animals fed green tea polyphenols.8 A fairly high dose of EGCG has been reported to be required in order to maintain the plasma concentration of EGCG at a level sufficient to exert antioxidant activities.14 However, dose escalation increases the risk of toxicity and may also cause unpleasant flavors.15,16 With the aim of developing an effective oral delivery system that reduces degradation and increases absorption of EGCG, nanoparticle delivery systems are considered a feasible approach.17 Nanoparticle strategies to improve the pharmacokinetics and pharmacodynamics of EGCG include polysaccharide-based nanoparticles,18 lipid nanocapsules,19 protein-based nanovehicles,20 and liposomes.21,22 Improved antiproliferation capacity against cancer cells in vitro of EGCG carriers was reported by Rocha et al.18 and Ru et al.12 Encapsulation or matrix embedding also reduces undesirable bitterness and astringency of EGCG as shown by β-lactoglobulin nanoparticles.18 However, the protection of EGCG against the alkaline pH environment and enzymatic degradation in the gastrointestinal tract were still not sufficiently provided by these systems. The EGCG embedded in gum Arabic and maltodextrin, while stable in aqueous media,

INTRODUCTION (−)-Epigallocatechin gallate (EGCG), a water-soluble flavonoid that is the most prominent component of green tea,1 has been reported to possess anticancer, antiatherogenic, anti-inflammatory, and cardiovascular preventive properties.2,3 The intake of EGCG is considered safe since green tea is a food that has been consumed for centuries. The health benefits of EGCG have been associated with its antioxidant properties4 and the ability to interact with signaling pathways related to energy metabolism, inflammation, and cancer.5 For example, EGCG was found to reduce free radical mediated oxidative DNA damage through its radical scavenging ability.6 EGCG’s cancer-preventive properties are supported by multiple in vitro, in vivo, epidemiological, and clinical studies.7 Because of its safety and useful bioactive properties, academic and industrial researchers consider it a promising phytomedicine to prevent cancer, inflammation, cardiovascular, and neurodegenerative disorders. However, EGCG is unstable in the digestive tract and poorly absorbed across the intestinal epithelium cells.8 Overcoming these hurdles to bioavailability has been a substantial challenge.9−11 In the intestinal tract EGCG is degraded by the alkaline pH and dissolved oxygen. The absorption of EGCG in the intestine might be related to Lipinski’s rule of five, which states that compounds with a molecular weight greater than 500 g/mol and 5 or more hydrogen bond donors do not easily pass through the plasma membrane.12 Research supports Lipinski’s rule and has shown that the high hydrophilicity of © XXXX American Chemical Society

Received: Revised: Accepted: Published: A

September 18, 2016 November 14, 2016 November 16, 2016 November 16, 2016 DOI: 10.1021/acs.jafc.6b04147 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry was totally released within 3 h in the intestinal tract.18 Nanoparticles of beta-lactoglobulin are resistant to gastric digestion but susceptible to digestion by intestinal enzymes.20 A similar result was also obtained from liposomes.21 Chitosan is another biopolymer widely used for encapsulation, but it is soluble only in acidic environments, which would result in dissolution in the stomach and decreased availability of EGCG in the intestinal tract.23 Therefore, a novel approach for oral delivery of EGCG with stability within the gastrointestinal tract is needed. Niosomes are a vesicular system structurally analogous to liposomes composed of nonionic surfactants and cholesterol with their hydrophilic ends exposed on the inner and outer surfaces of the vesicle, while the hydrophobic chains face each other within the bilayer. Hence, hydrophilic molecules may be enclosed within the inner aqueous space, while hydrophobic molecules may be embedded within the bilayer membrane.24 With the property of penetration enhancer of nonionic surfactant,25 the niosomal systems exhibit marked improvement for the bioavailability of a variety of drugs, such as paclitaxel,26 griseofulvin,27 and valsartan28 by facilitating the transfer of the compounds across the intestinal epithelial membrane. EGCG niosome prepared by Span 60 and cholesterol was also verified to enhance uptake and transportation of EGCG by intestinal cells.13 According to the research from Wilkhu et al.,29 niosomes could also enhance the stability of oral vaccines in the gastrointestinal tract since nonionic surfactants are more resistant to the intestinal enzymatic degradation; and recently modified niosomes with second layer by sodium carboxymethyl cellulose were produced to further improve the resistance of EGCG niosomes to pH variation and thermal and osmotic stress.30 However, the stability of EGCG niosomes in the gastrointestinal tract is still unknown until now. Hence, in this research, an in vitro digestion model is used to investigate the stability of EGCG niosomes to reveal the possibility of their application to oral administration. Tween-60 as a common nonionic surfactant with the permission to be safely used in food and pharmaceutical products by FDA was chosen to prepare niosome in this study. The characterizations of droplet size, morphology, encapsulation efficiency, and in vitro gastrointestinal stability of EGCG loaded niosomes were investigated. The antioxidant activity of EGCG niosomes during intestinal digestion was also evaluated using ferric reducing antioxidant power (FRAC) and cellular antioxidant activity (CAA).



