Article Cite This: J. Agric. Food Chem. 2019, 67, 6476−6486
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Enhanced Antiarthritic Efficacy by Nanoparticles of (−)-Epigallocatechin Gallate−Glucosamine−Casein Yafang Zheng,† Lizheng Xiao,‡ Chenhuan Yu,§ Peng Jin,† Dingkui Qin,† Yongquan Xu,∥ Junfeng Yin,∥ Zhonghua Liu,*,‡ and Qizhen Du*,†
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†
The Key Laboratory for Quality Improvement of Agricultural Products of Zhejiang Province, The College of Agricultural and Food Sciences, Zhejiang A & F University, Linan 311300, China ‡ Key Lab of Education Ministry for Tea Science, National Research Center of Engineering Technology for Utilization of Botanical Functional Ingredients, Hunan Agricultural University, Changsha 410128, China § Experimental Animal Center of the Zhejiang Academy of Medical Sciences, Hangzhou 310013, China ∥ Tea Research Institute Chinese Academy of Agricultural Sciences, Key Laboratory of Tea Biology and Resources Utilization, Ministry of Agriculture, 9 South Meiling Road, Hangzhou 310008, China S Supporting Information *
ABSTRACT: This work aims to improve the antiarthritic activity of (−)-epigallocatechin gallate (EGCG) and glucosamine (GA) through fabrication and optimization of casein protein nanoparticles (EGC-NPs). Optimized EGC-NPs were obtained with a EGCG/GA/casein ratio of 1:2:8 (w/w/w). The EGC-NPs gave a mean size of 186 ± 3.5 nm and an entrapment efficiency of 86.8 ± 2.7%, and they exhibited a greater inhibitory activity against human fibroblast-like synoviocytesosteoarthritis cells and human fibroblast-like synoviocytes-rheumatoid arthritis cells compared with that of the EGCG-GA mixture by 33.5% and 20.8%, respectively. Freeze-dried EGC-NPs stored at 25 °C during 12 months showed high dispersion stability. Moreover, the redispersion of the freeze-dried EGC-NPs produced almost no significant changes in their physicochemical properties and bioactivity. Rat experiments demonstrated that the antiarthritis effect of the EGC-NPs was significantly higher than that of EGCG-GA mixture, as assessed through an analysis of anti-inflammatory efficacy, radiographic images and histopathological assessments of paw joints, and immunohistochemical changes in serum cytokines. The enchanced antiarthritic activity in vivo was consistent with that in vitro. The EGC-NPs demonstrate potential as a food supplement for the treatment of arthritis. KEYWORDS: (−)-epigallocatechin gallate, glucosamine, caseins, nanoparticle, arthritis
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and IL-6 to produce an anti-inflammatory effect.6,7 The antiinflammatory effects of EGCG have been demonstrated in many pathological studies related to inflammation. Kim et al. found that EGCG inhibited the production of IL-8 by respiratory epithelial cells, thereby reducing the severity of respiratory inflammation.8 Yan Tang et al. demonstrated that EGCG could block the JAK1/2 tyrosine kinase signaling pathway in vascular endothelial cells and block P2X4 receptormediated proinflammatory gene expression through IFN-γ.9 EGCG can also inhibit the production of enzymes that destroy cartilage, thereby reducing cartilage loss. Moreover, EGCG can induce osteoclast apoptosis and inhibit the formation of osteoclasts by blocking the production of NFκB and IL-1β and reduce bone resorption by inhibiting the formation of osteoclasts. In addition, during the development of osteoclasts, receptor activator of nuclear factor-κB ligand (RANKL) can up-regulate the expression of NF-ATc1 and induce the production of specific genes such as telomeric repeats amplification protocol (TRAP) and calcitonin receptor
INTRODUCTION Arthritis is an inflammatory disease that occurs in human joints or surrounding tissues and is caused by infection, trauma, and other factors.1 The clinical symptoms of arthritis are joint swelling, fever, pain, and even deformity, which usually lead to joint dysfunction.2 Patients with serious arthritis may develop joint disability, which not only shortens their life span by 10 to 15 years but also has a very serious impact on their quality of life.3 At present, the primary food supplement used to alleviate arthritis is glucosamine (GA), which mainly exists in cartilage between bones and joints and is an important component of polyglucosamine in cartilage matrix and synovial fluid.4 (−)-Epigallocatechin-3-gallate (EGCG), a major polyphenolic component of green tea, accounts for approximately 40%−60% of green tea polyphenols and has been investigated by researchers worldwide.5 EGCG can reduce NO3−/NO2− conversion from NO and decrease the level of reactive oxygen species (ROS)/reactive nitrogen species (RNS) to inhibit the activation of nuclear factor kappa-B (NF-κB) and activator protein 1 (AP-1), thereby down-regulating the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reducing the production of inflammatory factors. EGCG can also reduce the expression of inflammatory factors such as Toll-like receptor-4 and the release of TNF-α, IL-1β, © 2019 American Chemical Society
Received: Revised: Accepted: Published: 6476
April 2, 2019 May 7, 2019 May 20, 2019 May 22, 2019 DOI: 10.1021/acs.jafc.9b02075 J. Agric. Food Chem. 2019, 67, 6476−6486
Article
