Utilization of albumin fraction from defatted rice bran to stabilize and deliver (-)-epigallocatechin gallate

Food Chemistry xxx (xxxx) xxxx

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Utilization of albumin fraction from defatted rice bran to stabilize and deliver (-)-epigallocatechin gallate Meng Shia, Ze-Shi Wanga, Long-Yue Huanga, Jun-Jie Dongb, Xin-Qiang Zhenga, Jian-Liang Lua, ⁎ ⁎ Yue-Rong Lianga, , Jian-Hui Yea, a b

Tea Research Institute, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310013, China Zhejiang Camel Transworld (Organic Food) Co., Ltd., 16 Chachang Road, Yuhang District, Hangzhou 310000, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Rice bran albumin EGCg Fluorescence quenching Stability In vitro digestion Anti-inflammation

This work aims to use defatted rice bran albumin (RBA) for delivering epigallocatechin gallate (EGCg). The mode of RBA particle size shifted from 142 nm to 164 nm upon interaction with EGCg. Hydrophobic interaction is the major force between EGCg and RBA resulted in the formation of EGCg-RBA complex based on fluorescence quenching. Upon incorporation into RBA, the recovery of EGCg in pH 7.4 phosphate buffer was elevated by 2 folds. The recovery of EGCg in EGCg-RBA was 18.9% after 2 h intestinal digestion, being higher than 7.6% of native EGCg. The pretreatments of HT-29 cells with EGCg, RBA and EGCg-RBA significantly repressed the transcriptional activation of mitogen-activated protein kinase 14, nuclear transcription factor-κB, and activators of transcription 3 as stimulated with interleukin-1β afterwards, leading to attenuated expressions of corresponding downstream genes. Antioxidant ability importantly functioned in anti-inflammation. RBA is a promising vehicle with inherent anti-inflammatory property for stabilizing and delivering EGCg.

1. Introduction Rice bran is the common agro-food by-product of white rice polishing, which has a potential annual production of 60 million tons worldwide (Zarei et al., 2018). Rice bran contains lipids (15–22%), carbohydrates (34.1–52.3%), fiber (7–11.4%) and protein (10–16%) (Juliano, 1985; Saunders, 1990). Despite the rich nutrients, the utilization of rice bran is mainly confined to animal feedstuff and rice bran oil (Saunders, 1990). The commercialized production of rice bran oil results in a vast amount of defatted rice bran waste after lipid extraction. The redundant rice bran and defatted rice bran biomasses are submitted to landfilling or even directly disposed in the open, becoming hazards to environment. The development of comprehensive utilization methods of rice milling by-products is desirable worldwide to reduce food-derived contaminants, transform agro-food residues and add new value to agro-food wastes. New attempts are made to exploit the application field of rice bran/defatted rice bran and its underlying value. Both rice bran and defatted rice bran are the rich source for extraction of bioactives, such as phenolic compounds, tocopherols and dietary fiber (Sohail, Rakha, Butt, Iqbal, & Rashid, 2017). However, these attempts operate at a low profit or only utilize a small portion of the nutrients in rice bran, while the value of some abundant component like

⁎

protein is not fully excavated. Food-grade biopolymers, derived from plants, dairy products and agro-food wastes, are explored as carriers for delivering bioactives with the advantages of rich source, low cost and no adverse effects. Protein is an ideal biopolymeric soft material due to the possession of hydrophobicity and hydrophilicity, which is conducive to emulsification. Milk proteins (caseins and whey proteins), gelatin as well as some plant proteins from maize, sorghum, soybean and grains have been used to transport and protect bioactives (Ye & Augustin, 2018). Rice bran protein consists of albumin, globulin, glutelin and prolamin (Betschart, Fong, & Saunders, 1977). Albumin and globulin are the major soluble proteins, accounting for 29% and 23% of the total protein in rice bran (Landers & Hamaker, 1994). Rice bran protein that is rich in essential amino acids (e. g. lysine) has been associated with superior nutrition and many bioactivities such as hypoallergenicity and antioxidant effects (Sohail et al., 2017), which makes rice bran protein a superior ingredient for nutritional foods and a protein supplement. Dietary polyphenols, such as epigallocatechin gallate (EGCg), function in the control of signaling and inflammation due to their antioxidant property (Romier, Schneider, Larondelle, & During, 2009), which might be good candidates for preventing or reducing the occurrence probability of inflammatory bowel diseases (IBD). However,

Corresponding authors. E-mail addresses: [email protected] (Y.-R. Liang), [email protected] (J.-H. Ye).

https://doi.org/10.1016/j.foodchem.2019.125894 Received 8 August 2019; Received in revised form 9 November 2019; Accepted 11 November 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Meng Shi, et al., Food Chemistry, https://doi.org/10.1016/j.foodchem.2019.125894