Hanks’ balanced salt solution (HBSS), and phosphate buffer solution (PBS) (1×) were purchased from GIBCO (Grand Island, NY). Other chemical reagents used in this study were analytical grade. Preparation of EGCG-Loaded Niosomes. Niosomes were prepared by the ethanol injection method developed by Fan et al.31 and Pratap et al.27 with slight modifications. A total amount of 200 mg of Tween-60 and cholesterol was mixed at a 4:1 molar ratio and dissolved at 60 °C in 1 mL of absolute ethyl alcohol containing a predefined concentration of EGCG (25 mg/mL). The resulting solution was slowly and uniformly injected into 18.5 mL of deionized water by syringe (2 mL) at 50 °C in a beaker under constant mild stirring using a magnetic stirrer. Absolute ethyl alcohol was then removed by reduced-pressure evaporation working at 0.1 MPa. Characterizations of EGCG-Loaded Niosomes. Z-average diameter and polydispersity index (PDI) of the niosomes were determined by dynamic light scattering (DLS) on a Zetasizer NanoZS (Malvern Instrument Ltd., U.K.), according to methods in our previous report.32 Briefly, 100 μL aliquots of the vesicle suspension were added into 10 mL of deionized water, and the mixture was transferred to the cuvette. The initial niosomal shape and size and the changes during the digestive process were visualized by transmission electron microscopy (TEM, H-7650, HITACHI, Japan) according to our previously reported method with a slight modification.32 A fresh copper mesh grid was placed onto droplets containing prediluted EGCG noisome suspension, and excess liquid was removed with filter paper after 4 min. Samples were air-dried at room temperature, and the morphology of the EGCG niosomes was recorded by TEM at a voltage of 80 kV. Determination of Encapsulation Efficiency of EGCG in the Niosomes. Encapsulated EGCG was separated from free EGCG by ultracentrifugation as described by Suwakul et al.33 with minor modification. Briefly, EGCG niosomal dispersion (5 mL) was diluted with ultrapure grade water to 25 mL in a centrifuge tube, and ultracentrifuged at 200000g for 30 min at 4 °C (L 80, Beckman, USA). The supernatant was collected as the free EGCG. Total EGCG in niosomal dispersions was released by complete disruption of the vesicles using Triton X-100. The amount of free and total encapsulated EGCG was quantified by reversed phase high-performance liquid chromatography (RP-HPLC) using the Hitachi Chromaster HPLC system (Hitachi HighTechnologies Corporation, Japan) based on the method reported by Li et al.34 Briefly, sterile syringe filters (0.22 μm) were used to sterilize each sample prior to analysis. Chromatographic separation was performed on a Waters Symmetry C18 (4.6 × 250 mm, 5 μm) column at 25 °C. The mobile phase was a mixture of solvent A (v/v/v, 0.05% phosphoric acid:80% methanol:20% ultrapure grade water) and solvent B (v/v/v, 0.05% phosphoric acid:10% methanol:90% ultrapure grade water). The flow rate was 1 mL/min. The linear gradients were as follows: solvent A 20% to 80% in 15 min, reduced to 20% in 2 min, and held at 20% for 3 min until injection of the next sample. The injection volume was 20 μL, and the detection wavelength of DAD spectrum was set at 274 nm. The HPLC assay for EGCG was linear in the range of 5−100 μg/mL. The regression equation of the calibration curve was calculated based on peak area versus standard concentrations. The encapsulation efficiency (EE) was calculated according to the following equation:

MATERIALS AND METHODS

Materials. EGCG (98%) and 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) were purchased from Shanghai Canspec Scientific Instruments Co., Ltd. (Shanghai, China). Triton X-100, phosphoric acid, absolute ethyl alcohol, FeCl3, DMSO, Tween-60, cholesterol, and methanol of HPLC grade were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ultrapure grade water purchased from A.S. Watson Group, Ltd. (Hong Kong, China) was used for HPLC tests, and deionized water from a Milli-Q water purification system (Millipore Co., Bedford, MA) was used in other experiments. 2′,7′-Dichlorofluorescein diacetate (DCFH-DA), 2,2′-azobis(2-amidinopropane) dihydrochloride (ABAP), pepsin from porcine gastric mucosa (P7000, enzymatic activity of 738 U/mg), bile extract porcine (B8631), and pancreatin from porcine pancreas (P7545, 8 × USP specifications) were purchased from Sigma-Aldrich Co. LLC (St. Louis, MO). The human hepatocellular carcinoma (HepG2) cell line was a gift from Dr. Xing-Guo Wang (Jiangnan University, Wuxi, China). Dulbecco’s modified Eagle’s medium (DMEM) (containing 4.5 g/L D-glucose and GlutaMAX), nonessential amino acid (100×), penicillin and streptomycin (100×), fetal bovine serum (FBS), trypsin,

EE % =

1 − Cfree × 100% C total

where Cfree and Ctotal are the concentrations of free and total EGCG concentration in the prepared niosomal dispersions, respectively. EGCG Remaining under Simulated Gastrointestinal pH through Dialysis Method. The EGCG remaining of EGCG loaded niosomes under simulated gastrointestinal pH was investigated via the dialysis method developed by Zou et al.35 with slight modifications. EGCG solution was used as the control. In brief, 2 mL of EGCG niosome suspension was loaded into a cellulose membrane dialysis bag (Mw cutoff of 3 kDa, Sinopharm Co., China) and immersed in 100 mL of acetate buffer solution (0.05M) at pH 2 or PBS (0.05 M) at pH 7.4 which represented the pH for gastric and small intestinal environment, B

DOI: 10.1021/acs.jafc.6b04147 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry with constant stirring at 25 °C. Samples in the dialysis bags were collected at predetermined time intervals (0.5, 1.5, 2.5, 3.5, 5, 7, 10, and 24 h) and analyzed for the content of EGCG by HPLC. The EGCG remaining was calculated according to the following equation:

EGCG remaining % =

niosomes and EGCG solution plus 25 μM DCFH-DA dissolved in treatment medium. After seeding for 1 h, wells were then washed with 100 μL of PBS, and 600 μM ABAP in 100 μL of HBSS was poured onto the 96-well plate. Finally, the 96-well microplate was placed into a microplate reader (Spectramax M5, Molecular Devices, USA) at 37 °C. Emission at 538 nm was measured with excitation at 485 nm every 5 min for 1 h. In addition, EGCG niosomes and EGCG solution after simulated intestinal digestion were collected and diluted to various concentrations for CAA assay under the same conditions. Each plate also included six replicates of control wells containing cells treated with DCFH-DA and ABAP and blank wells containing cells treated with DCFH-DA without ABAP for EGCG solution; DCFHDA without ABAP and blank niosomes without EGCG for EGCG niosomes; DCFH-DA without ABAP and digested solution without EGCG for digested EGCG solution; DCFH-DA without ABAP and digested blank niosomes without EGCG for digested EGCG niosomes, respectively. After blank subtraction from the fluorescence readings, the area under the curve of fluorescence versus time was integrated to calculate the CAA value according to Wolfe and Liu37 as follows:

Ci × 100% C0

where Ci is the EGCG concentration of samples in the dialysis bag at interval times, and C0 is the EGCG concentration of samples at initial time. In Vitro Digestion Stability of EGCG Niosomes. The in vitro digestion stability of EGCG niosomes was assessed in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) separately according to procedures reported by Liu et al.21 with minor modifications. The SGF was prepared by placing 2 g of NaCl, 7 mL of HCl (37 wt %), and 3.2 mg/mL pepsin in 1 L of water and adjusting pH to 2.0 using 1.0 M HCl. SIF was prepared consisting of bile salts (7 mg/mL) and pancreatin (1 mg/mL) in PBS (0.05 M, pH 7.4). An EGCG solution was used as the control in both SGF and SIF digestions. The SGF and SIF solutions were prewarmed to 37 °C prior to incubating the samples. Briefly, sample solution was mixed with SGF or SIF with a volume ratio of 2:5. The mixed solution was then incubated at 37 °C with mild agitation (100 rpm), and subsamples were taken for analysis of Z-average diameter at different time intervals (10, 20, 30, 40, 50, and 60 min for SGF and 10, 30, 60, 90, 120 min for SIF). In order to evaluate the impact of alkaline condition of SIF, bile salts, and pancreatin in SIF on EGCG niosomes, morphology evaluation and HPLC analysis were performed on EGCG niosomes incubated in PBS (pH 7.4) only and specific SIF with bile salts or pancreatin separately. The EGCG remaining was calculated according to the following equation:

⎛ ∫ SA − ∫ BA ⎞ ⎟ × 100 CAA unit = 100 − ⎜⎜ ⎟ ⎝ ∫ CA − ∫ BA ⎠ where ∫ SA is the integrated area under the sample fluorescence versus time curve, ∫ BA is the integrated area from the blank curve, and ∫ CA is the integrated area under the control curve. The median effective dose (EC50) was determined for free EGCG and EGCG loaded f

niosomes from the median effect plot of log( a ) versus log (dose), where fu

fa is the fraction affected and f u is the fraction unaffected by the treatment, f

and EC50 was determined from the plot of log( a ) = log 1 = 0.

C EGCG remaining % = i × 100% C0

fu

Statistical Analysis. All measurements were performed at least in triplicate. The values are expressed as mean ± standard deviation (SD). Comparisons between two means were performed using unpaired Student’s t tests. When there were more than two means, differences were detected by one way analysis of variance (ANOVA) test followed by Duncan’s multiple range test using the statistical software package SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Differences were considered to be significant when p < 0.05.

where Ci is the EGCG concentration of samples at interval times in SGF or SIF, and C0 is the EGCG concentration in the digestive fluid at initial time. Antioxidant Activities of EGCG Niosomes during Digestion. The antioxidant properties of EGCG loaded niosomes in SIF were determined by ferric reducing antioxidant power (FRAP)36 and compared with free EGCG solution. Briefly, the working FRAP reagent was freshly prepared by mixing 25 mL of acetate buffer (0.3 M) at pH 3.6 with 2.5 mL of 10 mM TPTZ (2,4,6-tri(2-pyridyl)-s-triazine) in 40 mM HCl and 2.5 mL of 20 mM FeCl3·6H2O at 37 °C. Then 0.2 mL of the sample from SIF incubation was added to 4.8 mL of FRAP reagent. The reaction mixture was incubated in a water bath for 10 min at 37 °C. The absorbance of each sample was recorded at 593 nm via UV−vis spectrophotometry, and the difference in absorbance between sample (EGCG loaded niosomes and free EGCG solution in SIF) and blank (SIF with empty niosomes and blank SIF) was used to calculate the FRAP value. FRAP value was expressed in terms of the equivalent content of EGCG, using the EGCG standard curve. Cellular Antioxidant Activity (CAA) Assay of EGCG Niosomes before and after Digestion. The cell antioxidant ability of EGCG niosomes was determined by cellular antioxidant activity (CAA) assay according to the method of Wolfe and Liu,37 taking EGCG solution as a control group. Human hepatocellular carcinoma HepG2 cells were routinely maintained in complete DMEM medium in Corning cellculture flasks in a humidified incubator with 5% CO2 and 95% relative humidity. Complete DMEM medium was supplemented with 10% fetal calf serum, 1% penicillin−streptomycin−glutamine, and 1% nonessential amino acids. Culture medium was changed every 2 days when reaching 80−90% confluence. The cells used in the present study were between passage 15 and 30. 100 μL of HepG2 cells were grown on a 96-well plate at a density of 6 × 104 cells/well, except for the outside wells on account of much more variation from them.37 After 24 h incubation, the supernatant was removed and the wells were washed with PBS. Six replicates of wells were treated with 100 μL of EGCG



RESULTS AND DISCUSSION Characterization of EGCG Niosomes. Particle size, PDI, encapsulation efficiency, and morphology of EGCG niosomes are important characteristics of niosomes as nanodelivery vehicles. As shown in Figure 1A, the Z-average diameter of EGCG niosomes prepared by the ethanol injection method was approximately 60 nm with a PDI value around 0.110 by using dynamic laser light scattering (DLS), indicating that the vesicle size was relatively homogeneous. Compared to other EGCG niosomes prepared by thin film hydration method (∼100 nm),13 the particle size of EGCG niosomes prepared by ethanol injection was smaller. Except for the preparation method, the components incorporated in niosome and the type of emulsifiers would also affect the droplet size.28,38,39 The surface morphology and vesicle shape of EGCG niosomes were observed by TEM. As presented in Figure 1B, the TEM image showed that the vesicles were small and mainly spherical in shape. The size by TEM (∼60 nm) correlates well with the results by DLS. The TEM image also showed the distribution of the aqueous phase (dark area) surrounded by a unilamellar lipid membrane (light area). The aqueous core is about 50 nm in diameter surrounded by a 5 nm thick membrane. The encapsulation efficiency of EGCG loaded niosomes was 76.4 ± 0.8%, which is higher than that of β-Lg-EGCG nanoparticles of 60−70% at similar loading capacity of 12%20 C