Journal of Agricultural and Food Chemistry (CTR) in osteoclasts.10 EGCG can inhibit the signal transduction of RANKL through a variety of mechanisms, thereby avoiding the up-regulation of nuclear factor of activated T cells 1 (NF-ATc1) by RANKL and inhibiting osteoclast differentiation.10,11 EGCG can also promote the formation of mineralized bone nodules and preosteoblasts.12,13 Moreover, EGCG can affect osteogenic differentiation by regulating the expression of bone morphogenetic protein 2 (BMP-2).13 Under EGCG stimulation, runt-related transcription factor 2 (Runx2) expression is continuously upregulated, demonstrating the role of EGCG in the process of osteogenic differentiation.14 The combination of tricalcium phosphate granules and EGCG could significantly stimulate bone regeneration in rat skull defects.15 EGCG (5 μM) could significantly promote the differentiation of human bone marrow mesenchymal stem cells into osteocytes.14 In vitro studies have also shown that EGCG has different regulatory effects on cartilage, bone, and synovial fibroblasts.16 The combination of EGCG and methotrexate can inhibit the progress of arthritis in rats.17 Oral administration of EGCG can significantly inhibit pristane-induced arthritis.18 The effectiveness of GA for the treatment of arthritis could be improved, although it currently shows positive effects. On the basis of the hypothesis that EGCG and GA may bring about a synergistic antiarthritic efficacy, the present study aims to investigate the antiarthritis effects of a combination of EGCG and GA encapsulated in casein NPs. Encapsulation has been extensively investigated as a strategy to enhance the stability and effectiveness of EGCG which is vulnerable to oxidation because of its physical and chemical characteristics at physiological pH.19−21
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specimen grid. Excess sample was removed through blotting, and the grid was covered with a small drop of staining solution (2% w/v phosphotungstic acid). The staining solution was left on the grid for a few minutes, and then the excess solution was drained. The sample was allowed to air-dry thoroughly and was then examined using a transmission electron microscope. The NP dispersion was snap-frozen using liquid nitrogen and then freeze-dried. The freeze-dried powder was subjected to scanning electron microscopy. Determination of Entrapment Efficiency and Stability. To confirm the stability of the NP dispersions, the samples were placed in sealed amber-colored glass vials and stored at 2−8 °C, 25 or 37 °C. The NP size, polydispersity index (PDI), zeta potential (ZP), and entrapment efficiency (EE) of these dispersions were measured at predetermined time intervals and compared with values obtained with the fresh dispersions.22 The EE was obtained by determining the free EGCG and GA in the NP dispersions, which were separated using an Amicon Ultra-3K centrifugal filter device (Millipore Corp., Billerica, MA, U.S.A.) consisting of a centrifuge tube and a filter unit with a low-binding Ultracel membrane (3000 MWCO). After centrifugation at 4000g for 30 min, the EGCG-GA-casein NPs remained in the filter unit and the free EGCG and GA penetrated through the Ultracel membrane into the centrifuge tube. EGCG and GA were detected using high-performance liquid chromatography (HPLC), which was performed using a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) consisting of two LC-10A pumps, an SIL-10Avp autosampler, an SPD-M10Avp UV detector and a Symmetry C18 (5 μm, 4.6 mm × 250 mm) column. The elution of EGCG required a mobile phase composed of methanol/water/phosphoric acid (30:70:0.01) at a flow rate of 1 mL/min at 30 °C, and monitoring using UV absorbance at 280 nm, and the elution of GA required a mobile phase composed of 0.1 M sodium acetate/methanol (80:20) at a flow rate of 1 mL/min at 30 °C, and monitoring using UV absorbance at 340 nm. Storage Stability Assay of EGCG in EGC-NPs. The prepared EGC-NP dispersions were stored at 4 and 25 °C for predetermined times (0, 15, 30, and 45 days), and color changes were observed and recorded. The remaining EGCG in the dispersions was analyzed using the above HPLC method. To evaluate the EGCG stability in the freeze-dried EGC-NPs, the samples were stored for 3, 6, 9, and 12 months, and then the remaining EGCG in the NPs was measured through HPLC. Stability Assay of EGCG in EGCG-GA-Casein NPs in Simulated Gastrointestinal Fluids. The in vitro gastric digestion phase was mimicked by simulated gastric fluid (SGF), which was prepared by dissolving pepsin (3.2 mg/mL) in PBS (pH 2.1). The simulated intestinal fluid (SIF) consisting of PBS (pH 7.2), pancreatin (0.16 mg/mL), and bile salts (5 mg/mL) was used to assess the intestinal fate of EGC-NPs.23 The amount of EGCG remaining in the EGC-NPs in SGF and SIF after 0−2 h was determined as reported by Krook and Hagerman.24 Briefly, EGC-NPs were added to SGF or SIF, to a final EGCG concentration of 0.25 mg/mL (0 h), and then incubated in a water bath at 37 °C with stirring (100 rpm). After 30, 60, 90, and 120 min of incubation, 1 mL of the incubated solution was sampled, and the EGCG concentration in this aliquot was determined using HPLC as described above. The remaining amount of EGCG with different time reflects the stability of the EGCG in the EGC-NPs during digestion in vitro. Hydrolysis Analysis of Caseins in EGC-NPs Using Pepsin. The in vitro digestibility of the EGC-NPs was performed on the basis of a previously reported method.25 Pepsin was dissolved at 0.1 g/mL in distilled water and adjusted to pH 2.1 as a stock solution. For the simulated gastric digestion, EGC-NPs samples (40 mg/mL) were preadjusted to pH 2.1 using 1.0 M HCl, followed by the addition of the pepsin stock solution to obtain a pepsin concentration of about 3.2 mg/mL. Samples were incubated at 37 °C in a water bath for 3 h with gentle stirring. The proteolytic reaction was terminated by raising the pH to 7.0 using 1.0 M NaOH. Then, 50 μL of the sample was withdrawn for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein bands were stained using Coomassie Brilliant Blue G-250.
