International Journal of Biological Macromolecules 72 (2015) 519–525
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Cheonggukjang polysaccharides enhance immune activities and prevent cyclophosphamide-induced immunosuppression Chang-Won Cho a , Chun-ji Han b , Young Kyoung Rhee a , Young-Chul Lee a , Kwang-Soon Shin c , Ji-Sun Shin d , Kyung-Tae Lee d , Hee-Do Hong a,∗ a
Division of Strategic Food Research, Korea Food Research Institute, Gyeonggi 463-746, Republic of Korea Department of Preventive Medicine, Yanbian University, Jilin 133002, China c Department of Food Science and Biotechnology, Kyonggi University, Gyeonggi 443-760, Republic of Korea d Department of Pharmaceutical Biochemistry, Kyung Hee University, Seoul 130-701, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 9 April 2014 Accepted 8 September 2014 Available online 16 September 2014 Keywords: Cheonggukjang Polysaccharide Immunostimulatory activity
a b s t r a c t Cheonggukjang is a traditional Korean fermentation product prepared from soybean that is reported to have various biological functions. We previously reported that the polysaccharides from Cheonggukjang (PSCJ) have immunostimulatory activities in RAW 264.7 macrophages and primary cultured splenocytes. In this study, the immunostimulatory activities of the PSCJ were investigated further using various experimental models such as in vitro, ex vivo, and in vivo. The PSCJ was able to stimulate the complement system (ITCH50 : 30.6%). In primary cultured mouse peritoneal macrophages, the PSCJ was found to significantly increase nitric oxide and immunostimulatory cytokines (IL-6 and IL-12) production in a concentration-dependent manner (1–100 g/mL). In the normal mice model, the oral administration of the PSCJ increased the weight of spleen (p < 0.05 at 100 and 200 mg/kg) and improved the phagocytic rates of peritoneal macrophages (p < 0.05 at 200 mg/kg) and lymphocytes proliferation (p < 0.05 at 100 and 200 mg/kg). Similarly, the PSCJ markedly restored the decreased lymphocytes proliferation (p < 0.01 at 200 mg/kg), natural killer cell activity (p < 0.01 at 200 mg/kg), and white blood cell count (p < 0.01 at 100 and 200 mg/kg) in the cyclophosphamide-induced immunosuppressed mice model. These results suggest that the PSCJ could be utilized as an effective immunostimulatory agent. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The modulation of immune response to prevent diseases has long been one of the primary concerns of many researchers [1]. It was recently proven that the use of immunomodulators to enhance the host defense responses can be an effective way to increase resistance to disease [2]. Polysaccharides separated from natural substances including food ingredients and medicinal plants have been regarded as important immunostimulant candidates due to their broad spectrum of immunostimulatory activities with relatively low toxicity and adverse effects [3]. Polysaccharides from natural sources have been known to improve the immune functions of the body by activating immune-related cells, such as
∗ Corresponding author at: Division of Strategic Food Research, Korea Food Research Institute, 1201-62 Anyangpangyo-ro, Bundang, Seongnam, Gyeonggi 463746, Republic of Korea. Tel.: +82 31 780 9285; fax: +82 31 709 9876. E-mail address: [email protected] (H.-D. Hong). http://dx.doi.org/10.1016/j.ijbiomac.2014.09.010 0141-8130/© 2014 Elsevier B.V. All rights reserved.
macrophages, lymphocytes, and natural killer (NK) cells, and by promoting cytokine secretion, antibody production, and activation of the complement system [4]. Therefore, many natural polysaccharides are receiving increasing attention in the fields of therapeutics and functional foods. Cheonggukjang is a traditional Korean fermentation product prepared from soybean and is also called Natto, Tempeh, and Douchi in other Asian countries [5]. Cheonggukjang is manufactured by the short-term fermentation of soybeans using Bacillus subtilis, which contains many enzymes, microorganisms, and bioactive compounds that are absent from unfermented soybeans [6]. Cheonggukjang is known for its functionality, including antioxidant, anti-inflammatory, anti-hypertensive, and anti-diabetic activities [7], in addition to its nutritional value. Most previous functionality studies of Cheonggukjang used the whole product or a crude extract [5–8]. However, few studies have reported the activity of polysaccharides from Cheonggukjang. In the studies that investigated the biological activities of polysaccharides prepared from the traditional Korean fermentation products, it was reported
520
C.-W. Cho et al. / International Journal of Biological Macromolecules 72 (2015) 519–525
that the polysaccharides from persimmon vinegar, soy sauce, and makgeolli, which is traditional Korean rice wine, have an effect on immune activity [9–11]. In a previous study, we isolated and characterized polysaccharides from Cheonggukjang (PSCJ), and investigated their immunostimulatory activity and underlying molecular mechanisms on recombinant interferon (rIFN)-␥-primed RAW 264.7 macrophages and primary cultured splenocytes [12]. To further investigate the immunostimulatory activities of the PSCJ, the present study explores the effect of the PSCJ on the complement system and primary cultured peritoneal macrophages and subsequently in normal mice and cyclophosphamide (CY)-induced immunosuppressed mice models. 2. Materials and methods 2.1. Materials and chemicals Soybeans were purchased from Namboeun NongHyup (Boeun, Korea) and commercial Cheonggukjang starter culture (B. subtilis 10% and soybeans 90%) was purchased from NUC Corp. (Daegu, Korea). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), concanavalin A (ConA), dimethyl sulfoxide (DMSO), Griess reagent, lipopolysaccharide (LPS), phosphate buffered saline (PBS), thioglycollate, and trypan blue solution (0.4%) were purchased from Sigma–Aldrich (St. Louis, MO, USA). CVT-E002TM (sold commercially as COLD-FX® ) was purchased from Afexa Life Sciences Inc. (Edmonton, AB, Canada). Rosewell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from GIBCO BRL (Grand Island, NY, USA). Enzyme linked immunosorbent assay (ELISA) kits for interleukin (IL)-6 and IL-12 were obtained from BD Biosciences (San Diego, CA, USA). Cyclophosphamide (CY) was purchased from Jiangsu Heungrui Medicine Co., Ltd. (Lianyungang, China). 2.2. Preparation of the PSCJ Whole soybeans (1000 g) were washed five to six times and soaked with filtered water at 4 ◦ C for 24 h and then steamed for 40 min at 121 ◦ C. The steamed soybeans were maintained at 50 ◦ C to allow them to cool down. The cooked soybeans were then inoculated with 0.5% (w/w) Cheonggukjang starter culture and fermented for 48 h at 40 ◦ C in an incubator. The Cheonggukjang samples were freeze-dried and stored at −20 ◦ C. Lyophilized Cheonggukjang powder was extracted with 10 volumes of distilled water at 90 ◦ C for 3 h. The extracts were centrifuged at 6500 × g for 20 min and filtered through Advantec filter paper No. 2 (5 m, Toyo Roshi Kaisha, Ltd., Tokyo, Japan) to remove any insoluble particles. Polysaccharides were precipitated from the extracts by the addition of four volumes of 95% ethanol. The precipitate was dissolved in distilled water and dialyzed using Spectra/Por (MWCO; 6000–8000, Spectrum Laboratories Inc., Rancho Dominquez, CA, USA) for three days. Finally, the high-molecular-weight solution was lyophilized. The lyophilized powder (denoted as PSCJ) was stored in the dark at 4 ◦ C. 2.3. Anti-complementary activity of the PSCJ (in vitro) The anti-complementary activity was measured by the complement fixation test based on complement consumption and the degree of red blood cell lysis by residual complement [13]. Normal human serum (NHS) was obtained from volunteer adults. A 50 L aliquot of 1000 g/mL PSCJ was mixed with equal volumes of NHS and gelatin veronal-buffered saline (GVB2+ , pH 7.4) containing 500 mM Mg2+ and 150 mM Ca2+ , respectively. The mixtures were pre-incubated at 37 ◦ C for 30 min and the residual
total hemolytic complement (TCH50 ) was determined using IgM hemolysin-sensitized sheep erythrocytes (1 × 108 cells/mL). The NHS was incubated with water and GVB2+ to provide a control. The anti-complementary activity of the PSCJ is expressed as the percent inhibition of the control TCH50 (ITCH50 ) of polysaccharide K (PSK), which is a known immunoactive polysaccharide from Coriolus versicolor that was used as a positive control [14]. ITCH50 (%) = [TCH50 (control) − TCH50 (treated with sample)]/TCH50 (control) × 100. 2.4. Immunostimulatory activity of the PSCJ on primary cultured mouse peritoneal macrophages (ex vivo) 2.4.1. Primary culture and cytotoxicity test Specific pathogen-free (SPF), 5-week-old male C57BL/6 mice were purchased from Hanlim Experimental Research Institute (Hwaseong, Korea). The peritoneal macrophages of C57BL/6 mice were harvested from 5% thioglycollate-treated mice as described previously [15]. The peritoneal macrophages were resuspended in RPMI 1640–FBS (containing 10% FBS) medium and then plated into 96 well-culture plates (2 × 105 cells/well). After incubation for 2 h, the non-adherent cells were removed by PBS washing, and the adherent macrophages were stimulated with various concentrations of the PSCJ for 24 h. The macrophages were incubated with a MTT solution for 4 h at 37 ◦ C under 5% CO2 . The MTT-containing medium was removed, and the cells were solubilized in DMSO. The absorbance of each well at 540 nm was measured using a microplate reader (TECAN, Männedorf, Switzerland). 