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Figure 2. EGCG remaining within dialysis tubes for EGCG solution (ES) and EGCG loaded niosomes (EN) under simulated gastrointestinal pH 2 and 7.4.

of EGCG outside the dialysis bag. The ability of niosomes to prevent EGCG leakage was greater than that of some other delivery systems, for example, the leakage ratio of EGCG chitosan−calcium polyphosphate (CS-CPP) nanoparticles is about 25−50% in 24 h at pH 6.2 as reported by Hu et al.40 Polysaccharide nanoparticles reported by Rocha et al. leaked about 65% of EGCG in the first 30 min, and EGCG was completely released in 3 h at pH 7.4.18 But compared to the novel dextran sulfate coated amphiphilic chitosan derivative nanoliposomes with leakage level of 20.2% at pH 5.5 reported by Zou et al.,41 it was a little bit weaker. However, compared to free EGCG solution, the amount of EGCG remaining of niosome in the first 3 h (2.8% and 6.2% at pH 2 and 7.4) was dramatically increased, which indicated that niosome may be an efficient system to control the leakage of ECGC within the 3 h digestion time in the GI tract; and the characterization of this system in simulated SGF and SIF digestion was examined further. Stability of EGCG Loaded Niosomes and Free EGCG during Transit through the Digestive System. The stability of EGCG loaded niosomes during in vitro digestion was followed by measuring the Z-average diameter, the amount of EGCG remaining, and the morphology of the niosomes during the digestive tract. Niosomes were incubated in SGF using free EGCG as the control group. Figure 3A is a plot of the Z-average diameter and PDI of the EGCG loaded niosomes after SGF incubation over time. The Z-average diameter remained constant at about 60 nm over 1 h. These results were in good agreement with the findings from Chang et al.42 that emulsions stabilized by Tween 80 were resistant to droplet aggregation under gastric conditions. The retention rate of both EGCG loaded niosomes and free EGCG solutions (Figure 3B) were unchanged over this time period. Retention was greater than 99% and 96% for the EGCG niosomes and free EGCG solution, respectively. These results show that free EGCG and EGCG niosome are both stable during the 1 h period in SGF. However, in SIF the average diameter of EGCG loaded niosomes increased from 60 to 149 nm within 10 min and continued to increase to 178 nm in 2 h (as shown in Figure 4A, columns with red color). The retention rates of EGCG loaded in niosomes and solution were around 49% and 3% respectively after 2 h in SIF (shown in Figure 4B), indicating a protection capacity of niosomes for EGCG. Others have observed the same stability of EGCG in acid fluids and rapid degradation

Figure 1. (A) Size distribution of EGCG niosomes; (B) TEM morphology of EGCG loaded niosomes.

and similar to that of liposomes of 70−80% at the same loading capacity of 12.5%.22 These results may be attributed to the specific structure characteristics of EGCG. The galloyl group of EGCG facilitates its absorption by the lipophilic segments of surfactants while the hydrophilic group of EGCG facilitates partition to the inner water phase.35 EGCG Remaining under Simulated Gastrointestinal pH through Dialysis Method. The EGCG remaining of bulk solution and niosomes at pH 2 and 7.4 through a 3 kDa dialysis membrane is presented in Figure 2. It is noted that only 10% of EGCG remained in the dialysis bag after 3.5 h at pH 2 and after 1.5 h at pH 7.4 for EGCG solutions. A possible reason for the rapid diffusion of EGCG solution at pH 7.4 was that EGCG tends to degrade into smaller molecules under alkaline conditions.20 With the decreasing of EGCG concentration in the solutions, EGCG would be pushed to diffuse from the dialysis bag. However, EGCG niosomes exhibited a relatively high amount remaining at the same conditions. Only 21.3% and 30.9% of EGCG was leaked from niosomes at pH 2 and 7.4, respectively, after 24 h dialysis. The EGCG remaining in acidic environment after 24 h matches well with the EE % of EGCG niosomes, indicating that free EGCG in niosomal vesicles was principally diffused across the dialysis membrane with limited EGCG leakage from vesicles. The reduced EGCG remaining that was found in pH 7.4 may also be caused by the degradation D

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Figure 3. (A) Z-average diameter and polydispersity index of EGCG niosomes during SGF incubating (mean ± STD, n = 3; p < 0.05). (B) EGCG remaining of EGCG solution (ES) and EGCG loaded niosomes (EN) incubated in SGF at set times.

from 97.9% to 3.4% within 1.5 h in neutral or alkaline solutions.35 Compared to liposomes with residual EGCG at 22.5% after incubation in SIF for 2 h,35 the protective ability of niosomes is greater. In order to understand the reason for the loss of EGCG during simulated intestinal digestion, specific SIF conditions with PBS (pH 7.4) only or PBS (pH 7.4) containing bile salts or pancreatin separately were used to do the digestion again. It has been observed that EGCG niosomes incubated in PBS exhibited no change of droplet size around 60 nm (Figure 4A, columns with blank color) and high ECGC remaining around 95% (Figure 4C) which indicated that EGCG niosome was stable under pH 7.4 for 2 h. However, the droplet size of EGCG niosome increased to 133 nm when incubated in PBS incorporated with bile salts and 162 nm with pancreatin (Figure 4A); and EGCG retention rate decreased to 87% when incubated in PBS with bile salts only and 65% with pancreatin only (Figure 4C). These results show that both bile salts and pancreatin in SIF have an impact on the stability of niosomes and pancreatin has a greater influence on the niosomal stability compared to bile salts. PDI value variations of EGCG niosomes under different SIF conditions also support this inference. As shown in Table 1, the PDI values of samples incubated in different conditions increased in the order PBS > bile salts > pancreatin > bile salts + pancreatin. Compared to the consistent PDI values of samples in PBS and PBS with bile salts only, the PDI values increased significantly after 2 h incubation