MATERIALS AND METHODS
Chemicals. EGCG, food grade GA, United States pharmacopeia grade pepsin from pig (1:30000), HPLC-grade acetonitrile and methanol, and analytical grade formic acid were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Complete Freund’s adjuvant, incomplete Freund’s adjuvant, bovine type II collagen, human vascular endothelial growth factor ELISA kit, human interleukin-6 (IL-6) ELISA kit, human IL-2L ELISA kit, human TNF-α ELISA kit, and human IFN-γ ELISA kit were purchased from Sigma-Aldrich (Shanghai, China). Arthritis Cells. Human fibroblast-like synoviocytes-osteoarthritis cells (HFLS-OA) and human fibroblast-like synoviocytes-rheumatoid arthritis cells (HFLS-RA) were obtained from Shanghai Cells Bank, Institute of Life Sciences, Chinese Academy of Sciences. Preparation of EGCG-GA-Casein Nanoparticles (EGC-NPs). Sodium caseinate (50 mg/mL) and EGCG (4 mg/mL) were fully dissolved in deionized water, respectively, and GA (50 mg/mL) was dissolved in a solution of 10 mM NaHCO3. The obtained GA solution and the EGCG solution were added into a sodium caseinate solution in proportions of EGCG: GA: caseins (w/w/w) = 1:2:4, 1:2:6, 1:2:8 or 1:2:10, respectively. Thereafter, the mixture was diluted with deionized water and the final casein concentration was adjusted to 10 mg/mL. The diluted sample was then vortexed for 2 min to make sure it was fully mixed. Subsequently, the dispersion solution was ultrasonicated for 5 min. After ultrasonication, the denatured protein was removed through centrifugation at 12000 g for 10 min. Finally, an EGCG-GA-casein NP dispersion was obtained. Characterization of Nanoparticles. The size distribution of the NPs was analyzed using a zeta potential measurement (Zetasizer ZSE, Malvern Instruments, Malvern, U.K.). The morphology and size of the NPs were further assessed using transmission electron microscopy (Hitachi, H-9500E) and scanning electron microscopy (Hitachi, SU9000). A dispersion of NPs diluted with pure water was adsorbed onto a carbon-coated Formvar film that was attached to a metal 6477
DOI: 10.1021/acs.jafc.9b02075 J. Agric. Food Chem. 2019, 67, 6476−6486
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Journal of Agricultural and Food Chemistry
including digits; and 4, paws with deformity or ankylosis.27 The maximum score for a single paw was 4, and for a single rat, the maximum score was 16; arthritis scores for all four paws of each rat were summed as the arthritis index. In a given group, the mean arthritis score for each group was calculated as the mean of the total arthritis scores of all rats within the group. The arthritis index was assessed under blinded conditions. Radiographic Assessments. At day 63 after the first immunization, the rats were sacrificed via anesthesia, and the hind paw was obtained from the control and treated rats. Hind paw images were obtained using a Radiographic System (MRAD-D50S RADREXI; Toshiba Medical Manufacturing Co, Ltd., Tokyo, Japan) to observe the radiologic changes. The X-ray parameters were 40 kV, 100 mA, and 0.02 ms. Images were assessed independently in a blinded fashion, and the radiologic score was assessed in accordance with the following criteria: 0 = no radiologic changes were observed; 1 = mild changes, with tissue swelling and edema; 2 = moderate changes, with joint erosion and disfiguration; and 3 = severe changes, with bone erosion and osteophyte formation.2 The total radiologic scores were calculated from the sum of both hind paws in each rat; the maximum value was 6. Histopathologic Assessments. For histologic analysis, the joints of the hind paw were removed and fixed in 4% paraformaldehyde for a week. The joints of the hind paw were then decalcified for 30 days in 10% EDTA, which was changed every 4 days. The paws were embedded in paraffin blocks and longitudinally cut into 4-pin sections using microtome. More than three serial sections were cut to ensure extensive evaluation of the arthritic joints. The sections were then splayed on a wet surface, mounted on microscope slides, and stained using H&E to observe the degree of synovitis and bone erosions through a blinded microscopic evaluation. Histologic scores were obtained on the basis of inflammatory cell infiltration, synovial hyperplasia, cartilage destruction, and bone erosion.28 The rating criteria are as follows: (1) inflammatory cell infiltration: 0 = normal, 1 = mild infiltration, 2 = moderate infiltration, and 3 = severe infiltration (large number of inflammatory cells were observed); (2) synovial hyperplasia: 0 = no hyperplasia, 1 = mild hyperplasia, 2 = moderate hyperplasia, and 3 = severe hyperplasia; (3) destruction of cartilage: 0 = no destruction, 1 = mild destruction, 2 = moderate destruction, and 3 = severe destruction plus vasculogenesis; and (4) erosions of bone: 0 = no erosions, 1 = mild erosions, 2 = moderate erosions, and 3 = severe erosions (extended erosions and destruction of bone). Measurement of Serum Cytokines. At day 63 (the end of 9 weeks) after the first immunization, serum samples were collected and stored at −80 °C until use. TNF-α, IL-1β, IL-6, and IL-8 levels in the serum were measured using rat TNF-α, IL-1β, IL-6, and IL-8 ELISA kits (R&D Systems, Minneapolis, MN) in accordance with the manufacturer’s instructions. Statistical Analyses. Data are expressed as means and standard deviation (means ± SD) for all groups. Statistically significant differences among groups were determined using one-way analysis of variance (SPSS software version 19.0) followed by Dunnett’s posthoc test. Differences with P < 0.05 were considered significant.