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2.2. Formulation and characterization of EGCg-RBA complex

the application of EGCg in clinical area is retarded by its low stability, low bioavailability, poor membrane permeability and low cell uptake (Ye & Augustin, 2018). Stabilization of EGCg is effective and essential way for exploring the application of EGCg. Our previous studies indicated binding to rice bran protein isolate improved the stability of EGCg in the digestion fluids (Shi et al., 2015, 2017), which lent more bioactive EGCg potentially available to the gut. However, the cytotoxicity of rice bran protein as carrier and the impacts of rice bran protein incorporation on the bioactivity and pharmacological behavior of EGCg are still unclear. It needs basic investigations before the largescale utilization of a new delivery material. In the present study, it was hypothesized that rice bran albumin (RBA) obtained from defatted rice bran would be an effective vehicle for delivery of EGCg. The complex of EGCg-RBA was characterized, and the interaction mechanism between EGCg and RBA was studied based on fluorescence quenching. Further, we evaluated the impacts of RBA carrier on the stability of EGCg in pH 7.4 phosphate buffer saline (PBS) and simulated digestion fluids, as well as the anti-inflammatory property of EGCg using the inflammatory model of HT-29 human colon cancer cells. The basic biochemical properties of RBA were also investigated in terms of antioxidant ability, cytotoxicity and anti-inflammatory effect.

The albumin fraction was obtained from defatted rice bran according to the reported method (Adebiyi, Adebiyi, Hasegawa, Ogawa, & Muramoto, 2009). Briefly, defatted rice bran was extracted with water twice (a solid–liquid ratio of 1:10, room temperature, 1 h). The proteincontaining extracts were combined and submitted to centrifugation (5000 rpm, 4 °C, 15 min). The collected supernatant was adjusted to pH 4.1 with 0.1 M HCl. The mixture was kept at 4 °C for 60 min to precipitate protein. After centrifugation (5000 rpm, 4 °C, 15 min) again, the obtained protein isolate was rinsed by water, and then was freeze dried and stored at 4 °C. The soluble protein content of the protein isolate was measured by the Bradford assay. The soluble protein was profiled by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (Shi et al., 2017), and the result showed that albumin was the dominating soluble protein component of the protein isolate. To prepare the stock solution of RBA, 100 mg of the obtained protein isolate was dispensed in 1 mL water, ultrasonicated for 5 min three times, centrifuged (8000 rpm, 4 °C, 5 min), and the supernatant was filtered through 0.22 µm membrane. EGCg stock solution (20 mM) was prepared with water. The RBA solution (2000 µL) was mixed with 10 µL of 20 mM EGCg solution, incubating at 25 °C for 30 min. The particle size distribution of the obtained 100 µM EGCg-RBA complex were measured by a LitesizerTM500 particle analyzer (Anton Paar) at 25 °C in triplicates. The surface morphologies of RBA and 100 µM EGCg-RBA complex were characterized by scanning electron microscope (SEM, HITACHI SU-8010, 3.0 kV).

2. Materials and methods 2.1. Materials, chemicals and reagents Rice bran was obtained from a local rice producer (Hangzhou, Zhejiang province, China). EGCg (950 mg g−1) was provided by Orient Tea Development Co. Ltd. (Hangzhou, Zhejiang province, China). 30% Acrylamide/Bis Solution (29:1) was purchased from BioRad Laboratories Co., Ltd. (Shanghai, China). Sodium dodecyl sulfate was purchased from Amresco, LLC (Solon, Ohio, USA). 2 × Protein loading buffer, 4 × Tris-HCl (pH 8.8 and pH 6.8), 10 × Tris-Glycine gel running buffer pH 8.3, N,N,N″,N″-Tetramethylethylenediamine, Coomassie Brilliant Blue R-250 and PBS were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Molecular weight markers were purchased from BBI Life Sciences Corporation (Hongkong, China). The HPLC-grade acetonitrile and acetic acid were obtained from Jinmei Biotech Corporation (Tianjin, China). NaOH, HCl and NaHCO3 were from Sinopharm chemical reagent Co., Ltd. (Shanghai, China). Fluorescein sodium salt, 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), pepsin from porcine gastric mucosa (3200–4500 units/mg), pancreatin from porcine pancreas (4 × USP specification), and bile salts were purchased from Sigma-Aldrich (Shanghai, China). HT-29 cells were purchased from Chinese Academy of Sciences Kunming Cell Bank (Kunming, Yunnan province, China). Dulbecco’s modified eagle medium, fetal bovine serum and penicillin streptomycin were purchased from Gibco Laboratories (Life Technologies Corpration, Grand Island, NY, USA). Interleukin-8 (IL-8) and prostaglandin E2 (PGE2) ELISA Kits were purchased from Shanghai Xinyu Biotech Co., Ltd (Shanghai, China). Cell Malondialdehyde (MDA) assay kit (Colorimetric method) and superoxide dismutase (SOD) activity assay kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu province, China). Trizol kit was purchased from Vazyme Biotech Co., Ltd (Nanjing, Jiangsu province, China). cDNA was synthesized by PrimeScript Reagent Kit (TaKaRa, Dalian, Liaoning province, China). Primer sets was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) (MTT) assay kit was purchased from Jiangsu Kai Ji Biotechnology Co., Ltd (Nanjing, Jiangsu province, China). The Milli-Q water was prepared by an EASYPure II UV UltraPure Water System (Barnstead International, Dubuque, IA, USA).