2.4.2. Quantification of nitric oxide and cytokine production Peritoneal macrophages were seeded in 96-well plates (2 × 105 cells/well) and stimulated with various concentrations of the PSCJ. After 24 h, the culture supernatants were collected, and the nitric oxide (NO) concentration was measured using the Griess reagent. Equal volumes of the Griess reagent (1% sulfanilamide, 0.1% naphthylenediamine dihydrochloride, and 0.5% H3 PO4 ) and the sample were incubated together at room temperature for 10 min. The absorbance at 540 nm was measured using a microplate reader (TECAN). The concentrations of cytokines (IL-6 and IL-12) in the culture supernatants were determined using ELISA kits according to the manufacturer’s protocol. 2.5. Immunostimulatory activity of the PSCJ in vivo 2.5.1. Animals SPF KM male mice (7–8 weeks of age) were obtained from the Experimental Animal Center of Yanbian University College of Medicine (Yanji, China). The mice were maintained under constant conditions (temperature: 22 ± 2 ◦ C, humidity: 40–60%, light and dark cycle: 12 h) and allowed free access to water and food. All of the animal experiments were performed according to the instructions of the Medical Ethics Committee for the Use of Experimental Animals at Yanbian University. 2.5.2. Animal experiments The animal experiments were conducted using both normal and immunosuppressed mice. For the animal experiments with normal mice, the mice were randomly divided into three groups (ten mice in each group). From day 1 to 28, the three different groups of mice were orally treated with saline or the PSCJ at 100 and 200 mg/kg body weight on a daily basis. Twenty-four hours after the last administration, the mice were weighed and sacrificed by cervical dislocation. The spleen and thymus were immediately removed and weighted. The spleen and thymus index were calculated as the spleen and thymus weight/body weight, respectively. The spleen sample was used for lymphocyte proliferation.
C.-W. Cho et al. / International Journal of Biological Macromolecules 72 (2015) 519–525
For the experiments with the immunosuppressed mice, the mice were randomly divided into five groups (ten mice in each group). One group of mice was used as a vehicle-treated control without any treatment for immunosuppression. The other four groups of mice were subjected to immunosuppression through intraperitoneal treatment with CY (100 mg/kg/day) from day 1 to 3. Among these groups, one group of CY-treated mice was used as an immunosuppressed model group. CVT-E002TM (CVT), an immunostimulatory polysaccharide-rich extract of North American ginseng (Panax quinquefolium), was used as a positive control [16]. From day 4 to 17, the five different groups of mice were administered the following: vehicle-treated control group, saline; immunosuppressed model group, saline; positive control group, 200 mg/kg body weight of CVT; and the PSCJ groups, 100 or 200 mg/kg body weight of the PSCJ. All of the mice were treated daily by oral administration. Twenty-four hours after the last administration, blood was collected from the mice for white blood cell (WBC) counts. The mice were subsequently weighed and sacrificed by cervical dislocation. The collected spleen samples were used to measure the lymphocyte proliferation and NK cell activity. The WBC number was measured using a semi-automatic blood cell analyzer (KCTH04, Guangzhou Kingcare Medical Equipment Co., Ltd., Guangzhou, China). 2.5.3. Phagocytic activity assay of peritoneal macrophages Twenty-four hours after the last administration, the mice were injected intraperitoneally with 1 mL of 20% chicken red blood cells (CRBCs). Thirty minutes later, the mice were killed by cervical dislocation, and 2 mL of PBS was used for peritoneal lavage. The cell suspension was smeared on glass slides and incubated for 30 min at 37 ◦ C in a humidified atmosphere with 5% CO2 . After incubation, the non-phagocytosed CRBCs and other cells were removed by washing with PBS. The macrophages were fixed with acetone:methanol (1:1, v/v) and stained with Giemsa solution. The phagocytic rate was determined by counting the number of macrophages phagocytosing CRBCs in a population of 100 macrophages. The phagocytic index was determined by counting the number of phagocytosed CRBCs per 100 macrophages [17]. 2.5.4. Lymphocyte proliferation assay The spleens obtained from the mice sacrificed under aseptic conditions were washed with RPMI 1640 medium and crushed to isolate the splenocytes. The splenocytes were passed through a 200-mesh stainless steel sieve to obtain a homogeneous cell suspension. The splenocyte suspension was washed twice with RPMI 1640–FBS medium and with centrifugation at 1000 rpm for 5 min. The recovered splenocytes were resuspended in Trisbuffered ammonium chloride solution (NH4 Cl, pH 7.2) for 5 min to remove the red blood cells. After centrifugation, the harvested splenocytes were resuspended in RPMI 1640–FBS medium, and the cell numbers were measured with a hemocytometer using trypan blue dye exclusion. The splenocytes were seeded in 96-well plates (3.0 × 105 cells/well) for the lymphocytes proliferation assays with ConA (5 g/mL) or LPS (10 g/mL). After incubation, MTT solution was added to each well, and the plates was incubated for 4 h. DMSO solution was added to resolve the formazan. The absorbance was measured in a microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 540 nm. 2.5.5. NK cell-mediated cytotoxicity assay NK-mediated cytotoxicity was measured in YAC-1 cells (NKsensitive cell line) and primary cultured splenocytes from the PSCJ-treated mice. Splenocyte suspensions were added to the YAC1 cells (1 × 105 cells/mL) to obtain an effector-to-target cell ratio of 50:1 in 96-well U-bottom culture plates. The cells were then incubated at 37 ◦ C in a humidified atmosphere containing 5% CO2 for 4 h. Following incubation, the culture supernatants were mixed
521
Fig. 1. Anti-complementary activities of the PSCJ. The anti-complementary activity was expressed as the inhibition of 50% total complement hemolysis by Mayer’s method [13]. The concentrations of PSK and PSCJ were 1000 g/mL. PSK was used as a positive control. The data are expressed as the means ± SD of three separate experiments. ***p < 0.001 vs. control.
with a lactate dehydrogenase (LDH) solution (Roche, Mannheim, Germany), and the absorbance value of each well was measured at 490 nm [18]. The NK cell cytotoxicity (%) = [(experimental release − spontaneous release)/(maximum release − spontaneous release)] × 100. 2.6. Statistical analysis All of the statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) version 17.0 (SPSS Inc., Chicago, IL, USA). The quantitative data are expressed as the means ± SD. All statistical comparisons were performed using a one-way ANOVA test followed by Duncan’s multiple range tests. p-Values less than 0.05 were considered statistically significant. 3. Results 3.1. Effect of the PSCJ on the complement system activation The chemical composition of the PSCJ was reported in our previous report, and it was proven that the immunostimulatory effect of the PSCJ did not result from LPS contamination [12]. The PSCJ was consisted of protein (6.52%) and polysaccharide (93.48%, w/w), and polysaccharide was shown to be the neutral (24.02%) and acidic (69.46%) polysaccharide. Neutral polysaccharides in the PSCJ mainly comprised of galactose (39.9 mole%), arabinose (31.9 mole%), glucose (8.8 mole%), xylose (8.2 mole%), rhamnose (5.7 mole%), fucose (3.3 mole%), and mannose (2.2 mole%) [12]. The complement system plays an important role in host defense, inflammation, and allergic reactions. Thus, anti-complementary activity is considered an immunomodulating activity that is responsible for immunological defense [19]. To evaluate the biological activity of the PSCJ as it relates to the complement cascade reaction, the anti-complementary activity was tested. As shown in Fig. 1, the anti-complementary activity of the PSCJ was 30.3%. PSK was used as a positive control. 3.2. Effect of the PSCJ on primary cultured mouse peritoneal macrophage To determine whether the immunostimulatory activity effects of the PSCJ observed in RAW 264.7 macrophages also occur in
522
C.-W. Cho et al. / International Journal of Biological Macromolecules 72 (2015) 519–525
Fig. 2. Effects of the PSCJ on NO, IL-6, and IL-12 productions in primary cultured mouse peritoneal macrophages. Mouse peritoneal macrophages were stimulated with the PSCJ (1, 10, or 100 g/mL) or LPS (2 g/mL) for 24 h. LPS was used as a positive control. (A) NO production was determined by measuring the nitrite accumulation in the culture medium. (B and C) The amount of IL-6 and IL-12 was measured by ELISA assay. (D) The cell viability was determined by the MTT assay. The data are expressed as the means ± SD of three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.
primary cells, we examined the effect of the PSCJ on the productions of NO and cytokines, such as IL-6 and IL-12 in peritoneal macrophages isolated from C57BL/6 mice. In these cells, the PSCJ was found to significantly increase the secretion of NO, IL-6, and IL-12 in a concentration-dependent manner (Fig. 2A–C). LPS was used as a positive control for NO and cytokine productions. When we examined the toxicity of the PSCJ on primary cultured mouse peritoneal macrophages using the MTT assay, it was found that the PSCJ (1–100 g/mL) did not affect the cell viability compared to the control cells (Fig. 2D).