Viability of Arthritic Cells in Culture in Vitro. The effects of EGCG, GA, EGCG-GA, and EGC-NPs on the viability of osteoarthritis cells (HFLS-OA) and rheumatoid arthritis cells (HFLS-RA) were assessed as an evaluation of in vitro arthritis inhibition. HFLS-OA and HFLS-RA were cultured in DMEM high sugar medium mixed with 10% fetal bovine serum and 1% penicillin in a cell incubator at 37 °C and 5% CO2. Cells were maintained as monolayer cultures in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. When the cell growth reached approximately 70%, they were used in experiments. A cell suspension was digested using trypsin and diluted to prepare the required concentration. Then the cells were inoculated into 96-well plates (200 μL/well) for 24 h. When the cells adhered to the wells and grew to approximately 70% confluency, 20 μL of drug at a given concentration was added to the wells. After the cells were cultured in the incubator for another 48 h, MTT solution was added for 4 h of the reaction, followed by 150 μL of dimethyl sulfoxide to dissolve the purple-colored formazan precipitate. The absorbance at 490 nm was recorded using a Bio-Rad micro plate reader with a reference serving as a blank. Arthritis Inhibition Experiments in Vivo. Female Wistar rats, 6 weeks old (130−170 g), were provided by the Experimental Animal Center of Medical Academy of Zhejiang Province (Hangzhou, China). Five animals were housed per cage in a clear and ventilated environment maintained under laboratory conditions (temperature 22 ± 1 °C, relative humidity 50 to 70%, and a 12 h light/dark cycle). Standard food and water were provided ad libitum throughout the experiments. Animals were acclimated to their surroundings over 5 days to eliminate the effect of stress prior to initiation of the experiments. All animal experiments were performed in accordance with relevant experimental animal rules and ethical guidelines in Zhejiang Academy of Medical Sciences, China (No. SCXK (Zhejiang) 2018-0007). Collagen-induced arthritis (CIA) was used for the arthritis inhibition experiments in vivo. CIA was induced as described previously, with minor modifications.26 Briefly, bovine type II collagen (Chondrex, Redmond, WA) was dissolved at 2 mg/mL in 0.05 M acetic acid with gentle stirring overnight at 40 °C. Collagen solutions were emulsified with an equal volume of complete Freund’s adjuvant (Chondrex) using a homogenizer (Ronghua Instrument Manufacturing Co, Jiangsu, China) in an ice water bath. An emulsion containing 200 mg of collagen (0.2 mL) was subcutaneously injected into the right hind paw. To ensure a high incidence and severity of arthritis, a booster injection was administered on day 7 after initial immunization, using 0.1 mL (100 mg of collagen) of Freund’s incomplete adjuvant (Chondrex) as a secondary immunization. In this model, the onset of arthritis in rats occurs within 1 week after the second immunization. Starting on the day after the booster injection, the rats were regularly monitored for the development and severity of paw inflammation. The primary immunization day was defined as day 0. The body weight of the rats was measured using an electronic scale every week from the primary immunization, and hind paw thickness was measured using electronic digital calipers every week. On day 16 after the primary immunization, rats displaying the onset of arthritis (arthritis index >2) were randomly assigned to the following groups (n = 6): (1) rats with CIA receiving physiologic saline (0.1 mL/100 g once daily oral administration); (2) rats with CIA receiving EGC-NPs (corresponding to EGCG 80 mg/kg + GA 800 mg/kg); (3) rats with CIA receiving EGCG-GA (EGCG 80 mg/kg + GA 800 mg/kg); (4) rats with CIA receiving celecoxib (25 mg/kg). Intragastric administration for all drugs was carried out for 9 weeks. EGC-NPs, EGCG-GA, or celecoxib were suspended in physiologic saline prior to experimentation. Clinical Assessment of Arthritis. Rats were inspected daily to assess the onset of arthritis in the paws from the second immunization. Macroscopic signs of clinical arthritis were assessed using a qualitative clinical score method every 3 days beginning on the day when arthritic signs were first visible. Each paw was scored in accordance with the following criteria: 0, normal; 1, mild redness and swelling of ankle or wrist joints; 2, moderate redness and swelling of ankle or wrist joints; 3, severe redness and swelling of the entire paw
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RESULTS AND DISCUSSION Preparation and Characterization of EGCG-GA-Casein NPs (EGC-NPs). EGCG-GA with the mass ratio of 0.4:0.8, corresponding the sample containning 0.4 mg/mL EGCG and 0.8 mg/mL GA, exhibited the highest inhibitory effects on HFLS-OA cells (OA cells) and HFLS-RA cells (RA cells) in our preliminary experiments. However, the instability of EGCG during storage and oral administration limits its commercial application and results in low bioavailability. According to the results of other studies, encapsulation of EGCG may be an effective method to fight against adverse environmental conditions.29,30 Therefore, casein micelles were used as a nanocarrier to load the EGCG-GA mixtures. In this study, various concentrations of caseins were tested for the 6478
DOI: 10.1021/acs.jafc.9b02075 J. Agric. Food Chem. 2019, 67, 6476−6486
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Journal of Agricultural and Food Chemistry
Table 1. Effects of the Concentration of Caseins (CN) on the Mean Diameter (MD), Zeta-Potential (ZP), Polydispersity Index (PDI), and Entrapment Efficiency (EE) of the Nanoparticlesa EGCG:GA:CN (w/w/w) 1:2:4 1:2:6 1:2:8 1:2:10
MD (nm) 151 153 186 212
± ± ± ±
0.8a 1.8a 3.5b 3.7c
ZP (mV) −30.2 −31.1 −35.8 −36.7
± ± ± ±
PDI
2.9a 2.9a 0.7b 1.2b
0.14 0.13 0.10 0.18
± ± ± ±
0.06a 0.03a 0.03a 0.09b
EE of EGCG (%) 47.9 57.8 86.8 88.2
± ± ± ±
0.9a 1.3b 2.7c 2.2c
EE of GA (%) 30.1 73.9 98.2 98.3
± ± ± ±
1.4a 2.5b 3.7c 3.6c
Mean ± SD, n = 3. Values in a column followed by different letters are significantly different (p < 0.05).
a
Figure 1. Transmission electron microscopic and scanning electron microscopic images of EGCG-GA-casein nanoparticles (EGC-NPs).