Figure 4. (A) Changes in Z-average diameter of EGCG niosomes in PBS (pH 7.4) only or PBS (pH 7.4) with both pancreatin and bile salts or pancreatin and bile salts alone; (B) EGCG remaining of EGCG solution (ES) and EGCG loaded niosomes (EN) incubated in SIF with pancreatin and bile salts at set times; (C) EGCG remaining of EGCG niosomes incubated in PBS (pH 7.4) only or PBS (pH 7.4) with both pancreatin and bile salts or pancreatin and bile salts alone at set times.

in digestion fluids incorporated with pancreatin. The morphology of EGCG loaded niosomes after incubation in specific SIF with bile salts or pancreatin separately were also observed by TEM. As shown in Figures 5B-1 and 5C-1, the interior of the incubated niosomes in SIF with bile salts or pancreatin shows irregular distribution of light and dark areas unlike the freshly prepared niosomes with the light surfactant membrane surrounding the dark aqueous core. Coalescence of two or more particles was observed for the samples incubated with bile salts, as shown in Figure 5B-2. Some droplet appeared to be disintegrating when incubated in SIF with pancreatin E

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Table 1. Changes in PDI Values of EGCG Niosomes in PBS (pH 7.4) Only or PBS (pH 7.4) with Both Pancreatin and Bile Salts or Pancreatin and Bile Salts Alonea time (min) 10 30 60 90 120

PBS 0.119 0.111 0.115 0.116 0.119

± ± ± ± ±

0.012 0.014 0.024 0.019 0.029

pancreatin and bile salts A,b A,b A,b A,b A,b

0.227 0.241 0.247 0.273 0.286

± ± ± ± ±

0.023 0.009 0.005 0.007 0.010

C,b D,b D,b D,c D,c

bile salts 0.150 0.158 0.152 0.158 0.156

± ± ± ± ±

0.009 0.016 0.007 0.027 0.011

pancreatin B,b B,b B,b B,b B,b

0.194 0.201 0.218 0.222 0.236

± ± ± ± ±

0.023 0.020 0.016 0.005 0.008

C,b C,b C,bc C,bc

C,c

a

Values of means followed by different uppercase letters (A−D) in the same rows are significantly different (p < 0.05). Values of means followed by different lowercase letters (b, c) in the same column are significantly different (p < 0.05).

micelles or undigested niosomes which were not observed in TEM would lead to lower Z-average diameters of niosomal vesicles by using DLS. There are reports that surfactants (such as polysorbate) bind proteins and enzymes44 and their complexes may destabilize the niosomes. Bile acid micelles bind cholesterol,45 and Tween also interacts with bile salts46 through its hydrophobic tail. These observations suggest that the niosomal membrane consisting of Tween 60 and cholesterol may be disturbed by the bile salts and pancreatin in SIF. Both bile salts and pancreatin appeared to fuse or coalesce niosomes into larger particles. However, while pancreatin resulted in breakdown of the fused particles, bile salts did not. The mechanism of the structural change of EGCG niosomes in the gastrointestinal tract could be illustrated through Figure 6. As shown in the figure,

Figure 5. TEM morphology of EGCG loaded niosomes after SIF incubation: (A) EGCG loaded niosomes after digestion with pancreatin and bile salts; (B) EGCG loaded niosomes after digestion in SIF with bile salts only; (C) EGCG loaded niosomes after digestion in SIF with pancreatin only.

Figure 6. Illustration of the preparation, oral administration of EGCG loaded niosomes in vitro, and effects of pancreatin and bile salts on the noisome membrane under intestinal tract conditions.

(Figure 5C-2) which corresponded to the nonspherical niosome observed in Figure 5A marked with arrows. The diameters of nanoparticles visualized by TEM for samples were varied from 136 to 181 nm, 133 to 160 nm, and 166 to 200 nm for niosomes incubated in SIF with both bile salts and pancreatin or bile salts and pancreatin only, which corresponded to the increased PDI values in Table 1. Compared to the Z-average diameters obtained in Figure 4, the droplet diameters were a little bit larger by using TEM. Similar results were also observed by Zou et al.43 which may also be caused by the inhomogeneity of samples after digestion. Smaller digestive

bile salts would insert into the niosomal membrane and cause the coalescence of droplets, while pancreatin may disrupt the bilayer membrane, leading to partial release of entrapped EGCG. Chemical Antioxidant Activity of EGCG Loaded Niosomes and Free EGCG. The in vitro antioxidant activity of EGCG during SIF incubation was found to be related to the digestion time. As shown in Figure 7, the FRAP values of EGCG and EGCG loaded niosomes showed a continual decrease F

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Journal of Agricultural and Food Chemistry

Figure 7. FRAP values of EGCG solution (ES) and EGCG loaded niosomes (EN) during SIF digestion at set times.