2.3. Interaction based on fluorescence quenching The twenty-fold diluted RBA solution (1980 µL) was mixed with 20 µL of excessive EGCg solution (0–10 mM) to achieve the final concentrations of EGCg at 0, 12.5, 25, 50, 75 and 100 µM. These mixtures were placed in a Thermo-shaker (Hangzhou AOSheng Instrument Co., Ltd.) at 150 rpm and various temperatures (288 K, 298 K and 308 K) for 30 min. The fluorescence emission spectra (310 nm ~ 500 nm) of RBA and EGCg − RBA mixtures were measured by a Synergy H1 microplate reader (BioTek Instruments, Inc., USA), under the excitation wavelength (λex) of 280 nm. 2.4. Stability evaluation 2.4.1. Stability in pH 7.4 PBS Four hundred microliters of 100 μM EGCg-RBA were added to 1.6 mL of 2 × PBS, using 100 μM EGCg solution as control. The mixtures were incubated at 37 °C and sampled at 0, 5, 15, 30 and 60 min. The pH value of samples (200 μL) was immediately adjusted below 5 through adding 1.2 μL of 1 M HCl to stop reaction before HPLC analysis. 2.4.2. Stability in digestive fluids An in vitro digestion system was employed to study the impact of RBA incorporation on the digestive stability of EGCg using modified procedures (Shi et al., 2017). EGCg solution (100 µM) and 100 µM EGCg-RBA were preheated (37 °C, 120 rpm) for 10 min. The prewarmed sample (1 mL) was added to 4 mL gastric fluid (7 g L−1 pepsin, 0.1 M HCl, pH 2.0). After purging with nitrogen, the mixture was closed tightly and incubated for 1 h and 2 h in dark (37 °C, 120 rpm), and the gastric digestion fluid was sampled for HPLC analysis. Afterwards, the pH value of the rest digestive fluid was gradually adjusted to 6.5 with 1 M NaOH, and then mixed with 4 mL intestinal fluid (7 g L−1 pancreatin, 7.4 g L−1 bile salts, 0.1 M NaHCO3) for intestinal digestion. After purging with nitrogen, the mixture was incubated in dark (37 °C and 120 rpm), and sampled at 1 h and 2 h. The pH value of 200 μL sample was adjusted below 5 by adding 1.2 μL of 1 M HCl to terminate reaction before HPLC analysis. 2

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incubating at 37 °C until ~80% confluence was reached. The pretreatment of cells with inhibitors were based on the reported method (Mascia et al., 2010). After removal of supernatant, the cells were pretreated with different inhibitors in PBS: 50 μg/mL RBA, 40 μM EGCg and EGCg-RBA (40 μM EGCg, 50 μg/mL RBA), using PBS as Blank. No obvious cell apoptosis was observed for different groups (Fig. S3). After 2 h inhibitor pretreatment, the supernatant was discarded and the cells were stimulated with 35 ng/mL IL-1ß in full cell culture medium for 24 h. Then, the transcriptional expressions of inflammatory biomarkers were analyzed. The cell/supernatants were collected for the measurements of IL-8, PGE2 and MDA levels as well as SOD activity.

2.5. HPLC analysis of EGCg All samples were centrifuged at 12,000 rpm and 4 °C for 10 min, and the supernatants were submitted to HPLC analysis according to the modified method (Shi et al., 2017). The HPLC conditions were: Injection volume 10 μL, Agilent TC-C18 column (4.6 mm × 250 mm, 5 μm), column temperature 32 °C, mobile phase A = acetonitrile/acetic acid/ water (6:1:193, v/v), mobile phase B = acetonitrile/acetic acid/ water (60:1:139, v/v), gradient elution from 40%A/60%B (v/v) to 0%A/ 100%B (v/v) during early 10 min and then 40%A/60%B(v/v) till 15 min, flow rate 1 mL min−1, Shimadzu SPD ultraviolet detector at 280 nm.