Figure 2. Effects of EGCG (a), glucosamine (b), the combination of EGCG and glucosamine (c), and EGC-NPs (d) on HFLS-RA and HFLS-OA cells. Values in the same color bars followed by different lowercase letters are significantly different (p < 0.05).
construction and optimization of EGC-NPs. The particle sizes, PDI, and ZP values are shown in Table 1. With an increase in
the casein concentration from 1.6 to 4.0 mg/mL, the particle size and ZP gradually increased from 150 to 211 nm and −30.2 6479
DOI: 10.1021/acs.jafc.9b02075 J. Agric. Food Chem. 2019, 67, 6476−6486
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Journal of Agricultural and Food Chemistry to −36.7 mV, respectively. The size of the particles is an important property because it has direct relevance to their stability, cellular uptake, in vivo distribution, and drug release.29 ZP is also an important parameter reflecting the physicochemical and biological stability of NPs in suspension. A high absolute ZP value helps the particles to repel each other, which ensures long-term stability and avoids particle aggregation.30 The addition of casein significantly increased negative charge ( 0.05) in terms of inhibitory activity agaist OA and RA cells (Figure S1). This indicated that freeze-drying had no effect on the biological activity of the EGC-NPs. These results indicate that the freezedrying process is conducive to the long-term and stable storage of EGC-NPs. The EGCG remaining in the EGC-NPs was assessed in simulated gastrointestinal fluids and compared with an EGCGGA mixture (Table 3). After incubation for 120 min in SIF, the relative EGCG remaining in the EGC-NPs was 32.4%, while that in the mixture rapidly decreased to 3.3% after incubation, showing a very significant difference (P < 0.01). A similar phenomenon was observed in the gastric environment, and the EGCG remaining in the NPs and the mixture was 83.6% and 52.9%, after a 120 min incubation, respectively. The stability of the EGCG under gastric conditions was significantly higher than that under intestinal conditions. EGCG has a stronger stability at low pH and is more conducive to maintaining its 6480
DOI: 10.1021/acs.jafc.9b02075 J. Agric. Food Chem. 2019, 67, 6476−6486
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Journal of Agricultural and Food Chemistry Table 3. % EGCG Remaining in an EGC-NP Dispersion in Simulated Gastric Fluid and Simulated Intestinal Fluida % EGCG remaining sample in simulated gastric fluid EGCG+GA EGC-NPs in simulated intestinal fluid EGCG+GA EGC-NPs
0
30
60
120
100 ± 8.1 100 ± 8.5
94.5 ± 6.5a 81.1 ± 5.9b
85.6 ± 7.4a 72.4 ± 4.3b
80.0 ± 4.4a 63.9 ± 7.1b
100 ± 7.8 100 ± 7.1
81.1 ± 9.5b 45.6 ± 5.1c
69.4 ± 8.0b 11.8 ± 1.2c
47.4 ± 4.4b 5.90 ± 0.64c
Mean ± SD, n = 3. Values in a column followed by different letters are significantly different (p < 0.05).
a
Figure 3. Profile of inflammatory and radiographic changes in the joints of arthritic control and treated rats. (a) Representative photographs of the arthritic paws. (b) Evaluation by arthritis scores; ** significantly different (p < 0.01). (c) Evaluation by radiographic scores; values in the same color bars followed by different lowercase letters are significantly different (p < 0.05), and values followed by a lowercase letter and a different capital letter are significantly different (p < 0.01). (d) Radiographic changes in joints of arthritic control and treated rats.
bioactivity.29 This result is consistent with previous research, in which Hong et al. found that the loss of EGCG in NPs was only 10−15% less than that of free EGCG at pH 2.1.22 In contrast, EGCG was more unstable in intestinal fluid (pH 7.2) than in gastric fluid (pH 2.1), especially at 2 h, and up to 93% of free EGCG was degraded; however, the level of EGCG remaining in the NP formulation was still maintained at a high level within the SIF. Therefore, NPs can significantly protect drug components from destruction.36
The stability of protein-based NPs during digestion is an important characteristic for the oral delivery of drugs/ nutrients.37 Therefore, to more directly show that NPs can help to resist the digestive effect of pepsin in gastric fluid, SDSPAGE was used to examine the degradation of casein protein in EGC-NPs compared to native caseins during incubation (Figure S3). The results showed that the native casein protein was obviously degraded after a 30 min incubation, while the distinguished degradation time for caseins in EGC-NPs was 90 min. The delayed proteolytic degradability of caseins indicated 6481
DOI: 10.1021/acs.jafc.9b02075 J. Agric. Food Chem. 2019, 67, 6476−6486
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Journal of Agricultural and Food Chemistry
Figure 4. Profile of the histopathological changes in collagen-induced arthritis in rat paw joints. (a) Effects of EGCG-GA, EGC-NP, and celecoxib treatment on joint histology. Evaluation of inflammatory cells, synovial hyperplasia, cartilage damage, and bone erosion in rat right (b) and left (c) paws, as assessed using histologic scores. Values in the same color bars followed by different lowercase letters are significantly different (p < 0.05), and values followed by a lowercase letter and a different capital letter are significantly different (p < 0.01).