over time, which corresponds with the results of EGCG degradation in the SIF. Finally, the FRAP values of EGCG solution and EGCG niosomes were reduced 42% and 12%, respectively, after 2 h SIF incubation. It is remarkable that EGCG loaded niosomes displayed a lower FRAP value than free EGCG in the initial state, and a slower reduction rate (k = −0.0143) than EGCG solution (k = −0.185). These results correspond well with the higher stability of EGCG loaded niosomes in the intestinal tract. The higher FRAP value of EGCG solution than EGCG loaded niosomes before SIF incubation was possibly due to the shielding effect of the niosomal bilayer membrane, which impeded the contact of EGCG with FRAP reagent. Cell Antioxidant Activity (CAA) Assay of EGCG Loaded Niosomes and Free EGCG. The results of FRAP assay have shown that niosomal encapsulation provides good protection to EGCG throughout the intestinal tract, due to the niosomal membrane barrier protecting the EGCG core from the alkaline environment. Nevertheless, it is unclear that whether EGCG loaded niosomes have antioxidant properties in biological systems. The CAA assay developed by Wolfe and Liu37 is a biological approach to antioxidant activity. Human hepatocellular carcinoma (HepG2) cells used in the CAA assay have been reported to have many similar biological activities as normal live cells in humans (L02).47 So first the CAA values and median effect plots determined from the data from DCF fluorescence in HepG2 cells cultured with EGCG loaded niosomes and free EGCG are shown in Figure 8A−C. All the studies in this research were based on the concentration of EGCG within the safety range (according to the MTT results shown in Figure S1). Both free and encapsulated EGCG quenched the ABAP induced peroxyl radical reaction with reduced fluorescence units (as shown in Figure S2). CAA values of EGCG loaded niosomes increased significantly (p < 0.05) compared to that of free EGCG at the same concentration (Figure 8A). The higher CAA value of EGCG loaded niosomes may suggest that niosomal encapsulation increases the cellular antioxidant activity of EGCG. The EC50 value was calculated based on the linear regression of the median effect curve presented in Figure 8B,C. The EC50 value of free EGCG was 10.4 μg/mL, and was decreased to 6.9 μg/mL after incorporated in niosomes. The CAA of EGCG was increased about 1.5 fold by encapsulation. Hu et al.40 also observed that the EC50 of EGCG decreased, from 15.25 to 12.60 μg/mL, as it was encapsulated in CS-CPP nanoparticles.

Figure 8. (A) Cellular antioxidant activities of EGCG solution (ES) and EGCG loaded niosomes at different EGCG concentrations (mean ± STD, n = 6). (*) Significant difference (p < 0.05). (B, C) Median effect plot for inhibition of peroxyl radical induced DCFH oxidation by free EGCG (B) and EGCG loaded niosomes (C).

Since EGCG is administered orally, the CAA after digestion needs to be determined. EGCG loaded niosomes were collected after simulated intestinal digestion, and then diluted into various concentrations used for CAA assay. As presented in Figure 9, CAA values of EGCG loaded niosomes and free EGCG both decreased compared to the results before digestion, indicating that EGCG would be degraded in passing through the digestive tract. However, compared to the significant decrease in CAA value of free EGCG after digestion, the value of EGCG loaded in niosomes did not change significantly. The CAA assay agrees with the previous results that niosomes appear to protect EGCG against degradation. G

DOI: 10.1021/acs.jafc.6b04147 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Notes

The authors declare no competing financial interest.



Figure 9. Cellular antioxidant activities of free EGCG and EGCG loaded niosomes at different EGCG concentrations after SIF digestion (mean ± STD, n = 6). (*) Significant difference (p < 0.05).

According to the enhanced uptake of EGCG in niosomal formulation from Song et al.,13 the improved CAA values for EGCG niosome could be also attributed to the increased intercellular EGCG levels compared to free EGCG. Since the CAA tests address complicated issues including uptake, distribution, and metabolism of compounds in cells, the mechanism of EGCG working to the intrinsic antioxidant enzymes is still unclear. More cellular tests and animal tests relating to toxicity and bioavailability of EGCG niosomes will be further investigated in our following studies. In summary, niosomes prepared by the ethanol injection method were used to encapsulate EGCG to enhance digestive stability and antioxidant activity in vitro. The relatively high EGCG remaining after simulated intestinal digestion reveals the slow leakage characteristics of the niosomal system and the potential of niosomes to protect EGCG during passage through the digestive system. The results of TEM morphology, DLS particle size distribution, and EGCG remaining studies show that EGCG loaded niosomes remained intact after SGF incubation, but were partly ruptured during SIF incubation. Niosomal encapsulation improved the antioxidant activity of EGCG, even after digestion, indicating that niosomal encapsulation is a potential delivery system that could be used to enhance the digestive stability and bioavailability of EGCG.



ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b04147. Methods, results, discussion, and graph of in vitro cytotoxicity of EGCG loaded niosomes (PDF)