2.6.3. Quantitative real-time PCR (qPCR) analysis The total RNA of cells was isolated by Trizol kit (Vazyme Biotech Co., Ltd) and cDNA was synthesized by PrimeScript Reagent Kit (TaKaRa, Dalian, China). Primer sets specific for mitogen-activated protein kinase 14 (MAPK14), NF-κB, signal transducers and activators of transcription 3 (STAT3), tumor necrosis factor α (TNF-α), CXCL8, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), nuclear factor erythroid 2-related factor 2 (Nrf2) and vascular endothelial growth factor (VEGF) were shown in Table S2 based on literatures, using β-actin as an endogenous control. A StepOnePlus Real-time PCR platform (Thermo Fisher Scientific) was used to run PCR reactions as follows: 95 °C for 2 min, 94 °C for 3 s and 40 cycles at 60 °C for 30 s, annealing at 95 °C for 15 s, 60 °C for 60 s, 95 °C for 15 min. The relative expression levels were obtained from the average threshold cycle of triplicate using the 2−ΔΔCt method.

2.6. In vitro anti-inflammatory activity study 2.6.1. Establishment of HT-29 inflammatory model HT-29 cells were seeded (2 × 105 cells/mL) and incubated at 37 °C until ~80% confluence was reached (about 24 h). In a preliminary study, the cytotoxicities of EGCg, RBA and EGCg-RBA to HT-29 cells were evaluated by MTT assay. The IC50 values of EGCg and RBA were 204 μM and 511 μg/mL based on the MTT results (Fig. S1). EGCg concentration below 100 μM and RBA concentration below 50 μg/mL were selected and EGCg-RBA (20–80 μM EGCg, 50 μg/mL RBA) exerted no obvious impact on the cell viability (Fig. S1). The HT-29 cells were stimulated with the whole cell culture media containing 35 ng/mL IL1ß (Pepro Tech, USA) for 24 h. The inflammatory model was checked by the transcriptional expression of inflammatory biomarkers, compared with non-IL-1ß treated cells. Two hour pretreatments with EGCg (20–100 μM) and RBA (50–200 μg/mL) exerted inhibition on the expressions of inflammatory biomarkers nuclear transcription factor-κB (NF-κB), Cyclooxygenase-2 (COX-2) and chemokine (C-X-C motif) ligand 8 (CXCL8), while RBA pretreatments had lower anti-inflammatory effect compared with EGCg (Fig. S2). Therefore, RBA is a bioactive carrier with inherent anti-inflammatory property.

2.7. Data analysis The triplicates of tests were carried out and the value of mean ± standard deviation (SD) was presented. Statistical significance analysis was performed on the SAS System for Windows version 8.1 (SAS Institute Inc., Cary, NC, USA), using Turkey test. Statistical significance analysis of two groups was performed on the Origin Pro 8.5.1 software (Originlab Corporation, Northampton, MA, USA), using two-sample tTest.

2.6.2. Pretreatment with inhibitors: EGCg, RBA and EGCg-RBA Oxygen radical absorbance capacity (ORAC) was conducted to optimize the formula of EGCg-RBA for cell culture study. The ORAC values of EGCg (20, 40 and 60 μM), RBA (50 μg/mL) and the corresponding EGCg-RBA were evaluated according to the published method (Huang, Ou, Hampsch-Woodill, Flanagan, & Prior, 2002), using trolox (50–300 μM) as standard. The results were shown in Table S1. The formula of 40 μM EGCg and 50 μg/mL RBA, with high antioxidant ability and no obvious protein aggregation, was selected for the following cell culture study. HT-29 cells were transferred onto 6-well plates (2 × 105 cells/mL),

3. Results and discussion 3.1. Characteristics of EGCg-loaded RBA microparticles The protein isolate contained 300 mg/g soluble protein, and SDSPAGE showed the soluble protein patterns (Fig. 1a). Heavy bands at 14 and 17 k Da and light band at 32 k Da were observed in the L2 for the soluble proteins from protein isolate, which were attributed to albumin

Fig. 1. Characterization of RBA and 100 µM EGCg-loaded RBA complex. (a) SDS-PAGE profile of marker (L1) and RBA (L2), (b) Particle size distribution, (c) SEM image of EGCg-RBA complex. 3

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increased, with a main mode at around 164 nm (Fig. 1b), possibly due to the interaction between EGCg and RBA. The particle size analysis result was consistent with the SEM result of EGCg-RBA complex (Fig. 1c), which suggests that the albumin fraction prepared from defatted rice bran or even directly prepared from rice bran is a promising food-grade carrier for bioactives with micron-scale. 3.2. Interaction between EGCg and RBA by fluorescence spectroscopy Fluorescence spectroscopy is widely used for studies on the interactions of proteins with phenolic compounds (Bandyopadhyay, Ghosh, & Ghosh, 2012; Joye, Davidov-Pardo, Ludescher, & McClements, 2015), because the intrinsic fluorescence property of proteins is sensitively influenced by the microenvironment. Fig. 2a shows the fluorescence spectra of EGCg-RBA mixtures (λex = 280 nm, 298 K). Fluorescence quenching was observed that the maximum fluorescence intensity of the EGCg-RBA mixture declined appreciably as EGCg increased from 0 μM to 100 μM, suggesting the interaction occurred between EGCg and RBA. Stern–Volmer equation can be used for interpreting interaction types: binding-related quenching (complex formation) and collisional quenching (Joye et al., 2015):