maximum scores of 4.3, 2.7, 2.6, and 4.3 points were reached after 4 weeks, indicating that the tested drugs had significant alleviating effects on the CIA symptoms (P < 0.01). Remarkably, EGC-NP administration significantly suppressed arthritis aggravation and showed inhibitory effects that were similar to those of celecoxib (P > 0.05). However, EGCG-GA showed an arthritis alleviating effect that was obviously lower than that of the EGC-NPs (P < 0.01), which demonstrates that the EGC-NPs improved the efficiency of EGCG-GA. The nanosizing of the EGCG-GA formulation may play a key role in this improvement, since nanosizing can decrease the oxidation and degradation of EGCG in the gastrointestinal environment (Figure S1). A radiographic evaluation and scoring system was used to evaluate the difference between the different treatments in terms of affecting the progression of the degenerative arthritis. As shown in Figure 4c, in the model group, the radiographic scores of both paws reached 3.82 and 3.75 points, respectively. In the EGCG-GA group, EGC-NP group, and celecoxib group, the radiographic scores of the right paws were 3.12, 1.28, and 1.16 points, respectively, but those of almost all the left paws were close to 0. The results indicate that the EGCG-GA, EGCNP, and celecoxib treatments were more efficient in affecting mild symptoms compared with severe arthritis symptoms. Moreover, the images showed the serious development of paw bone erosions in the model group (Figure 3d), which were not present in the treatment groups, indicating that the treatments had a protective effect on the bone. Notably, the therapeutic effect of the EGC-NPs was significantly better than that of the
that part of the EGC-NPs might reach the adsorption place in intestinal tract which was helpful for improve the bioavailability of EGCG. Antiarthritis Effect of EGC-NPs in Vivo. Animal experiments were carried out to investigate the biological effects of the EGC-NP delivery system in CIA. Paw swelling was examined by measuring thickness changes in the paws. The swelling of the adjuvant-injected right hind paws increased rapidly after the injections and reached maximal arthritis symptoms at day 7. In contrast, the swelling of the adjuvantuninjected left hind paws gradually increased from day 5, but the symptoms were significantly less than that of the right hind paws. After 9 weeks of drug administration through gavage, the body weight of the rats in the EGC-NP group, EGCG-GA group, celecoxib group, and arthritis model group were significantly affected compared with the control group (Figure S4). The body weight of rats in the celecoxib group and model group decreased by 66% and 50%, respectively, which indicate that CIA and celecoxib could lead to side-effects in rats. The body weight of rats in the EGC-NP group and the EGCG-GA group decreased by 27% and 31%, respectively, which suggested that EGCG or GA could alleviate the effects of CIA. The therapeutic efficacy of adjuvant arthritis was evaluated through direct observation (Figure 3a); an arthritis score based on the severity and extent of erythema and swelling of the periarticular tissues; and the enlargement, distortion, or ankylosis of the joints (Figure 3b,c).38,39 As shown in Figure 4b, in the model group, the arthritis score increased progressively, and reached 7 points after 5 weeks, while in the EGCG-GA group, EGC-NP group, and celecoxib groups, 6482
DOI: 10.1021/acs.jafc.9b02075 J. Agric. Food Chem. 2019, 67, 6476−6486
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Journal of Agricultural and Food Chemistry Table 4. Expression Levels of Cytokines in CIA-Induced Arthritis Cells after 9 Weeksa group
TNF-α
IL-1β
IL-6
IL-8
model EGCG-GA EGC-NPs celecoxib
100C 56.8 ± 3.0B 22.4 ± 1.1A 20.8 ± 2.1A
100C 69.2 ± 2.4B 32.1 ± 3.9A 30.4 ± 1.4A
100C 75.3 ± 1.9B 29.1 ± 1.3A 32.8 ± 0.7A
100C 49.5 ± 2.2B 13.1 ± 1.7A 12.8 ± 1.3A
The relative expression is represented as the percentage of the model group (100%). Mean ± SD, n = 3. Values in a column with capital superscripts are significantly different (p < 0.01).
a
between joint arthritis and the expression of IL-1β, IL-6, and IL-8. A significant decrease in immunopositivity was observed following EGC-NP (P < 0.01), EGCG-GA mixture (P < 0.01), and celecoxib (P < 0.01) treatment. Compared with the model group, the expression of TNF-α, IL-1β, IL-6, and IL-8 showed a moderate decrease, following EGCG-GA mixture after the treatment of 9 weeks. However, a significant difference in cytokine expression did not occurr between the EGC-NP group and the celecoxib group. These results suggest that EGC-NPs suppressed the expression of TNF-α, IL-1β, IL-6, and IL-8 in arthritic rats more effectively than EGCG-GA mixtures, and approached the effect of celecoxib. EGCG could prevent collagen-induced arthrisis in mice.41,42 Up to this point, a series of studies have been conducted to elucidate the underlying molecular mechanisms regulating the antiarthritic activity of EGCG. Overall, the antiarthritic function of EGCG may be related to the following actions: (1) inhibition on BAFF and PI3K/Akt/m TOR signaling and consequent promotion of the apoptosis of B lymphocytes;43 (2) down-regulation of COX-2 expression/PGE2 production via up-regulation of has-miR-199a-3p expression in human OA chondrocytes;44 (3) hampered expression of IL-1p-induced ADAMTS5 (a disintegrin and metalloprotease with