REFERENCES

(1) Fujiki, H.; Suganuma, M. Green tea: An effective synergist with anticancer drugs for tertiary cancer prevention. Cancer Lett. 2012, 324, 119−125. (2) Nagle, D. G.; Ferreira, D.; Zhou, Y. D. Epigallocatechin-3-gallate (EGCG): Chemical and biomedical perspectives. Phytochemistry 2006, 67, 1849−1855. (3) Zaveri, N. T. Green tea and its polyphenolic catechins: Medicinal uses in cancer and noncancer applications. Life Sci. 2006, 78, 2073− 2080. (4) Yi, J.; Lam, T. I.; Yokoyama, W.; Cheng, L. W.; Zhong, F. Betacarotene encapsulated in food protein nanoparticles reduces peroxyl radical oxidation in Caco-2 cells. Food Hydrocolloids 2015, 43, 31−40. (5) Wiseman, S.; Mulder, T.; Rietveld, A. Tea flavonoids: Bioavailability in vivo and effects on cell signaling pathways in vitro. Antioxid. Redox Signaling 2001, 3, 1009−1021. (6) Lambert, J. D.; Elias, R. J. The antioxidant and pro-oxidant activities of green tea polyphenols: A role in cancer prevention. Arch. Biochem. Biophys. 2010, 501, 65−72. (7) Singh, B. N.; Shankar, S.; Srivastava, R. K. Green tea catechin, epigallocatechin-3-gallate (EGCG): Mechanisms, perspectives and clinical applications. Biochem. Pharmacol. 2011, 82, 1807−1821. (8) Kim, S. B.; Lee, M. J.; Hong, J. I.; Li, C.; Smith, T. J.; Yang, G. Y.; Seril, D. N.; Yang, C. S. Plasma and tissue levels of tea catechins in rats and mice during chronic consumption of green tea polyphenols. Nutr. Cancer 2000, 37, 41−48. (9) Sang, S. M.; Lambert, J. D.; Yang, C. S. Bioavailability and stability issues in understanding the cancer preventive effects of tea polyphenols. J. Sci. Food Agric. 2006, 86, 2256−2265. (10) Chow, H. H. S.; Hakim, I. A.; Vining, D. R.; Crowel, J. A.; Ranger-Moore, J.; Chew, W. M.; Celaya, C. A.; Rodney, S. R.; Hara, Y.; Alberts, D. S. Effects of dosing condition on the oral bioavailability of green tea catechins after single-dose administration of Polyphenon E in healthy individuals. Clin. Cancer Res. 2005, 11, 4627−4633. (11) Green, R. J.; Murphy, A. S.; Schulz, B.; Watkins, B. A.; Ferruzzi, M. G. Common tea formulations modulate in vitro digestive recovery of green tea catechins. Mol. Nutr. Food Res. 2007, 51, 1152−1162. (12) Ru, Q. M.; Yu, H. L.; Huang, Q. R. Encapsulation of Epigallocatechin-3-gallate (EGCG) Using Oil-in-Water (O/W) Submicrometer Emulsions Stabilized by iota-Carrageenan and betaLactoglobulin. J. Agric. Food Chem. 2010, 58, 10373−10381. (13) Song, Q.; Li, D.; Zhou, Y.; Yang, J.; Yang, W.; Zhou, G.; Wen, J. Enhanced uptake and transport of (+)-catechin and (−)-epigallocatechin gallate in niosomal formulation by human intestinal Caco-2 cells. Int. J. Nanomed. 2014, 9, 2157−65. (14) Lambert, J. D.; Sang, S.; Lu, A. Y. H.; Yang, C. S. Metabolism of dietary polyphenols and possible interactions with drugs. Curr. Drug Metab. 2007, 8, 499−507. (15) Xie, Y. L.; Kosinska, A.; Xu, H. R.; Andlauer, W. Milk enhances intestinal absorption of green tea catechins in in vitro digestion/Caco2 cells model. Food Res. Int. 2013, 53, 793−800. (16) Wang, D. X.; Taylor, E. W.; Wang, Y. J.; Wan, X. C.; Zhang, J. S. Encapsulated nanoepigallocatechin-3-gallate and elemental selenium nanoparticles as paradigms for nanochemoprevention. Int. J. Nanomed. 2012, 7, 1711−1721. (17) Wang, S.; Su, R.; Nie, S. F.; Sun, M.; Zhang, J.; Wu, D. Y.; Moustaid-Moussa, N. Application of nanotechnology in improving bioavailability and bioactivity of diet-derived phytochemicals. J. Nutr. Biochem. 2014, 25, 363−376. (18) Rocha, S.; Generalov, R.; Pereira, M. D.; Peres, I.; Juzenas, P.; Coelho, M. A. N. Epigallocatechin gallate-loaded polysaccharide nanoparticles for prostate cancer chemoprevention. Nanomedicine 2011, 6, 79−87. (19) Barras, A.; Mezzetti, A.; Richard, A.; Lazzaroni, S.; Roux, S.; Melnyk, P.; Betbeder, D.; Monfilliette-Dupont, N. Formulation and

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Corresponding Author

*Tel: +86 13812536912. E-mail: [email protected]. ORCID

Rong Liang: 0000-0002-1578-2047 Funding

This research was supported by the National Natural Science Foundation of China (No. 31401533, 31571891) and the National Thirteenth-Five Year Research Program of China (2016YFD0400801) and the Fundamental Research Funds for the Central Universities (No. JUSRP11422, JUSRP51507). H

