Research Article Received: 6 February 2015

Revised: 4 May 2015

Accepted article published: 29 May 2015

Published online in Wiley Online Library: 1 July 2015

(wileyonlinelibrary.com) DOI 10.1002/jsfa.7287

Combination of selenium-enriched green tea polysaccharides and Huo-ji polysaccharides synergistically enhances antioxidant and immune activity in mice Chengfu Yuan,a* Zhihong Li,a Fan Peng,a Fangxiang Xiao,a Dongming Ren,a Hui Xue,a Tao Chen,a Gohar Mushtaqb and Mohammad Amjad Kamalc,d Abstract BACKGROUND: The aim of this study was to investigate the influence of a combination of selenium-enriched green tea polysaccharides (Se-GTP) and Huo-ji polysaccharides (HJP) on the immune function and antioxidant activity in mice. RESULTS: The results showed that the indices of spleen and thymus were markedly increased, and the activity of natural killer (NK) cell was promoted in mice treated with the combination of Se-GTP and HJP. The combined treatment of Se-GTP and HJP also reduced the content of tumour necrosis factor-𝜶 (TNF-𝜶) and interleukin-6 (IL-6) in splenocytes. In addition, the activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD) were remarkably enhanced, and malondialdehyde (MDA) levels were significantly reduced in mice treated with combination of Se-GTP and HJP. Furthermore, the combined treatment of Se-GTP and HJP increased nuclear factor erythroid 2-related factor (Nrf2) expression at mRNA and protein levels in splenocytes. The effects of the combination treatment of Se-GTP and HJP in mice were stronger than with Se-GTP or HJP treatment alone. CONCLUSION: Our study suggests that the combined administration of Se-GTP and HJP can synergistically improve immune function and decrease the oxidative stress by enhancing the mechanisms involved in the clearance of free radicals. © 2015 Society of Chemical Industry Supporting information may be found in the online version of this article. Keywords: selenium-enriched green tea; polysaccharides; Huo-ji; mice; antioxidant system; Nrf2

INTRODUCTION

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Correspondence to: Chengfu Yuan, Department of Biochemistry, College of Medical Science, China Three Gorges University, Yichang, HuBei 443002, China. E-mail: [email protected]

a College of Medical Science, China Three Gorges University, Yichang, HuBei, 443002, China b Department of Biochemistry, College of Science, King Abdulaziz University, Jeddah, 21589, Saudi Arabia c King Fahd Medical Research Center, King Abdulaziz University, Jeddah, 21589, Saudi Arabia d Enzymoic, 7 Peterlee Place, Hebersham, NSW 2770, Australia

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Oxidative stress plays a pivotal role in the pathology and development of numerous disorders, including cancer.1 The activation of reactive oxygen species (ROS), such as superoxide, hydroxyl radicals, and H2 O2 , can cause lipid peroxidation of cell membranes. These excessive free radicals bind to macromolecules (proteins, lipids, and nucleic acids), which eventually leads to tissue injury and various disorders.2,3 Natural antioxidants play important roles in the continuous balance between the removal and production of free radicals.4 Nowadays, it is well known that the natural components of some medicinal plants possess high antioxidant capacity.5 For this reason, much attention has been paid to natural antioxidants (such as polysaccharides) in order to prevent oxidative stress-related diseases. Polysaccharides can lower blood pressure and cholesterol levels, protect against infections and inflammation,6 and also serve as potent immune modulators.7,8 Much evidence has shown that polysaccharides are serving as promising options to treat oxidative stress-related diseases.9 Tea is the second commonly consumed drink worldwide next to water, and is particularly common in Asian countries.10 Studies

have discovered that green tea possesses antioxidant, anticancer and antimutagenic activities.11 The health benefits of green tea have been well-documented in previous reviews.12 – 14 Selenium (Se) is a trace element needed for humans and animals and plays an important role in antioxidant defence system because it is an essential component of certain antioxidant enzymes, such as glutathione peroxidase (GPx) that can eliminate peroxide radicals.15 Interestingly, Se-containing green tea exhibits higher antioxidant