thrombospondindomains; ADAMTS) via increasing the expression of hsa-miR-140−3p in human OA chondrocytes;45−47 (4) impeding the expression of pro-inflammatory cytokines, oxidative stress proteins, and STAT3;48 (5) suppressed STAT3 proteins and Th17-cell differentiation by inducing a higher Treg/Th17 cell ratio in CD4 (+) T-cell;49 (6) improving the IL-1β-induced expression of IL-8 through inhibition of the NF-κB, p38, and ERK pathways;50 (7) elicited indoleamine-2,3-dioxygenase-producing dendritic cells to increase frequencies of T regs and induce the activation of the nuclear factor-E2-related factor 2 (Nrf-2) antioxidant pathway;51 (8) inducted phosphorylated-extracellular signalregulated kinase, nuclear respiratory factor 2, and heme oxygenase-1 and inhibition on signal transducer and activator of transcription-3 activation;52 (9) enhanced proteasomeassociated deubiquitinase expression to rescue proteins from proteasomal degradation by reducing TAK1 phosphorylation and K48-linked polyubiquitination;53 and (10) interference on the IL-1β signaling pathway and regulating the expression of pro-inflammatory mediators (IL-6 and IL-8) and Cox-2 via controlling the TAK-1 activity.54 These investigations have further verified the activity of EGCG against arthrisis, which are important to develop the arthritic products of EGCG. Moreover, the molecular mechanism of the synergistic inhibition of GA and EGCG on arthritis merits further study. In conclusion, EGCG, as a plant polyphenol nutrient, shows anti-inflammatory effects and mitigates arthritis symptoms in a combination regimen with GA. Freeze-dried EGCG-GA NPs with casein micelles could be redispersed and demonstrated similar properties as a fresh dispersion of EGC-NPs. The EGC-
EGCG-GA mixture and comparable to the antiarthritic effect of celecoxib. The effect of EGC-NP administration on histopathological changes in the ankle joint cartilage is shown in Figure 4a. Compared with the control group, a large area of articular cartilage was missing and had been replaced by connective tissue in the model group, accompanied by angiogenesis, inflammatory cell infiltration (black arrow), and injury to cancellous bone (yellow arrow). Of note, necrotic and exfoliated tissue (red arrow) was visible in the articular lumen, with synovial hyperplasia and considerable inflammatory cell infiltration. With EGCG-GA treatment, these symptoms were slightly reduced, and synovial hyperplasia and inflammatory cell infiltration decreased significantly (yellow arrow), while EGC-NP and celecoxib administration strongly suppressed these changes in adjuvant arthritis rats. These changes in inflammatory cells, synovial hyperplasia, cartilage damage, and bone erosion were further evaluated using the Mankin score (Figure 4b,c). As expected, the scores of the right and left paws were significantly increased to >3.0 by CIA. Consistent with the histopathological changes, the scores were reduced in the EGCG-GA group, compared with the model group, although the difference in the right paws was not statistically significant. In contrast, the EGC-NP and celecoxib group had higher histopathological scores than that of the EGCG-GA group in terms of inflammatory cells, synovial hyperplasia, and cartilage destruction, with significant differences (P < 0.01) compared with the model and EGCGGA group. Overall, the histopathological scores of the left paws were significantly higher (P < 0.01) than those of the right paws, which is consistent with the inflammatory scores. Numerous studies have shown that GA, as an amino monosaccharide, may exhibit chondro-protective action by inhibiting the degradation and stimulating the synthesis of proteoglycans,32 restoring articular function,39 and suppressing the activation of inflammatory cells, chondrocytes, and synoviocytes.40 Combined with EGCG, GA can effectively promote its antiarthritis effects. However, because of poor bioavailability caused by low absorption and/or substantial loss in the gastrointestinal tract, this efficiency is greatly compromised. The current study is the first to investigate the effects of an EGC-NP formulation on CIA by evaluating changes in cartilage as well as histological criteria, and the formulation provides a promising alternative for arthritis treatment. Secreted inflammatory cytokines are among the critical mediators of the disturbed processes implicated in osteoarthritis pathophysiology.1 A qualitative measurement of the expression of pro-inflammatory cytokines such as TNF-α, IL1β, IL-6, and IL-8 using immunohistochemical techniques is shown in Table 4. A considerably high expression of TNF-α, an inflammatory cytokine, was detected in the tissue joint of arthritic rats (model group), together with a strong association 6483
DOI: 10.1021/acs.jafc.9b02075 J. Agric. Food Chem. 2019, 67, 6476−6486
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Journal of Agricultural and Food Chemistry