F0/F = 1 + KSV [Q] = 1 + K q τ0 [Q]

(1)

F0: fluorescence intensity of RBA solution; F: fluorescence intensity of RBA solution with certain level of EGCg; KSV: the Stern–Volmer quenching constant, [Q]: free EGCg concentration. Kq: quenching rate constant, τ0: average lifetime of the biopolymer fluorophore without EGCg. Fig. 2b shows the linearity of F0/F plot against [Q]. The values of apparent KSV slightly increased from 1.12 × 104 M−1 to 1.35 × 104 M−1 as temperature increased from 288 K to 308 K, indicating that high temperature (308 K) is favorable to the interaction. In general, the increase of KSV value with elevated temperature means collision-induced dynamic quenching was the primary process, otherwise complex formation is responsible for the fluorescence quenching of protein (Joye et al., 2015). However, in our case, the quenching constants Kq calculated from Stern–Volmer equation were much higher than the maximum value (1010 L mol−1 s−1) that is possible for the diffusion-induced fluorescence quenching. This suggests that the fluorescence quenching of RBA was ascribed to the formation of EGCg–RBA complex other than collision, despite the increase of KSV with temperature. Similar phenomenon was also reported in the complexation between resveratrol and gliadin that the KSV value increased with temperature, due to the hydrophobic interaction in between (Joye et al., 2015). For binding-related quenching, Eq. (2) can be used for the calculation:

log[(F0 − F)/F] = logKA + nlog[Q]

(2)

KA: apparent binding constant; n: the number of binding sites; the definition of [Q], F0 and F the same as Eq. (1). The values of KA and n at 288 K, 298 K and 308 K were present in Table 1 (R2 > 0.994), with KA increasing from 1.10 × 104 to 1.51 × 104 M−1 as temperature increased while all n values being close to 1. The KA values of our study are similar to the reported binding constants of EGCg-proteins listed by Bandyopadhyay et al. (2012). The highest binding constant KA at 308 K was in line with the previous result that high temperature is conducive to form EGCg–RBA complexes. The formation of 1:1 complex was

Fig. 2. Fluorescence quenching study on EGCg-RBA interaction. (a) Fluorescence spectra of EGCg-RBA mixtures (λex = 280 nm), with increasing EGCg concentrations (0–100 µM). (b) Stern − Volmer plots for EGCg-RBA interaction. (c) The Van’t Hoff plot for EGCg-RBA interaction.

Table 1 Apparent binding constants, binding sites, and ΔG values for EGCg–RBA interaction.

(Wang, Li, Xu, Hao, & Zhang, 2014). Hence, albumin was the dominating soluble protein component, and was used as RBA vehicles for carrying EGCg. The RBA vehicles were mainly distributed in the range of 50 nm–460 nm, with a main mode at around 142 nm. Upon interaction with EGCg, the particle size of EGCg-RBA complex slightly 4

Temperature (K)

KA (M−1)

n

R2

ΔG (kJ mol−1)

288 298 308

1.10 × 104 1.32 × 104 1.51 × 104

1.00 1.01 1.01

0.994 0.997 0.995

−22.3 −23.5 −24.6

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incorporation on the stability of EGCg in pH 7.4 PBS. A dramatic decline of EGCg in pH 7.4 PBS was observed along with incubation. At 60 min, there was only 1.38 µg EGCg preserved from 18.00 µg of added EGCg, with a recovery rate of 7.7%. This result is consistent with previous study that EGCg rapidly degraded at alkalescent condition (Shi et al., 2017). Upon incorporation into RBA, more EGCg was preserved with a recovery rate of 15.9% at 60 min, suggesting that RBA vehicles effectively stabilize EGCg at pH 7.4. The protective effect of human serum albumin on EGCg was also reported, which was attributed to the reduced exposure of EGCg to adverse condition via binding, as well as the sulfhydryl groups of albumin that function as an antioxidant to EGCg through scavenging reactive oxygen species or reducing catechol from quinone (Bae et al., 2009). Besides, the buffering effect of albumin to the system is nonnegligible.

proposed considering the values of n were all close to 1. Bose (2016) reviewed the interaction of tea polyphenols with serum albumins based on fluorescence spectroscopic analysis, and demonstrated a 1:1 complex of EGCg and serum albumin formed in most cases. The enthalpy change (ΔH) and entropy change (ΔS) can be calculated by the van’t Hoff equation to interpret the interaction mechanism:

lnKA =

ΔS ΔH − R RT

(3)