www.soci.org activity than regular green tea and Se.16 – 18 Enshi Yulu tea, a Se-enriched Camellia sinensis, is one of the notable Se-containing green teas in China,and, one of the ‘Top Ten Teas’ in China and the ‘Number 1 Historically Famous Tea’ in Hubei Province. Enshi Yulu Tea is processed with green tea leaves growing in the region of Enshi in Hubei Province, China, a place known as the ‘World Capital of Selenium’. The total selenium content of soils in Enshi has been estimated to be in the range of 20–60 mg kg−1 dry weight (DW), which is approximately 150–500 times higher than the average Se content (0.125 mg kg−1 DW) in Se-deficient areas and approximately 50–150 times higher than that (0.40 mg kg−1 DW) in Se-containing areas in China, respectively.19 It was reported that total soil Se was positively correlated with extractable Se in tea.20 Enshi Yulu Tea is one of a few steamed green teas preserved and processed with the technology and equipment discovered in ancient China and is widely distributed in the first seleniferous region (Enshi, Hubei Province) in the world. A number of studies in China have reported that Se-enriched Enshi green tea can decrease blood lipids,21 improve hepatic energy metabolism disorder, protect liver function, and prevent liver damage in non-alcoholic fatty liver disease (NAFLD) rats,22 decrease the oxidative stress,23,24 possesses anti-carcinogenic activity,16,25,26 and enhance the immune function in mice and rats.21 It has been reported that Se-enriched Enshi green tea has stronger effects on decreasing oxidative stress,27,28 and protective effects against hypoxia/reoxygenation injury in mice.29 Selenium-containing tea polysaccharides have been shown to possess inhibitory effects on human breast cancer MCF-7 cells30 and higher antioxidant activities compared to the ordinary polysaccharides.27 Huo-ji, also known as Pyracantha fortuneana (Maxim.) Li, (P. fortuneana) is a plant of Maloideae, and is mainly distributed in the north-west of China. Our previous study has shown that polysaccharides from P. fortuneana (Maxim.) Li can decrease the oxidative stress and enhance the immune function in mice.31 A recent study has shown that proanthocyanidins from P. fortuneana (Maxim.) Li can promote cellular antioxidant activity of quercetin in HepG2, leading to enhanced bioavailability of quercetin. There have been additive effects of proanthocyanidins and quercetin for their antioxidant benefits in vivo.32 In the present study, we first isolated selenium-enriched green tea polysaccharides (Se-GTP) and Huo-ji polysaccharides (HJP), and then investigated their effects in combination on antioxidative and immune functions in mice.

EXPERIMENTAL Materials Se-enriched Enshi green tea was purchased from Huazhi Tea Co., Ltd. (Enshi, Hubei Province, China). Huo-ji fruiting bodies were obtained from Shiyan of Hubei, China. The anti-Nrf2 (sc-13032) was provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA).

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Extraction of Se-GTP and HJP Se-GTP were extracted from Enshi green tea as previously described.33 Briefly, in order to remove lipophilic compounds, dried tea leaves were pretreated with 95% ethanol and then extracted with 5 L of H2 O at 100 ∘ C three times. After cooling, the whole extracts were pooled, filtered and centrifuged, and then the supernatant was precipitated with ethanol. The obtained precipitate was dissolved in Sevag reagent to remove proteins. Se-GTP was obtained after dialysis, condensation and lyophilisation.