3.3. Immunostimulatory effects of the PSCJ on normal mice The PSCJ was orally administered at doses of 100 and 200 mg/kg to normal mice, and its effects on the body mass gain and the weights of immune organs, such as the spleen and thymus, are shown in Table 1. Commonly, abnormal alterations in body weight have been used as indicators of the toxic effects of testing samples [20]. The ratios of the body mass gain were not significantly changed in the PSCJ-treated groups, suggesting that the PSCJ did not have adverse effects on the mice. We found that the spleen index was significantly increased in the PSCJ-treated groups (p < 0.05) compared with that found in the vehicle-treated control group. In addition, the thymus index tended to increase in the PSCJ-treated
groups compared with the control group, but the difference was not statistically significant. The phagocytic activity of macrophages in the PSCJ-treated groups was evaluated by the CRBCs uptake. The phagocytic rate and index obtained for the PSCJ-treated groups are presented in Table 2. The phagocytic rate was significantly increased in the 200 mg/kg PSCJ-treated group (p < 0.05). The phagocytic index was slightly increased, but the difference was not statistically significant. Because it is well known that ConA and LPS stimulate Tand B-lymphocyte proliferation [21], we tested whether the PSCJ influences the lymphocyte proliferation induced by ConA or LPS. As shown in Table 2, the PSCJ significantly increased the T- and Blymphocytes proliferation compared to the vehicle-treated control group, which suggests that the PSCJ is a direct mitogen for mouse lymphocytes. 3.4. Immunostimulatory effects of the PSCJ on cyclophosphamide-treated immunosuppressed mice To evaluate the effects of the PSCJ on cyclophosphamide (CY)-induced immunosuppression, we measured the lymphocyte proliferation, NK cell activity, and WBC counts. The intraperitoneal treatment of CY (100 mg/kg) inhibited the T- and B-lymphocyte proliferation, NK cell activity, and WBC counts (Fig. 3). As shown in Fig. 3A, the PSCJ significantly enhanced the proliferation of
C.-W. Cho et al. / International Journal of Biological Macromolecules 72 (2015) 519–525
523
Table 1 Effects of the PSCJ on immune organs in normal mice. Group
Number of animals
Initial body weight (g)
Final body weight (g)
Weight growth rate (%)
Spleen index (SI)a
Thymus index (TI)b
Control PSCJ 100 mg/kg PSCJ 200 mg/kg
10 10 10
22.00 ± 1.79 21.60 ± 1.84 22.60 ± 1.90
35.50 ± 4.38 33.50 ± 2.98 36.00 ± 4.00
61.36 55.09 59.29
0.65 ± 0.08 0.73 ± 0.06* 0.74 ± 0.03*
0.35 ± 0.13 0.38 ± 0.12 0.43 ± 0.13
The PSCJ (100 or 200 mg/kg) was administered daily for 28 days. The control group mice were treated with vehicle (normal saline solution). The data are presented as the means ± SD (n = 10). a Spleen weight/body weight. b Thymus weight/body weight. * p < 0.05 vs. the vehicle-treated control group.
T- and B-lymphocytes compared with those observed in the CY-treated immunosuppressed group. The administration of CY significantly decreased the activity of NK cells compared with the vehicle-treated control group. However, the activity of NK cells in the 200 mg/kg PSCJ-treated groups was significantly increased compared with that in the CY-treated immunosuppressed group (Fig. 3B). Additionally, the WBC counts were significantly restored in the PSCJ-treated groups compared with the CY-treated immunosuppressed groups (Fig. 3C). CVT, a positive control, also increased the T- and B-lymphocyte proliferation, NK cell activity, and WBC counts.
4. Discussion The immune system protects the organism from infectious disease and tumor through a layered defense with increasing specificity [22]. The role and necessity of the immune system are prominent during chemotherapeutic intervention for the treatment of many diseases [23]. In recent years, the use of immunomodulators to strengthen the host defense responses has been considered one of the most promising alternatives to classical drug treatment [24]. In a previous study, we isolated and characterized the PSCJ and showed its immunostimulatory activities [12]. The PSCJ exerts immunostimulatory activity resulting from nuclear factor-B (NFB) activation and thereby increases the gene expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-␣ in rIFN-␥-primed RAW 264.7 macrophages. The PSCJ also increases the proliferation, and IL-2 and IFN-␥ productions in primary cultured splenocytes. In addition, The PSCJ decreases immobility time and indicators related to fatigue in the forced swimming test animal model. In the present study, we attempted to further evaluate the immunostimulatory activities of the PSCJ, including its anticomplementary and peritoneal macrophage activation activities as well as its immune function enhancing activities in a normal mice model. We also investigated the protective effects of the PSCJ in immunosuppression caused by CY treatment.
The complement system plays an important role as a primary defense against bacterial and viral infections and is activated via the classical and alternative pathways [25]. Complement activation appears to be intrinsically associated with several immune reactions, including the activation of macrophages, lymphocytes, and immunopotentiation [26]. The anti-complementary activity of the PSCJ was measured by the complement fixation test. The PSCJ showed about 30% anti-complementary activity, whereas the known immunoactive polysaccharide Krestin (PSK), a -glucan from Coriolus versicolor [14] used as a positive control, showed 60% activity. However, the anti-tumor activity of PSK was evaluated in Japan for the prevention of esophageal, gastric and lung cancers in humans with promising results, and PSK is currently sold as a drug [27]. Thus, even though the activity of the PSCJ was about the half of that of PSK, our findings still suggest that the PSCJ is likely to be an effective complement activator. The activation of macrophages plays a key role in innate immunity for initiating and propagating defensive reactions against pathogens [1]. Activated macrophages induce the production of NO and various immunostimulatory cytokines, such as IL-6, IL12, and TNF-␣ [28]. The present results showed that the PSCJ significantly increases the production of NO and cytokines (IL-6 and IL-12) by primary cultured mouse peritoneal macrophages. NO can be used as a quantitative index of macrophage activation and has been shown to be the key molecule for killing pathogens and tumor cells [23]. Among the cytokines, IL-6 is one of the most important immune and inflammatory mediators that regulate diverse cell functions including proliferation and differentiation of T- and B-lymphocytes [29]. IL-12, which is mainly produced by activated macrophages, stimulates T-lymphocytes and NK cells. IL-12-mediated immunoregulation plays a pivotal role in cellmediated immunity against pathogens and tumors [30]. In our previous study, the PSCJ was found to stimulate the productions of NO and TNF-␣ in rIFN-␥-primed RAW 264.7 macrophages [12]. Taken together, these results may explain the immunostimulatory activities of the PSCJ on macrophages. To evaluate the immunostimulatory activity of the PSCJ on a normal mice model, we examined the increase in immune organ weights, phagocytic activity of macrophages, and mitogen-induced
Table 2 Effects of the PSCJ on immune functions in normal mice. Group
Number of animals
Phagocytic rate (%)a
Phagocytic indexb
Control PSCJ 100 mg/kg PSCJ 200 mg/kg
10 10 10
14.29 ± 3.90 19.63 ± 6.57 29.50 ± 9.17*
0.93 ± 0.17 1.18 ± 0.21 1.13 ± 0.17
Lymphocyte proliferationc T-lymphocyte
B-lymphocyte
0.26 ± 0.08 0.41 ± 0.07* 0.50 ± 0.09*
0.33 ± 0.09 0.44 ± 0.06* 0.53 ± 0.09*
The PSCJ (100 or 200 mg/kg) was administered daily for 28 days. The control group mice were treated with vehicle (normal saline solution). The data are presented as the means ± SD (n = 10). a The phagocytic rate was determined by counting the number of macrophages phagocytosing CRBCs in a population of 100 macrophages. b The phagocytic index was determined by counting the number of phagocytosed CRBCs per 100 macrophages. c The ConA-induced T-lymphocyte and LPS-induced B-lymphocyte proliferation was determined by the MTT assay. * p < 0.05 vs. the vehicle-treated control group.