T: temperature; KA: apparent binding constant; R: gas constant. The values of ΔH and ΔS calculated from Fig. 2c were 11.90 kJ mol−1 and 118.64 J mol−1 K−1. The positive values of ΔH and ΔS indicated that EGCg-RBA interaction was endothermic and the disorder degree of the system increased. Spontaneous interaction occurred to EGCg and RBA based on the negative value of ΔG (Table 1). Thereby it can be inferred that hydrophobic interaction is the predominant interaction force of incorporating EGCg into RBA. The same result was obtained for the interaction between EGCg and bovine serum albumin that was principally driven by hydrophobic force (Li & Hagerman, 2014; Li & Wang, 2015). The hydrophobic pockets of proteins are important binding sites for EGCg, possibly due to the interaction between aryl groups of polyphenols and hydrophobic surface/pockets of protein (Kawamoto, Mizutani, & Nakatsubo, 1997; Li & Hagerman, 2014). However, Zhou et al. (2017) reported an exothermic process for the interaction between EGCg and rice bran albumin dissolved in PBS, which suggests other interactions might occur to EGCg and rice bran albumin apart from hydrophobic interaction. Hydrogen bonding and van der Waals interactions were involved in the formation of EGCg-human serum albumin complex (Maiti, Ghosh, & Dasgupta, 2006). Thus, hydrophobic interaction, hydrogen bonding and van der Waals force are the common interaction forces of incorporating EGCg into albumin, which becomes the dominating force depending on the protein extraction condition (e. g. pH and temperature) and interaction circumstance (e. g. pH and PBS medium). Both thermal treatment of protein and pH value of medium affect the conformation of proteins resulted in different interaction mechanisms (Liang & Subirade, 2012). In our case, there is no need to precooling rice bran albumin extract and EGCg solution for industrial practice of incorporation, and a warm circumstance is proposed.

3.3.2. Stability of EGCg in digestive fluids Table 3 shows the digestion stability of EGCg carried by RBA. For the gastric digestion stage (0–2 h), EGCg was relatively stable (recovery rate > 79.6%) in both native EGCg system and EGCg-RBA complex. For the subsequent intestinal digestion stage, native EGCg severely degraded, with recovery rates being 13.7% and 7.6% for 1 h and 2 h in intestinal digestion. A remarkable decline of EGCg was reported during intestinal digestion (Shi et al., 2017). By contrast, the recovery rate of EGCg delivered by RBA vehicles was significantly elevated (46.4% and 18.9% for 1 h and 2 h, Table 3). Previous studies showed that binding with protein greatly enhanced the digestion stability of EGCg (Donsì, Voudouris, Veen, & Velikov, 2017; Shpigelman, Cohen, & Livney, 2012), since the interaction between EGCg and protein may lessen the exposure of EGCg to the adverse conditions resulted in the reduction of EGCg degradation (Puligundla, Mok, Ko, Liang, & Recharla, 2017). Besides, high antioxidant capacities of the hydrolysates or peptides from rice bran protein were reported (Chanput, Theerakulkait, & Nakai, 2009). The inherent antioxidant properties of RBA and its digestion products are conducive to stabilize EGCg. Considering albumin has similar structure, the albumin fraction from the different byproducts of cereal processing, e. g. oat bran and wheat bran, has the potential of protecting EGCg against degradation at alkalescent condition, which opens up a value-added utilization direction for the agro-food wastes in cereal processing.

3.3. Stabilization of EGCg via RBA vehicles 3.4. In vitro anti-inflammatory properties RBA vehicles were used for carrying EGCg so as to improve its stability at alkalescent condition and during gastrointestinal digestion.

3.4.1. Inhibitory effect on the activation of inflammation-related signal pathways MAPK14, also known as p38α, is one of the MAPKs which are activated by inflammation, cellular stress, and apoptosis (Choi, Jang, & Kim, 2011), while the activation of STAT3 is associated with IBD (Cénit, Alcina, Márquez, Mendoza, & Díaz-Rubio, 2010). Fig. 3a1-8 show the impacts of EGCg, RBA, and EGCg-RBA on the mRNA expressions of inflammation-related signal pathways MAPK14, NF-κB and STAT3 and relevant downstream genes. Upon stimulation with IL-1β, the expressions of MAPK14, NF-κB and STAT3 were significantly up-regulated compared to Blank, and NF-κB and STAT3 had higher fold changes of mRNA expression than that of MAPK14 (Fig. 3a1-3). Accordingly, the downstream genes of NF-κB pathway (TNFA, CXCL8, COX-2 and iNOS) and VEGF of STAT pathway were significantly up-regulated (P < 0.05, Fig. 3a4-8), suggesting a successful establishment of inflammatory model of HT-29 cells at the mRNA level. The pretreatments of HT-29 cells with EGCg, RBA and EGCg-RBA effectively repressed the IL-1βinduced transcription of CXCL8, TNFA, iNOS, COX-2 and VEGF, and the transcription of COX-2 was even lower than Blank. CXCL8 is the gene regulating the secretion of IL-8, and the transcriptional level of COX-2 is positively related with the secretion of PGE2 (Romier et al., 2009). The IL-8 levels of different pretreatments were: 148.3 ± 10.7 ng/L for Blank, 191.8 ± 20.1 ng/L for IL-1β group, 164.5 ± 4.6 ng/L for RBA, 82.3 ± 5.0 ng/L for EGCg, 109.5 ± 3.5 ng/L for EGCg-RBA. IL-1β