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HJP were extracted from Huo-ji using a previously described method.31 Briefly, dried and powdered fruiting bodies of Huo-ji were refluxed and degreased twice with 5 L of petroleum ether at 75 ∘ C (5 h for each time). After cooling, the extract was placed in hot water, followed by concentration and precipitation. The obtained precipitate was dissolved in Sevag reagent to remove proteins, which was followed by dialysis, condensation and lyophilisation to obtain HJP. Properties of Se-GTP and HJP The properties of the purified polysaccharides were investigated using phenol–sulfuric acid methods. The concentration of proteins was measured using the Bradford method.34 The uronic acid levels were measured according to the description given by Blumenkrantz and Asboe-Hansen.35 Atomic fluorescence spectroscopy was used to detect the Se level. Animal experiment Fifty Kunming mice (with weight of 19–23 g) were provided by Chongqing Medical University. Experimental procedures were approved by the Medical Ethics Committee of China Three Gorges University. Animals were randomly divided into five groups as follows: NC group (normal control); PC group (positive control); Se-GTP group; HJP group and Se-GTP + HJP group. Lingzhi polysaccharides were served as the positive control and given to the PC group at 0.3 g kg−1 body weight (BW) per day by gavage for 30 days. The Se-GTP group was given Se-GTP at 0.3 g kg−1 BW per day by gavage for 30 days. The HJP group was given HJP at 0.3 g kg−1 BW per day by gavage for 30 days. Se-GTP and HJP mixture (1:1) were given to the Se-GTP + HJP group at 0.3 g kg−1 BW per day by gavage for 30 days. The NC group was administered with vehicle alone for 30 days. The animals were maintained at 12 h/12 h light/dark cycle at room temperature (25 ∘ C) and 60 ± 5% of relative humidity with free access to food and water. Spleen and thymus indexes Mice were weighed and euthanised with CO2 after 30 days. Thymus and spleen were immediately removed and weighed. Thymus index was expressed as the thymus weight relative to total body weight. Spleen index was expressed as the spleen weight relative to total body weight. Determination of lymphocyte proliferation The spleens of mice were removed, and placed immediately in 0.1 mol L−1 cold phosphate-buffered saline (PBS), homogenised gently, and passed through a 200-mesh sieve to generate single-cell suspensions. Erythrocytes were washed by hypo-osmotic haemolysis. The remaining cells were suspended at a density of 2 × 105 cells mL−1 in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). Splenocytes (100 𝜇L well−1 ) were seeded onto a 96-well plate in the presence of ConA (8 μg mL−1 ).32 Splenocytes were cultured in 5% CO2 at 37 ∘ C for 72 h, and 10 μL of Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) was then added to each well. The plate was incubated for another 2.5 h. Finally, the absorbance was measured at 450 nm. Measurement of natural killer cell activity Natural killer (NK) cells are a type of cytotoxic lymphocytes playing critical role in the innate immune system. The activity of NK cells

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Se- GPT with HJP enhances antioxidant and immune activity was measured using CCK-8 kit. Splenocytes (2 × 105 cells mL−1 ) and YAC-1 cells (1 × 104 cells mL−1 ) were seeded. The ratio of effector to target was 50:1. After co-incubation with CCK-8 (10 μL well−1 ) at 37 ∘ C for 4 h, the optical density (OD450 ) was determined using a microplate reader. The absorbance of blank control, target cells and effector cells were recorded. The activity of NK cells was calculated by the formula: % NK cell activity = 1 − [(OD value of test samples − OD value of effector cell)/ OD value of target cell] × 100.31 Sub-grouping of spleen lymphocyte T-cells After treatment, mouse spleen was harvested into a tissue culture dish and was teased apart into a single cell suspension by pressing with the plunger of a 3 mL syringe. The cells were collected in 10 mL of flow cytometry staining buffer and pass cell suspension through a cell strainer to eliminate clumps and debris. The cell suspension was collected in a conical tube and centrifuged at 400 × g at 4 ∘ C for 5 min and the supernatant was discarded. The cell pellet was resuspended in appropriate volume of staining buffer, the final cell concentration is 1 × 107 cells mL−1 . Splenocytes were blocked with anti-CD8 or anti-CD4 (eBiosciences, San Diego, CA, USA) for 60 min. After washing, the cells were resuspended with paraformaldehyde. Histograms were gated on CD4+ and CD8+ sub-populations, which were counted by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). Measurement of IL-6, IL-2 and TNF-𝜶 Splenocytes were seeded onto a six-well plate at 2 × 105 mL−1 with ConA (8 μg mL−1 ) and incubated at 37 ∘ C for 72 h. The contents of proinflammatory cytokines, interleukin-6 (IL-6), interleukin-2 (IL-2) and tumour necrosis factor-𝛼 (TNF-𝛼), were assayed using the corresponding ELISA commercial kits (Life Science Technology, Shanghai, China).