524
C.-W. Cho et al. / International Journal of Biological Macromolecules 72 (2015) 519–525
Fig. 3. Effect of the PSCJ on a CY-induced immunosuppressed mice model. The mice were pretreated with CY (100 mg/kg for 3 days) for immunosuppression, and then orally administered the PSCJ (100 or 200 mg/kg for 14 days) or CVT (200 mg/kg for 14 days). CVT was used as a positive control. (A) The ConA-induced T-lymphocyte and LPS-induced B-lymphocyte proliferation was determined by the MTT assay. (B) The NK cell-mediated tumor cell cytotoxicity was determined in Yac-1 and primary cultured splenocytes from the treated mice. (C) The WBC number was measured using a semi-automatic blood cell analyzer. The data are expressed as the means ± SD (n = 10). ## p < 0.01 vs. vehicle-treated control group; **p < 0.01 vs. CY-treated immunosuppressed group.
T- and B-lymphocytes proliferation. The thymus and spleen, which are immune organs, play an important role in the body’s immune function. Their weights and organ indices are changed in response to the nonspecific immunity of the organism [31]. It has been reported that immune system activators can enhance the
weights of the thymus and spleen [32]. In this investigation, the relative spleen weights in the PSCJ-treated groups were significantly increased. This result supports the conclusion that the PSCJ stimulates the immune system. The immunostimulatory action of polysaccharides may begin with the activation of effector cells, including macrophages and lymphocytes. Therefore, we investigated the effects of the PSCJ on peritoneal macrophages and lymphocytes in the PSCJ-treated groups. Phagocytosis is the first step in the macrophage response to invading microorganisms, and the activation of phagocytosis elevates the innate immune response [32]. The present study showed that the PSCJ-treated groups enhanced the phagocytosis of peritoneal macrophages, suggesting that the PSCJ could enhance non-specific immune function. Splenocytes consist of various immune cells, including T- and B-lymphocytes, macrophages and dendritic cells [33]. To assess the immunostimulatory activity of the PSCJ on lymphocytes, we investigated the ConA- and LPS-induced splenocyte proliferation to evaluate the T- and B-lymphocyte proliferation activities. Lymphocyte proliferation is a pivotal event in the activation cascade of both cellular and humoral immune responses [34]. The PSCJtreated groups significantly increased the proliferation of T- and B-lymphocytes. In our previous study, we found that the PSCJ increases the proliferation, and IL-2 and IFN-␥ productions in primary cultured splenocytes [12]. Given these data, we speculated that the PSCJ exhibits immunostimulatory activity in lymphocytes. To evaluate the immunostimulatory activity of the PSCJ on a weakened immune system, we used the CY-treated mice model. It is well known that CY is an important chemotherapeutic drug in tumor treatment, but this compound has adverse effects on the immune system of an organism and leads to immunosuppression [35]. The decreased capacity of the immune functions such as lymphocyte proliferation, NK cell activity, and WBC counts in the CY-treated group in comparison to the vehicle-treated control group indicated that an immunosuppressed state was well established in our experiment. T- and B-lymphocyte proliferation is regarded as an indicator that reflects the cellular and humoral immunity state of an animal [36]. Treatment with CY inhibited T- and B-lymphocyte proliferation, and this effect was in line with previous results showing that CY treatment inhibits mitogen-stimulated lymphocyte proliferation [37]. In the present study, treatment with the PSCJ rescued the decrease in T- and Blymphocyte proliferation induced by CY treatment, which suggests a role for the PSCJ in the activation of lymphocytes. This result confirmed that the PSCJ-treated group can resist the CY-induced immunosuppression and exhibit enhanced cellular and humoral immune activities. As a type of lymphocyte, NK cells are involved in the early defense against external antigens, including various viruses and bacteria. NK cells also exhibit tumor cell killing activity. The group treated with 200 mg/kg PSCJ showed significantly higher toxicity to Yac-1 cells, a NK-sensitive mouse lymphoma cell line, compared to the CY-treated immunosuppressed group. Because IL12 is an important NK cell stimulatory factor, we thus speculated that the PSCJ-induced IL-12 production in macrophages may result in the enhancement of NK cell activity. Excessive CY administration is known to decrease the numbers of leukocytes and neutrophils by immunosuppression [38]. The number of WBC in the CY-treated group significantly decreased compared with the vehicle-treated control group, and the PSCJ-treated groups presented significantly increased WBC counts, thereby suggesting that the PSCJ can prevent CY-induced leukopenia. Because the reduction in the WBC count upon CY administration can cause severe immunodepression, various drugs have been developed to prevent leukopenia [39]. As such, the PSCJ has the potential to restore the immune activity inhibited by CY administration because it simultaneously increases lymphocyte proliferation, NK cell activity, and number of WBC in CY-treated immunosuppressed mice.
C.-W. Cho et al. / International Journal of Biological Macromolecules 72 (2015) 519–525
5. Conclusion In conclusion, the PSCJ exhibited potent immunostimulatory properties in vitro, ex vivo, and in vivo. The PSCJ was able to stimulate the complement system (in vitro), and the production of NO and immunostimulatory cytokines (IL-6 and IL-12) in primary cultured mouse peritoneal macrophages (ex vivo). The immunostimulatory activity of the PSCJ was also measured in normal and immunosuppressed mice models (in vivo). In the normal mice model, the PSCJ treatment increased the weight of immune organs (spleen and thymus), and improved the phagocytic rates of peritoneal macrophages and lymphocytes proliferation. Similarly, the PSCJ markedly restored the decreased lymphocyte proliferation, NK cell activity, and WBC count in the CY-induced immunosuppressed mice model. Taken together, we speculate that the PSCJ could be utilized as an effective immunostimulatory agent and that the PSCJ may have therapeutic potential in patients with inadequate immune functions. Acknowledgement This work was supported by research grants from the Korea Food Research Institute (Project No. E0132101). References [1] K.P. Mishra, Y.S. Padwad, M. Jain, D. Karan, L. Ganju, R.C. Sawhney, Immunopharmacol. Immunotoxicol. 28 (2006) 201–212. [2] T. Zhao, Y. Feng, J. Li, R. Mao, Y. Zou, W. Feng, D. Zheng, W. Wang, Y. Chen, L. Yang, X. Wu, Int. J. Biol. Macromol. 65 (2014) 33–40. [3] M.Y. Leung, C. Liu, J.C. Koon, K.P. Fung, Immunol. Lett. 105 (2006) 101–114. [4] M.H. Jiang, L. Zhu, J.G. Jiang, Expert Opin. Ther. Targets 14 (2010) 1367–1402. [5] M.J. Bae, H.S. Shin, H.J. See, O.H. Chai, D.H. Shon, J. Med. Food 17 (2014) 142–149. [6] I.S. Hwang, J.E. Kim, Y.J. Lee, M.H. Kwak, J. Go, H.J. Son, D.S. Kim, D.Y. Hwang, Nutr. Res. 34 (2014) 355–367. [7] J. Kim, J.N. Choi, J.H. Choi, Y.S. Cha, M.J. Muthaiya, C.H. Lee, Mol. Nutr. Food Res. 57 (2013) 1886–1891. [8] Y.J. Lee, J.E. Kim, M.H. Kwak, J. Go, D.S. Kim, H.J. Son, D.Y. Hwang, Int. J. Mol. Med. 33 (2014) 1185–1194. [9] Y.C. Hwang, K.S. Shin, Korean J. Food Sci. Technol. 40 (2008) 220–227. [10] H.R. Park, M.S. Lee, S.Y. Jo, H.J. Won, H.S. Lee, H. Lee, K.S. Shin, Korean J. Food Sci. Technol. 44 (2012) 228–234.