cancer chemopreventive polyphenol in green tea. Nutrients 2012, 4, 1679−1691. (6) Nagai, K.; Jiang, M.-H.; Hada, J.; Nagata, T.; Yajima, Y.; Yamamoto, S.; Nishizaki, T. (−)-Epigallocatechin gallate protects against NO stress-induced neuronal damage after ischemia by acting as an anti-oxidant. Brain Res. 2002, 956, 319−322. (7) Tedeschi, E.; Menegazzi, M.; Yao, Y.; Suzuki, H.; Forstermann, U.; Kleinert, H. Green tea inhibits human inducible nitric-oxide synthase expression by down-regulating signal transducer and activator of transcription-1alpha activation. Mol. Pharmacol. 2004, 65, 111−120. (8) Kim, I.-B.; Kim, D.-Y.; Lee, S.-J.; Sun, M.-J.; Lee, M.-S.; Li, H.; Cho, J.-J.; Park, C.-S. Inhibition of IL-8 production by green tea polyphenols in human nasal fibroblasts and A549 epithelial cells. Biol. Pharm. Bull. 2006, 29, 1120. (9) Tang, Y.; Matsuoka, I.; Ono, T.; Inoue, K.; Kimura, J. Selective up-regulation of P2 × 4-receptor gene expression by interferongamma in vascular endothelial cells. J. Pharmacol. Sci. 2008, 107, 419−427. (10) Takayanagi, H.; Kim, S.; Koga, T.; Nishina, H.; Isshiki, M.; Yoshida, H.; Saiura, A.; Isobe, M.; Yokochi, T.; Inoue, J. I.; et al. Induction and Activation of the Transcription Factor NFATc1 (NFAT2) Integrate RANKL Signaling in Terminal Differentiation of Osteoclasts. Dev. Cell 2002, 3, 889−901. (11) Morinobu, A.; Biao, W.; Tanaka, S.; Horiuchi, M.; Jun, L.; Tsuji, G.; Sakai, Y.; Kurosaka, M.; Kumagai, S. (−)-Epigallocatechin3-gallate suppresses osteoclast differentiation and ameliorates experimental arthritis in mice. Arthritis Rheum. 2008, 58, 2012−2018. (12) Vali, B.; Rao, L. G.; El-Sohemy, A. Epigallocatechin-3-gallate increases the formation of mineralized bone nodules by human osteoblast-like cells. J. Nutr. Biochem. 2007, 18, 341−347. (13) Chen, C. H.; Ho, M. L.; Chang, J. K.; Hung, S. H.; Wang, G. J. Green tea catechin enhances osteogenesis in a bone marrow mesenchymal stem cell line. Osteoporosis Int. 2005, 16, 2039−2045. (14) Jin, P.; Wu, H.; Xu, G.; Zheng, L.; Zhao, J. Epigallocatechin-3gallate (EGCG) as a pro-osteogenic agent to enhance osteogenic differentiation of mesenchymal stem cells from human bone marrow: an in vitro study. Cell Tissue Res. 2014, 356, 381−390. (15) Rodriguez, R.; Kondo, H.; Nyan, M.; Hao, J.; Miyahara, T.; Ohya, K.; Kasugai, S. Implantation of green tea catechin α-tricalcium phosphate combination enhances bone repair in rat skull defects. J. Biomed. Mater. Res., Part B 2011, 98B, 263−271. (16) Ahmed, T. A.; Hincke, M. T. Strategies for articular cartilage lesion repair and functional restoration. Tissue Eng., Part B 2010, 16, 305−329. (17) Roy, S.; Sannigrahi, S.; Vaddepalli, R. P.; Ghosh, B.; Pusp, P. A novel combination of methotrexate and epigallocatechin attenuates the overexpression of pro-inflammatory cartilage cytokines and modulates antioxidant status in adjuvant arthritic rats. Inflammation 2012, 35, 1435−1447. (18) Leichsenring, A.; Bäcker, I.; Furtmüller, P. G.; Obinger, C.; Lange, F.; Flemmig, J. Long-term effects of (−)-epigallocatechin gallate (EGCG) on pristane-induced arthritis (PIA) in female dark agouti rats. PLoS One 2016, 11 (3), No. e0152518. (19) Carbonaro, M.; Grant, G.; Pusztai, A. Evaluation of polyphenol bioavailability in rat small intestine. Eur. J. Nutr. 2001, 40, 84−90. (20) Wu, M.; Jin, J.; Jin, P.; Xu, Y.; Yin, J.; Qin, D.; Wang, K.; Du, Q. Epigallocatechin gallate-β-lactoglobulin nanoparticles improve the antitumor activity of EGCG for inducing cancer cell apoptosis. J. Funct. Foods 2017, 39, 257−263. (21) Yang, Y.; Jin, P.; Zhang, X.; Ravichandran, N.; Ying, H.; Yu, C.; Ying, H.; Xu, Y.; Yin, J.; Wang, K.; Wu, M.; Du, Q. New Epigallocatechin gallate (EGCG) nanocomplexes co-assembled with 3-mercapto-1-hexanol and β-lactoglobulin for improvement of antitumor activity. J. Biomed. Nanotechnol. 2017, 13, 805−814. (22) Hong, Z.; Xu, Y.; Yin, J. F.; Jin, J.; Jiang, Y.; Du, Q. Improving the effectiveness of (−)-epigallocatechin gallate (EGCG) against rabbit atherosclerosis by EGCG-loaded nanoparticles prepared from
NPs can significantly improve the stability of EGCG during storage and in simulated gastrointestinal conditions. The delayed degradation of casein protein in the EGC-NPs was observed, which may play a key role in their antiarthritis effects in vivo. The EGC-NPs demonstrate potential as a food supplement for alleviating arthritis as they are composed of food-grade materials (EGCG, GA, and casein) and exhibit antiarthritis efficacy in vitro and in vivo, since EGC-NPs suppressed the expression of TNF-α, IL-1β, IL-6, and IL-8 in arthritic rats more effectively than EGCG-GA mixture to alleviate arthritis of rats.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b02075.
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Stability evaluation of EGC-NPs dispersion stored at 4 and 25 °C for 1.5 month; inhibitory activity of freezedried EGC-NPs with different storage time against HFLS-OA and HFLS-RA cells viability; SDS-PAGE analysis of casein in simulated gastric fluid; and changes of the body weight of Sprague−Dawley rats treated with EGC-NPs, EGCG-GA mixture, and celexicob compared to the rats of arthritis model group (PDF)
AUTHOR INFORMATION
Corresponding Authors
*Tel.: +86-0731-84635304; Fax: +86-0731-84635306; E-mail:
[email protected] (Z. Liu). *Tel.: +86-571-15958126861; Fax: +86-571-88218710; Email:
[email protected] (Q. Du). ORCID
Qizhen Du: 0000-0003-2969-2840 Funding
This research was supported by the Key R&D Program Project of Zhejiang Province, China (Grant 2019C02072), the Fund for Distinguished Scholars of Zhejiang Agricultural and Forestry University (Grant 2014FR064), the Key R&D Program Project of China (Grant 2017YFD0400803) and Zhejiang Innovation discipline titled with Laboratory Animal Genetic Engineering (Grant 201604). Notes
The authors declare no competing financial interest.
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