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Article

Journal of Agricultural and Food Chemistry characterization of polyphenol-loaded lipid nanocapsules. Int. J. Pharm. 2009, 379, 270−277. (20) Shpigelman, A.; Cohen, Y.; Livney, Y. D. Thermally-induced beta-lactoglobulin-EGCG nanovehicles: Loading, stability, sensory and digestive-release study. Food Hydrocolloids 2012, 29, 57−67. (21) Liu, W. L.; Ye, A. Q.; Liu, C. M.; Liu, W.; Singh, H. Structure and integrity of liposomes prepared from milk- or soybean-derived phospholipids during in vitro digestion. Food Res. Int. 2012, 48, 499− 506. (22) Rashidinejad, A.; Birch, E. J.; Sun-Waterhouse, D.; Everett, D. W. Delivery of green tea catechin and epigallocatechin gallate in liposomes incorporated into low-fat hard cheese. Food Chem. 2014, 156, 176−183. (23) Peres, I.; Rocha, S.; Gomes, J.; Morais, S.; Pereira, M. C.; Coelho, M. Preservation of catechin antioxidant properties loaded in carbohydrate nanoparticles. Carbohydr. Polym. 2011, 86, 147−153. (24) Moghassemi, S.; Hadjizadeh, A. Nano-niosomes as nanoscale drug delivery systems: An illustrated review. J. Controlled Release 2014, 185, 22−36. (25) Marianecci, C.; Paolino, D.; Celia, C.; Fresta, M.; Carafa, M.; Alhaique, F. Non-ionic surfactant vesicles in pulmonary glucocorticoid delivery: Characterization and interaction with human lung fibroblasts. J. Controlled Release 2010, 147, 127−135. (26) Sezgin-Bayindir, Z.; Onay-Besikci, A.; Vural, N.; Yuksel, N. Niosomes encapsulating paclitaxel for oral bioavailability enhancement: preparation, characterization, pharmacokinetics and biodistribution. J. Microencapsulation 2013, 30, 796−804. (27) Jadon, P. S.; Gajbhiye, V.; Jadon, R. S.; Gajbhiye, K. R.; Ganesh, N. Enhanced Oral Bioavailability of Griseofulvin via Niosomes. AAPS PharmSciTech 2009, 10, 1186−1192. (28) Gurrapu, A.; Jukanti, R.; Bobbala, S. R.; Kanuganti, S.; Jeevana, J. B. Improved oral delivery of valsartan from maltodextrin based proniosome powders. Adv. Powder Technol. 2012, 23, 583−590. (29) Wilkhu, J. S.; McNeil, S. E.; Anderson, D. E.; Perrie, Y. Consideration of the efficacy of non-ionic vesicles in the targeted delivery of oral vaccines. Drug Delivery Transl. Res. 2014, 4, 233−245. (30) Feng, J.; Lin, C.; Wang, H.; Liu, S. Decoration of gemini alkyl O-glucosides based vesicles by electrostatic deposition of sodium carboxymethyl cellullose: Mechanism, structure and improved stability. Food Hydrocolloids 2016, 58, 284−297. (31) Fan, M. H.; Xu, S. Y.; Xia, S. Q.; Zhang, X. M. Preparation of salidroside nano-liposomes by ethanol injection method and in vitro release study. Eur. Food Res. Technol. 2008, 227, 167−174. (32) Yi, J.; Lam, T. I.; Yokoyama, W.; Cheng, L. W.; Zhong, F. Controlled Release of beta-Carotene in beta-Lactoglobulin-DextranConjugated Nanoparticles’ in Vitro Digestion and Transport with Caco-2 Monolayers. J. Agric. Food Chem. 2014, 62, 8900−8907. (33) Suwakul, W.; Ongpipattanakul, B.; Vardhanabhuti, N. Preparation and characterization of propylthiouracil niosomes. J. Liposome Res. 2006, 16, 391−401. (34) LeCorre, D.; Bras, J.; Dufresne, A. Influence of native starch’s properties on starch nanocrystals thermal properties. Carbohydr. Polym. 2012, 87, 658−666. (35) Zou, L. Q.; Peng, S. F.; Liu, W.; Gan, L.; Liu, W. L.; Liang, R. H.; Liu, C. M.; Niu, J.; Cao, Y. L.; Liu, Z.; Chen, X. Improved in vitro digestion stability of (−)-epigallocatechin gallate through nanoliposome encapsulation. Food Res. Int. 2014, 64, 492−499. (36) Zorilla, R.; Liang, L.; Remondetto, G.; Subirade, M. Interaction of epigallocatechin-3-gallate with beta-lactoglobulin: molecular characterization and biological implication. Dairy Sci. Technol. 2011, 91, 629−644. (37) Wolfe, K. L.; Liu, R. H. Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements. J. Agric. Food Chem. 2007, 55, 8896−8907. (38) Pando, D.; Beltrán, M.; Gerone, I.; Matos, M.; Pazos, C. Resveratrol entrapped niosomes as yoghurt additive. Food Chem. 2015, 170, 281−287.

(39) Junyaprasert, V. B.; Singhsa, P.; Suksiriworapong, J.; Chantasart, D. Physicochemical properties and skin permeation of Span 60/Tween 60 niosomes of ellagic acid. Int. J. Pharm. 2012, 423, 303−311. (40) Hu, B.; Ting, Y. W.; Zeng, X. X.; Huang, Q. R. Bioactive Peptides/Chitosan Nanoparticles Enhance Cellular Antioxidant Activity of (−)-Epigallocatechin-3-gallate. J. Agric. Food Chem. 2013, 61, 875−881. (41) Zou, L. Q.; Peng, S. F.; Liu, W.; Chen, X.; Liu, C. M. A novel delivery system dextran sulfate coated amphiphilic chitosan derivativesbased nanoliposome: Capacity to improve in vitro digestion stability of (−)-epigallocatechin gallate. Food Res. Int. 2015, 69, 114−120. (42) Chang, Y.; McClements, D. J. Influence of emulsifier type on the in vitro digestion of fish oil-in-water emulsions in the presence of an anionic marine polysaccharide (fucoidan): Caseinate, whey protein, lecithin, or Tween 80. Food Hydrocolloids 2016, 61, 92−101. (43) Zou, L.; Zheng, B.; Zhang, R.; Zhang, Z.; Liu, W.; Liu, C.; Xiao, H.; McClements, D. J. Food-grade nanoparticles for encapsulation, protection and delivery of curcumin: comparison of lipid, protein, and phospholipid nanoparticles under simulated gastrointestinal conditions. RSC Adv. 2016, 6, 3126−3136. (44) Chou, D. K.; Krishnamurthy, R.; Randolph, T. W.; Carpenter, J. F.; Manning, M. C. Effects of Tween 20 (R) and Tween 80 (R) on the stability of albutropin during agitation. J. Pharm. Sci. 2005, 94, 1368− 1381. (45) Posa, M.; Popovic, K.; Cirin, D.; Farkas, Z. Binary mixed micelles of polysorbates (Tween 20 and Tween 60) and bile salts (Nahyodeoxycholate and Na-cholate): Regular solution theory and change of pK(a) values of micellar bile acid - a novel approach to estimate of the stability of the mixed micelles. Fluid Phase Equilib. 2015, 396, 1−8. (46) Cirin, D. M.; Posa, M. M.; Krstonosic, V. S. Interactions between Sodium Cholate or Sodium Deoxycholate and Nonionic Surfactant (Tween 20 or Tween 60) in Aqueous Solution. Ind. Eng. Chem. Res. 2012, 51, 3670−3676. (47) Liu, S. M.; Huang, H. H. Assessments of antioxidant effect of black tea extract and its rationals by erythrocyte haemolysis assay, plasma oxidation assay and cellular antioxidant activity (CAA) assay. J. Funct. Foods 2015, 18, 1095−1105.

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DOI: 10.1021/acs.jafc.6b04147 J. Agric. Food Chem. XXXX, XXX, XXX−XXX