525
[11] C.W. Cho, C.J. Han, Y.K. Rhee, Y.C. Lee, K.S. Shin, H.D. Hong, Molecules 19 (2014) 5266–5277. [12] S.J. Lee, H.K. Rim, J.Y. Jung, H.J. An, J.S. Shin, C.W. Cho, Y.K. Rhee, H.D. Hong, K.T. Lee, Food Chem. Toxicol. 59 (2013) 476–484. [13] E.A. Kabat, M.M. Mayer, Experimental Immunochemistry, second ed., Charles C. Thomas, Springfield, IL, 1964. [14] H. Saito, H. Tomioka, K. Sato, J. Gen. Microbiol. 134 (1988) 1029–1035. [15] I. Saiki, S. Saito, C. Fujita, H. Ishida, J. Iida, J. Murata, A. Hasegawa, I. Azuma, Vaccine 6 (1988) 238–244. [16] M. Wang, L.J. Guilbert, L. Ling, J. Li, Y. Wu, S. Xu, P. Pang, J.J. Shan, J. Pharm. Pharmacol. 53 (2001) 1515–1523. [17] X.Y. Wu, G.H. Mao, Q.Y. Fan, T. Zhao, J.L. Zhao, F. Li, L.Q. Yang, Food Res. Int. 48 (2012) 935–939. ´ V. Juri´sic, ´ I. Spuzi ´ c, ´ J. Immunol. Methods 200 (1997) 199–201. [18] G. Konjevic, [19] S.H. Bae, J.W. Choi, K.S. Ra, K.W. Yu, K.S. Shin, S.S. Park, H.J. Suh, J. Sci. Food Agric. 92 (2012) 1765–1770. [20] V.B. Liju, K. Jeena, R. Kuttan, Food Chem. Toxicol. 53 (2013) 52–61. [21] F. Cerqueira, A. Cordeiro-Da-Silva, C. Gaspar-Marques, F. Simões, M.M. Pinto, M.S. Nascimento, Bioorg. Med. Chem. 12 (2004) 217–223. [22] Y.M. Ha, S.H. Chun, S.T. Hong, Y.C. Koo, H.D. Choi, K.W. Lee, Int. J. Biol. Macromol. 60 (2013) 399–404. [23] H.Y. Park, A.R. Yu, I.W. Choi, H.D. Hong, K.W. Lee, H.D. Choi, Int. Immunopharmacol. 17 (2013) 191–197. [24] A.O. Tzianabos, Clin. Microbiol. Rev. 13 (2000) 523–533. [25] V.M. Holers, Clin. Immunol. 107 (2003) 140–151. [26] S.H. Bae, E.Y. Jung, S.Y. Kim, K.S. Shin, H.J. Suh, J. Food Biochem. 34 (2010) 233–248. [27] V.E. Ooi, F. Liu, Curr. Med. Chem. 7 (2000) 715–729. [28] H. Wang, J.K. Actor, J. Indrigo, M. Olsen, A. Dasgupta, Clin. Chim. Acta 327 (2003) 123–128. [29] R.M. Sobota, P.J. Müller, C. Khouri, A. Ullrich, V. Poli, T. Noguchi, P.C. Heinrich, F. Schaper, Cell Signal. 20 (2008) 1385–1391. [30] S.J. Kim, J.S. Park, N.Y. Myung, P.D. Moon, I.Y. Choi, H.J. An, N.H. Kim, H.J. Na, D.H. Kim, M.C. Kim, N.H. An, I.K. Kim, J.Y. Lee, H.J. Jeong, J.Y. Um, H.M. Kim, S.H. Hong, Immunopharmacol. Immunotoxicol. 31 (2009) 246–252. [31] H.L. Cui, Y. Chen, S.S. Wang, G.Q. Kaia, Y.M. Fang, J. Sci. Food Agric. 91 (2011) 2180–2185. [32] X. Chen, W. Nie, S. Fan, J. Zhang, Y. Wang, J. Lu, L. Jin, Carbohydr. Polym. 90 (2012) 1114–1119. [33] T.T. Kawabata, K.L. White, Cancer Res. 47 (1987) 2317–2322. [34] C. Zhao, M. Li, Y. Luo, W. Wu, Carbohydr. Res. 341 (2006) 485–491. [35] H. Wang, M.Y. Wang, J. Chen, Y. Tang, J. Dou, J. Yu, Int. Immunopharmacol. 11 (2011) 1946–1953. [36] J.W. Hadden, Int. Immunopharmacol. 3 (2003) 1061–1071. [37] J. Moynihan, N. Cohen, Int. J. Immunopharmacol. 11 (1989) 517–527. [38] I. Shalit, Y. Kletter, D. Halperin, D. Waldman, E. Vasserman, A. Nagler, I. Fabian, Eur. J. Haematol. 66 (2001) 287–296. [39] H. Zhou, F. Sun, H. Li, S. Zhang, Z. Liu, J. Pei, C. Liang, Immunopharmacol. Immunutoxicol. 35 (2013) 426–433.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Cheonggukjang polysaccharides enhance immune activities and prevent cyclophosphamide-induced immunosuppression Chang-Won Cho a , Chun-ji Han b , Young Kyoung Rhee a , Young-Chul Lee a , Kwang-Soon Shin c , Ji-Sun Shin d , Kyung-Tae Lee d , Hee-Do Hong a,∗ a
Division of Strategic Food Research, Korea Food Research Institute, Gyeonggi 463-746, Republic of Korea Department of Preventive Medicine, Yanbian University, Jilin 133002, China c Department of Food Science and Biotechnology, Kyonggi University, Gyeonggi 443-760, Republic of Korea d Department of Pharmaceutical Biochemistry, Kyung Hee University, Seoul 130-701, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 9 April 2014 Accepted 8 September 2014 Available online 16 September 2014 Keywords: Cheonggukjang Polysaccharide Immunostimulatory activity
a b s t r a c t Cheonggukjang is a traditional Korean fermentation product prepared from soybean that is reported to have various biological functions. We previously reported that the polysaccharides from Cheonggukjang (PSCJ) have immunostimulatory activities in RAW 264.7 macrophages and primary cultured splenocytes. In this study, the immunostimulatory activities of the PSCJ were investigated further using various experimental models such as in vitro, ex vivo, and in vivo. The PSCJ was able to stimulate the complement system (ITCH50 : 30.6%). In primary cultured mouse peritoneal macrophages, the PSCJ was found to significantly increase nitric oxide and immunostimulatory cytokines (IL-6 and IL-12) production in a concentration-dependent manner (1–100 g/mL). In the normal mice model, the oral administration of the PSCJ increased the weight of spleen (p < 0.05 at 100 and 200 mg/kg) and improved the phagocytic rates of peritoneal macrophages (p < 0.05 at 200 mg/kg) and lymphocytes proliferation (p < 0.05 at 100 and 200 mg/kg). Similarly, the PSCJ markedly restored the decreased lymphocytes proliferation (p < 0.01 at 200 mg/kg), natural killer cell activity (p < 0.01 at 200 mg/kg), and white blood cell count (p < 0.01 at 100 and 200 mg/kg) in the cyclophosphamide-induced immunosuppressed mice model. These results suggest that the PSCJ could be utilized as an effective immunostimulatory agent. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The modulation of immune response to prevent diseases has long been one of the primary concerns of many researchers [1]. It was recently proven that the use of immunomodulators to enhance the host defense responses can be an effective way to increase resistance to disease [2]. Polysaccharides separated from natural substances including food ingredients and medicinal plants have been regarded as important immunostimulant candidates due to their broad spectrum of immunostimulatory activities with relatively low toxicity and adverse effects [3]. Polysaccharides from natural sources have been known to improve the immune functions of the body by activating immune-related cells, such as
∗ Corresponding author at: Division of Strategic Food Research, Korea Food Research Institute, 1201-62 Anyangpangyo-ro, Bundang, Seongnam, Gyeonggi 463746, Republic of Korea. Tel.: +82 31 780 9285; fax: +82 31 709 9876. E-mail address: [email protected] (H.-D. Hong). http://dx.doi.org/10.1016/j.ijbiomac.2014.09.010 0141-8130/© 2014 Elsevier B.V. All rights reserved.