www.soci.org Western blot for nuclear factor (erythroid-derived 2)-like 2 Splenocytes were lysed, and nuclear fractions were prepared as described previously.32 The protein levels of Nrf2 in splenocytes were assayed with western blot. Briefly, equal amounts (50 μg lane−1 ) of protein extract was denaturised at 95 ∘ C for 5 min, separated by 10% SDS-PAGE, and electrically transferred to PVDF membranes, which were blocked with 5% skim milk for 60 min followed by immunoblot with anti-Nrf2 antibody (1:500) or anti-𝛽-actin antibody overnight. Thereafter, the membranes were washed with agitation using TBS with Tween 20 (TBST) three times (10 min each time), incubated with horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG secondary antibodies at room temperature for 1 h. The membranes were washed three times using TBST (10 min each) and then blotted by ECL kits using chemiluminescent HRP substrate and the signal of substrates was developed with enhanced HRP, followed by exposure to X-ray film for autoradiography. Images were obtained from the films and the relative grey values of protein bands on the image were quantified by Quantity One software. The results were presented as the arithmetic mean values of triplicate data. 𝛽-Actin was used as an internal control. The expression level of Nrf2 was normalised to that of 𝛽-actin and expressed as the fold in relative to corresponding controls. Statistical analysis The experiments were performed in three replicates independently for each group. The data were presented as the mean ± standard deviation (SD). Significant differences among groups were determined by one-way ANOVA analysis of variance with the Bonferroni post hoc using SPSS11.5 software (SPSS Inc., Chicago, IL, USA). Statistical significance was set at P < 0.05.

RESULTS Determination of antioxidant activity Splenocytes were incubated in 5% CO2 for 72 h. The activities of GPx and SOD, as well as MDA levels were assayed using corresponding commercial kits (Nanjing Jiancheng Biological Institute, Nanjing, China), following the procedures provided in the kits.

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Effect of polysaccharides on spleen and thymus index Table 1 shows that the spleen and thymus index (100 × spleen or thymus weight/body weight) in the PC group (Lingzhi polysaccharides treated positive control) was significantly increased

Table 1. Effect of polysaccharides on thymus and spleen index Group NC PC Se-GTP HJP Se-GTP + HJP

Thymus index

Spleen index

0.201 ± 0.041 0.307 ± 0.053a 0.296 ± 0.101b 0.289 ± 0.027b 0.703 ± 0.107acd

0.321 ± 0.103 0.422 ± 0.067a 0.499 ± 0.098a 0.471 ± 0.102a 1.212 ± 0.088acd

Results are given as mean ± SD, n = 10. Thymus and spleen indices are expressed as 100 × spleen or thymus weight/body weight. a P < 0.001, compared with NC group. b P < 0.01, compared with NC group. c P < 0.001, compared with Se-GTP group. d P < 0.001, compared with HJP group.