3.3.1. Stability of EGCg in PBS PBS at pH 7.4 is normally used for cell culture, and its alkalescence is similar to human blood. Table 2 shows the effect of RBA Table 2 Effect of RBA vehicles on the stability of EGCg in pH 7.4 PBS (µg). Time

EGCg solution (Control)

EGCg-RBA

0

18.00 (100%) 7.07 ± 0.80a (39.3 ± 4.4%) 4.81 ± 0.18a (26.7 ± 1.0%) 3.05 ± 0.40a (16.9 ± 2.2%) 1.38 ± 0.14a (7.7 ± 0.8%)

18.00 (100%) 9.02 ± 1.08b (50.1 ± 6.0%) 8.36 ± 0.47b (46.4 ± 2.6%) 6.23 ± 0.35b (34.6 ± 1.9%) 2.86 ± 0.17b (15.9 ± 0.9%)

5 min 15 min 30 min 60 min

Different letters (a and b) indicate a significant difference in the same row at the 0.05 level. Data in the brackets are the recovery rates of EGCg, setting the initial amount of EGCg as 100%. 5

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Table 3 Effect of RBA carrier on the stability of EGCg in simulated gastric and intestinal fluids (µg). Initial amount of EGCg

Gastric digestion stage

Subsequent intestinal digestion stage

1h EGCg (Control) EGCg-RBAI

45.00 (100%) 45.00 (100%)

38.24 (85.0 40.17 (89.3

2h ± ± ± ±

a

1h a

35.84 ± 6.04 (79.6% ± 13.4%) 39.34 ± 0.51a (87.4 ± 1.1%)

0.38 0.8%) 1.21a 2.7%)

2h a

6.15 ± 0.20 (13.7% ± 0.4%) 20.90 ± 1.71b (46.4% ± 3.8%)

3.44 ± 0.10 a (7.6% ± 0.2%) 8.54 ± 1.46b (18.9% ± 3.2%)

Different letters (a and b) indicate a significant difference in the same column at the 0.05 level. The data in the brackets are the recovery of EGCg, setting the initial amount of EGCG as 100%.

Fig. 3. The effects of different pretreatments on the activation of signal pathways and downstream gene expressions. (a1-8) mRNA expressions of inflammationrelated signal pathways and the corresponding downstream genes. (b1-3) The indices of oxidative stress. 6

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In the stability study, EGCg (20 µM) greatly degraded in pH 7.4 PBS during 1 h incubation (Table 2), however that was not the same case as the cell culture study that up to 100 µM EGCg and 40 µM EGCg-RBA were used for inhibitor pretreatments (2 h incubation). Due to the relatively high concentration of EGCg and short incubation time, we consider native EGCg and delivered EGCg are the major antioxidants responsible for the anti-inflammatory activity in our study, despite that some bioactive byproducts might be derived from EGCg in cell culture, such as methylated EGCg, protein-bound EGCg, oxidized EGCg and aminated EGCg (Hatasa et al., 2016; Lotito, Zhang, Yang, Crozier, & Frei, 2011). Moreover, the inherent antioxidant and anti-inflammatory properties of RBA might also complement the bioactivity loss of EGCg due to binding. Considering the improved stability and bioaccessibility of EGCg delivered by RBA, our study shows RBA is a promising vehicle for delivering EGCg. The inherent antioxidant and the anti-inflammatory effects are the superiorities of the protein-based nano-/ micro-vehicles produced from cereal processing byproducts.