macrophages, lymphocytes, and natural killer (NK) cells, and by promoting cytokine secretion, antibody production, and activation of the complement system [4]. Therefore, many natural polysaccharides are receiving increasing attention in the fields of therapeutics and functional foods. Cheonggukjang is a traditional Korean fermentation product prepared from soybean and is also called Natto, Tempeh, and Douchi in other Asian countries [5]. Cheonggukjang is manufactured by the short-term fermentation of soybeans using Bacillus subtilis, which contains many enzymes, microorganisms, and bioactive compounds that are absent from unfermented soybeans [6]. Cheonggukjang is known for its functionality, including antioxidant, anti-inflammatory, anti-hypertensive, and anti-diabetic activities [7], in addition to its nutritional value. Most previous functionality studies of Cheonggukjang used the whole product or a crude extract [5–8]. However, few studies have reported the activity of polysaccharides from Cheonggukjang. In the studies that investigated the biological activities of polysaccharides prepared from the traditional Korean fermentation products, it was reported
520
C.-W. Cho et al. / International Journal of Biological Macromolecules 72 (2015) 519–525
that the polysaccharides from persimmon vinegar, soy sauce, and makgeolli, which is traditional Korean rice wine, have an effect on immune activity [9–11]. In a previous study, we isolated and characterized polysaccharides from Cheonggukjang (PSCJ), and investigated their immunostimulatory activity and underlying molecular mechanisms on recombinant interferon (rIFN)-␥-primed RAW 264.7 macrophages and primary cultured splenocytes [12]. To further investigate the immunostimulatory activities of the PSCJ, the present study explores the effect of the PSCJ on the complement system and primary cultured peritoneal macrophages and subsequently in normal mice and cyclophosphamide (CY)-induced immunosuppressed mice models. 2. Materials and methods 2.1. Materials and chemicals Soybeans were purchased from Namboeun NongHyup (Boeun, Korea) and commercial Cheonggukjang starter culture (B. subtilis 10% and soybeans 90%) was purchased from NUC Corp. (Daegu, Korea). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), concanavalin A (ConA), dimethyl sulfoxide (DMSO), Griess reagent, lipopolysaccharide (LPS), phosphate buffered saline (PBS), thioglycollate, and trypan blue solution (0.4%) were purchased from Sigma–Aldrich (St. Louis, MO, USA). CVT-E002TM (sold commercially as COLD-FX® ) was purchased from Afexa Life Sciences Inc. (Edmonton, AB, Canada). Rosewell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from GIBCO BRL (Grand Island, NY, USA). Enzyme linked immunosorbent assay (ELISA) kits for interleukin (IL)-6 and IL-12 were obtained from BD Biosciences (San Diego, CA, USA). Cyclophosphamide (CY) was purchased from Jiangsu Heungrui Medicine Co., Ltd. (Lianyungang, China). 2.2. Preparation of the PSCJ Whole soybeans (1000 g) were washed five to six times and soaked with filtered water at 4 ◦ C for 24 h and then steamed for 40 min at 121 ◦ C. The steamed soybeans were maintained at 50 ◦ C to allow them to cool down. The cooked soybeans were then inoculated with 0.5% (w/w) Cheonggukjang starter culture and fermented for 48 h at 40 ◦ C in an incubator. The Cheonggukjang samples were freeze-dried and stored at −20 ◦ C. Lyophilized Cheonggukjang powder was extracted with 10 volumes of distilled water at 90 ◦ C for 3 h. The extracts were centrifuged at 6500 × g for 20 min and filtered through Advantec filter paper No. 2 (5 m, Toyo Roshi Kaisha, Ltd., Tokyo, Japan) to remove any insoluble particles. Polysaccharides were precipitated from the extracts by the addition of four volumes of 95% ethanol. The precipitate was dissolved in distilled water and dialyzed using Spectra/Por (MWCO; 6000–8000, Spectrum Laboratories Inc., Rancho Dominquez, CA, USA) for three days. Finally, the high-molecular-weight solution was lyophilized. The lyophilized powder (denoted as PSCJ) was stored in the dark at 4 ◦ C. 2.3. Anti-complementary activity of the PSCJ (in vitro) The anti-complementary activity was measured by the complement fixation test based on complement consumption and the degree of red blood cell lysis by residual complement [13]. Normal human serum (NHS) was obtained from volunteer adults. A 50 L aliquot of 1000 g/mL PSCJ was mixed with equal volumes of NHS and gelatin veronal-buffered saline (GVB2+ , pH 7.4) containing 500 mM Mg2+ and 150 mM Ca2+ , respectively. The mixtures were pre-incubated at 37 ◦ C for 30 min and the residual
total hemolytic complement (TCH50 ) was determined using IgM hemolysin-sensitized sheep erythrocytes (1 × 108 cells/mL). The NHS was incubated with water and GVB2+ to provide a control. The anti-complementary activity of the PSCJ is expressed as the percent inhibition of the control TCH50 (ITCH50 ) of polysaccharide K (PSK), which is a known immunoactive polysaccharide from Coriolus versicolor that was used as a positive control [14]. ITCH50 (%) = [TCH50 (control) − TCH50 (treated with sample)]/TCH50 (control) × 100. 2.4. Immunostimulatory activity of the PSCJ on primary cultured mouse peritoneal macrophages (ex vivo) 2.4.1. Primary culture and cytotoxicity test Specific pathogen-free (SPF), 5-week-old male C57BL/6 mice were purchased from Hanlim Experimental Research Institute (Hwaseong, Korea). The peritoneal macrophages of C57BL/6 mice were harvested from 5% thioglycollate-treated mice as described previously [15]. The peritoneal macrophages were resuspended in RPMI 1640–FBS (containing 10% FBS) medium and then plated into 96 well-culture plates (2 × 105 cells/well). After incubation for 2 h, the non-adherent cells were removed by PBS washing, and the adherent macrophages were stimulated with various concentrations of the PSCJ for 24 h. The macrophages were incubated with a MTT solution for 4 h at 37 ◦ C under 5% CO2 . The MTT-containing medium was removed, and the cells were solubilized in DMSO. The absorbance of each well at 540 nm was measured using a microplate reader (TECAN, Männedorf, Switzerland). 2.4.2. Quantification of nitric oxide and cytokine production Peritoneal macrophages were seeded in 96-well plates (2 × 105 cells/well) and stimulated with various concentrations of the PSCJ. After 24 h, the culture supernatants were collected, and the nitric oxide (NO) concentration was measured using the Griess reagent. Equal volumes of the Griess reagent (1% sulfanilamide, 0.1% naphthylenediamine dihydrochloride, and 0.5% H3 PO4 ) and the sample were incubated together at room temperature for 10 min. The absorbance at 540 nm was measured using a microplate reader (TECAN). The concentrations of cytokines (IL-6 and IL-12) in the culture supernatants were determined using ELISA kits according to the manufacturer’s protocol. 2.5. Immunostimulatory activity of the PSCJ in vivo 2.5.1. Animals SPF KM male mice (7–8 weeks of age) were obtained from the Experimental Animal Center of Yanbian University College of Medicine (Yanji, China). The mice were maintained under constant conditions (temperature: 22 ± 2 ◦ C, humidity: 40–60%, light and dark cycle: 12 h) and allowed free access to water and food. All of the animal experiments were performed according to the instructions of the Medical Ethics Committee for the Use of Experimental Animals at Yanbian University. 2.5.2. Animal experiments The animal experiments were conducted using both normal and immunosuppressed mice. For the animal experiments with normal mice, the mice were randomly divided into three groups (ten mice in each group). From day 1 to 28, the three different groups of mice were orally treated with saline or the PSCJ at 100 and 200 mg/kg body weight on a daily basis. Twenty-four hours after the last administration, the mice were weighed and sacrificed by cervical dislocation. The spleen and thymus were immediately removed and weighted. The spleen and thymus index were calculated as the spleen and thymus weight/body weight, respectively. The spleen sample was used for lymphocyte proliferation.