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Real-time PCR Total RNA isolation and cDNA synthesis were carried out as our previous description.31,36,37 Briefly, the isolated total RNA was treated with RNase-free DNase I at 37 ∘ C for 30 min to remove the possible DNA residues. RNA quality was evaluated by using a 2100 Bioanalyzer (Agilent Technologies, Santa Cruz, CA, USA) and RNAse-free agarose gel electrophoresis and total RNA content was measured by a 2100 Bioanalyzer at 260 nm and 280 nm. Only those RNA samples whose 260 nm/280 nm ratio was greater than 1.8 were used for subsequent analyses. cDNA synthesis was conducted through reverse transcription with SuperScript® III RT (Life Science Technology). Real time PCR was performed using SYBR Green PCR Master Mix. The RT-qPCR system (25 μL) consisted of 1 μL of first-strand cDNAs, 0.5 μL of each primer (10 μmol L−1 ), 12.5 μL of 2 × SYBR FAST qPCR Master Mix. Briefly, after pre-denaturation at 50 ∘ C for 2 min, 95 ∘ C for 10 min, and then PCR amplification was performed with 40 cycles at 95 ∘ C for 15 s and 60 ∘ C for 1 min. The gene expression levels were calculated using 2−ΔΔCt method. GAPDH was used as an internal control. PCR primers are listed in Table S1 in the supporting information.

Characterisation of Se-GTP and HJP Table S2 in the supporting information summarises the carbohydrate content, uronic acid level, protein and Se concentrations of Se-GTP and HJP.

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Table 2. Effects of polysaccharides on the activity of NK cell and spleen lymphocyte proliferation

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Table 3. Comparison of spleen lymphocytes between groups CD4+

Group Group NC PC Se-GTP HJP Se-GTP + HJP

Lymphocyte proliferation (A450 ) 0.247 ± 0.088 0.633 ± 0.099a 0.523 ± 0.091a 0.491 ± 0.072a 1.301 ± 0.098acd

45.214 ± 6.104 61.213 ± 4.701a 53.170 ± 5.404b 56.711 ± 6.182b 127.041 ± 7.015acd

Results are given as mean ± SD, n = 10. a P < 0.001, compared with NC group. b P < 0.05, compared with NC group. c P < 0.01, compared with Se-GTP group. d P < 0.01, compared with HJP group.

(P < 0.001) compared with the NC group. The spleen and thymus indices in the Se-GTP and HJP groups were significantly (P < 0.01, P < 0.001) increased compared with the NC group. Moreover, Se-GTP + HJP treatment displayed a stronger influence on the spleen and thymus index than Se-GTP or HJP treatment alone (P < 0.001). Effects of polysaccharides on cellular immunity Our results showed that Lingzhi polysaccharides exerted strong effects on the activity of NK cells and spleen lymphocyte proliferation in the PC group compared with the NC group (P < 0.001). Se-GTP treatment markedly (P < 0.05) promoted the activity of NK cells and spleen lymphocyte proliferation in mice. HJP and Se-GTP + HJP treatments also markedly (P < 0.05) promoted the activity of NK cells and spleen lymphocyte proliferation. In addition, we found that Se-GTP + HJP administration displayed a stronger effect on the activity of NK cells and spleen lymphocyte proliferation compared with Se-GTP or HJP treatment alone (P < 0.05, Table 2). Effects of polysaccharides on CD8+ and CD4+ T cell counts Table 3 shows that there is no remarkable difference (P > 0.05) in the CD8+ T cell counts between the different groups (NC, PC, Se-GTP, HJP and Se-GTP + HJP). The CD4+ T cell counts in the NC group were markedly (P < 0.01) lower than those of the PC group. Compared with the NC group, Se-GTP, HJP and Se-GTP + HJP treatments enhanced the counts of CD4+ T cells (P < 0.05). In addition, the ratio of CD4+ /CD8+ T cells in the Se-GTP, HJP and Se-GTP + HJP groups was markedly increased compared with the NC group (Table 3, P < 0.001, P

Combination of selenium-enriched green tea polysaccharides and Huo-ji polysaccharides synergistically enhances antioxidant and immune activity in mice.

The aim of this study was to investigate the influence of a combination of selenium-enriched green tea polysaccharides (Se-GTP) and Huo-ji polysacchar...
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