group contained higher IL-8 level than Blank (P < 0.05), which verified the induction of inflammation at the metabolic level. The groups of EGCg and EGCg-RBA contained the lowest level of IL-8 (P < 0.05), followed by Blank, RBA and IL-1β groups. Thus, EGCg and EGCg-RBA exerted remarkable suppression on the production of IL-8, which was generally in an agreement with the expression of CXCL8 (Fig. 3a4). However, no PGE2 was detected in the supernatants of all samples (data not shown), possibly due to the low expression of COX-2 (Fig. 3a7). The angiogeneic balance in IBD is related with the expression of VEGF (Alkim et al., 2012). The present study shows EGCg-RBA exerted a better inhibitory effect on the expression of VEGF, compared with RBA and EGCg (P < 0.05). The induced oxidative stress at the inflamed sites stimulates the production of cytokines in a synergistic manner via a loop of ROS/RNScytokine-transcription factor (Biasi, Leonarduzzi, Oteiza, & Poli, 2013). Attenuation of cell oxidative stress is an important route for anti-inflammation. Fig. 3b1-3 shows the levels of oxidative status-related indexes, including the transcriptional level of (Nrf2), SOD activity and MDA concentration. Blank had the lowest transcriptional level of Nrf2, which indicated a low oxidative stress. Upon stimulation with IL-1β, the transcription of Nrf2 and SOD activity were significantly enhanced for the IL-1β group (P < 0.05), suggesting an increased oxidative stress in the inflammatory model of HT-29 cells. The oxidative stress was attenuated by the pretreatments of EGCg, RBA and EGCg-RBA due to their antioxidant properties, among which EGCg-RBA had the lowest transcriptional level of Nrf2, followed by RBA and EGCg (Fig. 3b1). Nrf2, a transcription factor, is involved in the protection against acute inflammation by up-regulating antioxidant enzymes (Osburn & Kensler, 2008). However, in the present study about the preventive effect on inflammation induction, the transcription of Nrf2 increased under higher oxidative stress, thus Nrf2 may play a role in a positive feedback loop in response to the up-regulation of NF-κB instead of up-regulation of antioxidative enzymes (Henning et al., 2018). This also might be a plausible explanation for the lower SOD activity observed in the groups of EGCg, RBA and EGCg-RBA than IL-1β group (Fig. 3b2), whereas all groups had similar MDA levels (Fig. 3b3). Fig. S4 gives a diagrammatic sketch of induced cellular inflammatory process. Briefly, IL-1β induced the transduction of MAPK, followed by the activation of its downstream pathway NF-κB, STAT3 and Nrf2. NF-κB, playing a central role in inflammation and immune responses (Surh, Chun, Cha, Han, Keum, Park, & Lee, 2001), is redoxsensitive and can be activated under oxidative/nitrosative stress. Up on activation, NF-κB was translocated to the nucleus and elevated the mRNA expressions of its downstream genes like TNFA, CXCL8, COX-2 and iNOS (P < 0.05). The transcription of CXCL8 led to the increased secretion of IL-8. The expression of VEGF was up-regulated along with the activation of STAT3 pathway, and SOD activity was increased in response to the high transcription of Nrf2 which behaved as feedback to the elevated expression of NF-κB. The pretreatments of EGCg, RBA and EGCg-RBA complex significantly attenuated the up-regulation of NF-κB pathway in the IL-1β-simulated HT-29 cells, resulting in the reduced expressions of the corresponding downstream genes and the induction of cytokines like IL-8. Similar result was also reported that the production of IL-8 was repressed by suppressing the transcription of MAPKs and NF-κB (Yang et al., 2018). Although the attenuating effect of EGCg on the existing inflammation was reported (Romier et al., 2009), our study showed that both RBA vehicles and EGCg complex had anti-inflammatory effects through suppressing the activation of relevant signal pathways, which might prevent the occurrence of inflammation at the first place. The anti-inflammatory activity of EGCg-RBA was comparable to EGCg, especially EGCg-RBA had a better inhibitory effect on the up-regulation of VEGF related with angiogenesis that is a dominant process in the pathogenesis of chronic inflammation (Alkim et al., 2012). It is worthwhile to note the stability of EGCg is concentration-dependent at low level of EGCg and high concentration is favorable for the stability of EGCg (Sang, Lee, Hou, Ho, & Yang, 2005).

4. Conclusions Rice bran is an abundant source of protein for producing albuminbased micron scale carriers for EGCg. The EGCg-RBA complexes have a mode at 164 nm. High temperature favors the binding between EGCg and RBA principally driven by hydrophobic interaction. The stability of EGCg in pH 7.4 PBS and in vitro digestion fluids is greatly improved by RBA carrier, potentially increasing the bioaccessibility of EGCg. RBA has the inherent antioxidant and anti-inflammatory effects. Incorporation of EGCg into RBA rarely exerts adverse impacts on the anti-inflammatory activity of EGCg. Therefore, this work provides a low cost, a sustainable and effective food-grade carrier for EGCg and can be used for industrial scale microencapsulation so as to increase the bioavailability and pharmaceutical values of the bioactive and promote its downstream applications. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the Fundamental Research Funds for the Central Universities (2017QNA6021) and China Agricultural Research System (Tea) (CARS-19). Author contributions M. Shi performed the major part of experimental work, with contributions from Z.S. Wang, L.Y. Huang and X. Q. Zheng. J. J Dong performed all computational calculations and analyses, guided by J. H. Ye. The manuscript was written by J. H. Ye, with contributions of all authors. J. L. Lu and Y. R. Liang guided the experimental work, reviewed and revised the manuscript. All authors approve the final version of the manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125894. References Adebiyi, A. P., Adebiyi, A. O., Hasegawa, Y., Ogawa, T., & Muramoto, K. (2009). Isolation and characterization of protein fractions from deoiled rice bran. European Food Research and Technology, 228(3), 391–401.

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