C.-W. Cho et al. / International Journal of Biological Macromolecules 72 (2015) 519–525
For the experiments with the immunosuppressed mice, the mice were randomly divided into five groups (ten mice in each group). One group of mice was used as a vehicle-treated control without any treatment for immunosuppression. The other four groups of mice were subjected to immunosuppression through intraperitoneal treatment with CY (100 mg/kg/day) from day 1 to 3. Among these groups, one group of CY-treated mice was used as an immunosuppressed model group. CVT-E002TM (CVT), an immunostimulatory polysaccharide-rich extract of North American ginseng (Panax quinquefolium), was used as a positive control [16]. From day 4 to 17, the five different groups of mice were administered the following: vehicle-treated control group, saline; immunosuppressed model group, saline; positive control group, 200 mg/kg body weight of CVT; and the PSCJ groups, 100 or 200 mg/kg body weight of the PSCJ. All of the mice were treated daily by oral administration. Twenty-four hours after the last administration, blood was collected from the mice for white blood cell (WBC) counts. The mice were subsequently weighed and sacrificed by cervical dislocation. The collected spleen samples were used to measure the lymphocyte proliferation and NK cell activity. The WBC number was measured using a semi-automatic blood cell analyzer (KCTH04, Guangzhou Kingcare Medical Equipment Co., Ltd., Guangzhou, China). 2.5.3. Phagocytic activity assay of peritoneal macrophages Twenty-four hours after the last administration, the mice were injected intraperitoneally with 1 mL of 20% chicken red blood cells (CRBCs). Thirty minutes later, the mice were killed by cervical dislocation, and 2 mL of PBS was used for peritoneal lavage. The cell suspension was smeared on glass slides and incubated for 30 min at 37 ◦ C in a humidified atmosphere with 5% CO2 . After incubation, the non-phagocytosed CRBCs and other cells were removed by washing with PBS. The macrophages were fixed with acetone:methanol (1:1, v/v) and stained with Giemsa solution. The phagocytic rate was determined by counting the number of macrophages phagocytosing CRBCs in a population of 100 macrophages. The phagocytic index was determined by counting the number of phagocytosed CRBCs per 100 macrophages [17]. 2.5.4. Lymphocyte proliferation assay The spleens obtained from the mice sacrificed under aseptic conditions were washed with RPMI 1640 medium and crushed to isolate the splenocytes. The splenocytes were passed through a 200-mesh stainless steel sieve to obtain a homogeneous cell suspension. The splenocyte suspension was washed twice with RPMI 1640–FBS medium and with centrifugation at 1000 rpm for 5 min. The recovered splenocytes were resuspended in Trisbuffered ammonium chloride solution (NH4 Cl, pH 7.2) for 5 min to remove the red blood cells. After centrifugation, the harvested splenocytes were resuspended in RPMI 1640–FBS medium, and the cell numbers were measured with a hemocytometer using trypan blue dye exclusion. The splenocytes were seeded in 96-well plates (3.0 × 105 cells/well) for the lymphocytes proliferation assays with ConA (5 g/mL) or LPS (10 g/mL). After incubation, MTT solution was added to each well, and the plates was incubated for 4 h. DMSO solution was added to resolve the formazan. The absorbance was measured in a microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 540 nm. 2.5.5. NK cell-mediated cytotoxicity assay NK-mediated cytotoxicity was measured in YAC-1 cells (NKsensitive cell line) and primary cultured splenocytes from the PSCJ-treated mice. Splenocyte suspensions were added to the YAC1 cells (1 × 105 cells/mL) to obtain an effector-to-target cell ratio of 50:1 in 96-well U-bottom culture plates. The cells were then incubated at 37 ◦ C in a humidified atmosphere containing 5% CO2 for 4 h. Following incubation, the culture supernatants were mixed
521
Fig. 1. Anti-complementary activities of the PSCJ. The anti-complementary activity was expressed as the inhibition of 50% total complement hemolysis by Mayer’s method [13]. The concentrations of PSK and PSCJ were 1000 g/mL. PSK was used as a positive control. The data are expressed as the means ± SD of three separate experiments. ***p < 0.001 vs. control.
with a lactate dehydrogenase (LDH) solution (Roche, Mannheim, Germany), and the absorbance value of each well was measured at 490 nm [18]. The NK cell cytotoxicity (%) = [(experimental release − spontaneous release)/(maximum release − spontaneous release)] × 100. 2.6. Statistical analysis All of the statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) version 17.0 (SPSS Inc., Chicago, IL, USA). The quantitative data are expressed as the means ± SD. All statistical comparisons were performed using a one-way ANOVA test followed by Duncan’s multiple range tests. p-Values less than 0.05 were considered statistically significant. 3. Results 3.1. Effect of the PSCJ on the complement system activation The chemical composition of the PSCJ was reported in our previous report, and it was proven that the immunostimulatory effect of the PSCJ did not result from LPS contamination [12]. The PSCJ was consisted of protein (6.52%) and polysaccharide (93.48%, w/w), and polysaccharide was shown to be the neutral (24.02%) and acidic (69.46%) polysaccharide. Neutral polysaccharides in the PSCJ mainly comprised of galactose (39.9 mole%), arabinose (31.9 mole%), glucose (8.8 mole%), xylose (8.2 mole%), rhamnose (5.7 mole%), fucose (3.3 mole%), and mannose (2.2 mole%) [12]. The complement system plays an important role in host defense, inflammation, and allergic reactions. Thus, anti-complementary activity is considered an immunomodulating activity that is responsible for immunological defense [19]. To evaluate the biological activity of the PSCJ as it relates to the complement cascade reaction, the anti-complementary activity was tested. As shown in Fig. 1, the anti-complementary activity of the PSCJ was 30.3%. PSK was used as a positive control. 3.2. Effect of the PSCJ on primary cultured mouse peritoneal macrophage To determine whether the immunostimulatory activity effects of the PSCJ observed in RAW 264.7 macrophages also occur in
522
C.-W. Cho et al. / International Journal of Biological Macromolecules 72 (2015) 519–525
Fig. 2. Effects of the PSCJ on NO, IL-6, and IL-12 productions in primary cultured mouse peritoneal macrophages. Mouse peritoneal macrophages were stimulated with the PSCJ (1, 10, or 100 g/mL) or LPS (2 g/mL) for 24 h. LPS was used as a positive control. (A) NO production was determined by measuring the nitrite accumulation in the culture medium. (B and C) The amount of IL-6 and IL-12 was measured by ELISA assay. (D) The cell viability was determined by the MTT assay. The data are expressed as the means ± SD of three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control.
primary cells, we examined the effect of the PSCJ on the productions of NO and cytokines, such as IL-6 and IL-12 in peritoneal macrophages isolated from C57BL/6 mice. In these cells, the PSCJ was found to significantly increase the secretion of NO, IL-6, and IL-12 in a concentration-dependent manner (Fig. 2A–C). LPS was used as a positive control for NO and cytokine productions. When we examined the toxicity of the PSCJ on primary cultured mouse peritoneal macrophages using the MTT assay, it was found that the PSCJ (1–100 g/mL) did not affect the cell viability compared to the control cells (Fig. 2D).
3.3. Immunostimulatory effects of the PSCJ on normal mice The PSCJ was orally administered at doses of 100 and 200 mg/kg to normal mice, and its effects on the body mass gain and the weights of immune organs, such as the spleen and thymus, are shown in Table 1. Commonly, abnormal alterations in body weight have been used as indicators of the toxic effects of testing samples [20]. The ratios of the body mass gain were not significantly changed in the PSCJ-treated groups, suggesting that the PSCJ did not have adverse effects on the mice. We found that the spleen index was significantly increased in the PSCJ-treated groups (p < 0.05) compared with that found in the vehicle-treated control group. In addition, the thymus index tended to increase in the PSCJ-treated
groups compared with the control group, but the difference was not statistically significant. The phagocytic activity of macrophages in the PSCJ-treated groups was evaluated by the CRBCs uptake. The phagocytic rate and index obtained for the PSCJ-treated groups are presented in Table 2. The phagocytic rate was significantly increased in the 200 mg/kg PSCJ-treated group (p < 0.05). The phagocytic index was slightly increased, but the difference was not statistically significant. Because it is well known that ConA and LPS stimulate Tand B-lymphocyte proliferation [21], we tested whether the PSCJ influences the lymphocyte proliferation induced by ConA or LPS. As shown in Table 2, the PSCJ significantly increased the T- and Blymphocytes proliferation compared to the vehicle-treated control group, which suggests that the PSCJ is a direct mitogen for mouse lymphocytes. 3.4. Immunostimulatory effects of the PSCJ on cyclophosphamide-treated immunosuppressed mice To evaluate the effects of the PSCJ on cyclophosphamide (CY)-induced immunosuppression, we measured the lymphocyte proliferation, NK cell activity, and WBC counts. The intraperitoneal treatment of CY (100 mg/kg) inhibited the T- and B-lymphocyte proliferation, NK cell activity, and WBC counts (Fig. 3). As shown in Fig. 3A, the PSCJ significantly enhanced the proliferation of
C.-W. Cho et al. / International Journal of Biological Macromolecules 72 (2015) 519–525
523
Table 1 Effects of the PSCJ on immune organs in normal mice. Group
Number of animals
Initial body weight (g)
Final body weight (g)
Weight growth rate (%)
Spleen index (SI)a
Thymus index (TI)b
Control PSCJ 100 mg/kg PSCJ 200 mg/kg
10 10 10
22.00 ± 1.79 21.60 ± 1.84 22.60 ± 1.90
35.50 ± 4.38 33.50 ± 2.98 36.00 ± 4.00
61.36 55.09 59.29
0.65 ± 0.08 0.73 ± 0.06* 0.74 ± 0.03*
0.35 ± 0.13 0.38 ± 0.12 0.43 ± 0.13
The PSCJ (100 or 200 mg/kg) was administered daily for 28 days. The control group mice were treated with vehicle (normal saline solution). The data are presented as the means ± SD (n = 10). a Spleen weight/body weight. b Thymus weight/body weight. * p < 0.05 vs. the vehicle-treated control group.
T- and B-lymphocytes compared with those observed in the CY-treated immunosuppressed group. The administration of CY significantly decreased the activity of NK cells compared with the vehicle-treated control group. However, the activity of NK cells in the 200 mg/kg PSCJ-treated groups was significantly increased compared with that in the CY-treated immunosuppressed group (Fig. 3B). Additionally, the WBC counts were significantly restored in the PSCJ-treated groups compared with the CY-treated immunosuppressed groups (Fig. 3C). CVT, a positive control, also increased the T- and B-lymphocyte proliferation, NK cell activity, and WBC counts.
4. Discussion The immune system protects the organism from infectious disease and tumor through a layered defense with increasing specificity [22]. The role and necessity of the immune system are prominent during chemotherapeutic intervention for the treatment of many diseases [23]. In recent years, the use of immunomodulators to strengthen the host defense responses has been considered one of the most promising alternatives to classical drug treatment [24]. In a previous study, we isolated and characterized the PSCJ and showed its immunostimulatory activities [12]. The PSCJ exerts immunostimulatory activity resulting from nuclear factor-B (NFB) activation and thereby increases the gene expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-␣ in rIFN-␥-primed RAW 264.7 macrophages. The PSCJ also increases the proliferation, and IL-2 and IFN-␥ productions in primary cultured splenocytes. In addition, The PSCJ decreases immobility time and indicators related to fatigue in the forced swimming test animal model. In the present study, we attempted to further evaluate the immunostimulatory activities of the PSCJ, including its anticomplementary and peritoneal macrophage activation activities as well as its immune function enhancing activities in a normal mice model. We also investigated the protective effects of the PSCJ in immunosuppression caused by CY treatment.
The complement system plays an important role as a primary defense against bacterial and viral infections and is activated via the classical and alternative pathways [25]. Complement activation appears to be intrinsically associated with several immune reactions, including the activation of macrophages, lymphocytes, and immunopotentiation [26]. The anti-complementary activity of the PSCJ was measured by the complement fixation test. The PSCJ showed about 30% anti-complementary activity, whereas the known immunoactive polysaccharide Krestin (PSK), a -glucan from Coriolus versicolor [14] used as a positive control, showed 60% activity. However, the anti-tumor activity of PSK was evaluated in Japan for the prevention of esophageal, gastric and lung cancers in humans with promising results, and PSK is currently sold as a drug [27]. Thus, even though the activity of the PSCJ was about the half of that of PSK, our findings still suggest that the PSCJ is likely to be an effective complement activator. The activation of macrophages plays a key role in innate immunity for initiating and propagating defensive reactions against pathogens [1]. Activated macrophages induce the production of NO and various immunostimulatory cytokines, such as IL-6, IL12, and TNF-␣ [28]. The present results showed that the PSCJ significantly increases the production of NO and cytokines (IL-6 and IL-12) by primary cultured mouse peritoneal macrophages. NO can be used as a quantitative index of macrophage activation and has been shown to be the key molecule for killing pathogens and tumor cells [23]. Among the cytokines, IL-6 is one of the most important immune and inflammatory mediators that regulate diverse cell functions including proliferation and differentiation of T- and B-lymphocytes [29]. IL-12, which is mainly produced by activated macrophages, stimulates T-lymphocytes and NK cells. IL-12-mediated immunoregulation plays a pivotal role in cellmediated immunity against pathogens and tumors [30]. In our previous study, the PSCJ was found to stimulate the productions of NO and TNF-␣ in rIFN-␥-primed RAW 264.7 macrophages [12]. Taken together, these results may explain the immunostimulatory activities of the PSCJ on macrophages. To evaluate the immunostimulatory activity of the PSCJ on a normal mice model, we examined the increase in immune organ weights, phagocytic activity of macrophages, and mitogen-induced
Table 2 Effects of the PSCJ on immune functions in normal mice. Group
Number of animals
Phagocytic rate (%)a
Phagocytic indexb
Control PSCJ 100 mg/kg PSCJ 200 mg/kg
10 10 10
14.29 ± 3.90 19.63 ± 6.57 29.50 ± 9.17*
0.93 ± 0.17 1.18 ± 0.21 1.13 ± 0.17
Lymphocyte proliferationc T-lymphocyte
B-lymphocyte
0.26 ± 0.08 0.41 ± 0.07* 0.50 ± 0.09*
0.33 ± 0.09 0.44 ± 0.06* 0.53 ± 0.09*
The PSCJ (100 or 200 mg/kg) was administered daily for 28 days. The control group mice were treated with vehicle (normal saline solution). The data are presented as the means ± SD (n = 10). a The phagocytic rate was determined by counting the number of macrophages phagocytosing CRBCs in a population of 100 macrophages. b The phagocytic index was determined by counting the number of phagocytosed CRBCs per 100 macrophages. c The ConA-induced T-lymphocyte and LPS-induced B-lymphocyte proliferation was determined by the MTT assay. * p < 0.05 vs. the vehicle-treated control group.
524
C.-W. Cho et al. / International Journal of Biological Macromolecules 72 (2015) 519–525
Fig. 3. Effect of the PSCJ on a CY-induced immunosuppressed mice model. The mice were pretreated with CY (100 mg/kg for 3 days) for immunosuppression, and then orally administered the PSCJ (100 or 200 mg/kg for 14 days) or CVT (200 mg/kg for 14 days). CVT was used as a positive control. (A) The ConA-induced T-lymphocyte and LPS-induced B-lymphocyte proliferation was determined by the MTT assay. (B) The NK cell-mediated tumor cell cytotoxicity was determined in Yac-1 and primary cultured splenocytes from the treated mice. (C) The WBC number was measured using a semi-automatic blood cell analyzer. The data are expressed as the means ± SD (n = 10). ## p < 0.01 vs. vehicle-treated control group; **p < 0.01 vs. CY-treated immunosuppressed group.
T- and B-lymphocytes proliferation. The thymus and spleen, which are immune organs, play an important role in the body’s immune function. Their weights and organ indices are changed in response to the nonspecific immunity of the organism [31]. It has been reported that immune system activators can enhance the
weights of the thymus and spleen [32]. In this investigation, the relative spleen weights in the PSCJ-treated groups were significantly increased. This result supports the conclusion that the PSCJ stimulates the immune system. The immunostimulatory action of polysaccharides may begin with the activation of effector cells, including macrophages and lymphocytes. Therefore, we investigated the effects of the PSCJ on peritoneal macrophages and lymphocytes in the PSCJ-treated groups. Phagocytosis is the first step in the macrophage response to invading microorganisms, and the activation of phagocytosis elevates the innate immune response [32]. The present study showed that the PSCJ-treated groups enhanced the phagocytosis of peritoneal macrophages, suggesting that the PSCJ could enhance non-specific immune function. Splenocytes consist of various immune cells, including T- and B-lymphocytes, macrophages and dendritic cells [33]. To assess the immunostimulatory activity of the PSCJ on lymphocytes, we investigated the ConA- and LPS-induced splenocyte proliferation to evaluate the T- and B-lymphocyte proliferation activities. Lymphocyte proliferation is a pivotal event in the activation cascade of both cellular and humoral immune responses [34]. The PSCJtreated groups significantly increased the proliferation of T- and B-lymphocytes. In our previous study, we found that the PSCJ increases the proliferation, and IL-2 and IFN-␥ productions in primary cultured splenocytes [12]. Given these data, we speculated that the PSCJ exhibits immunostimulatory activity in lymphocytes. To evaluate the immunostimulatory activity of the PSCJ on a weakened immune system, we used the CY-treated mice model. It is well known that CY is an important chemotherapeutic drug in tumor treatment, but this compound has adverse effects on the immune system of an organism and leads to immunosuppression [35]. The decreased capacity of the immune functions such as lymphocyte proliferation, NK cell activity, and WBC counts in the CY-treated group in comparison to the vehicle-treated control group indicated that an immunosuppressed state was well established in our experiment. T- and B-lymphocyte proliferation is regarded as an indicator that reflects the cellular and humoral immunity state of an animal [36]. Treatment with CY inhibited T- and B-lymphocyte proliferation, and this effect was in line with previous results showing that CY treatment inhibits mitogen-stimulated lymphocyte proliferation [37]. In the present study, treatment with the PSCJ rescued the decrease in T- and Blymphocyte proliferation induced by CY treatment, which suggests a role for the PSCJ in the activation of lymphocytes. This result confirmed that the PSCJ-treated group can resist the CY-induced immunosuppression and exhibit enhanced cellular and humoral immune activities. As a type of lymphocyte, NK cells are involved in the early defense against external antigens, including various viruses and bacteria. NK cells also exhibit tumor cell killing activity. The group treated with 200 mg/kg PSCJ showed significantly higher toxicity to Yac-1 cells, a NK-sensitive mouse lymphoma cell line, compared to the CY-treated immunosuppressed group. Because IL12 is an important NK cell stimulatory factor, we thus speculated that the PSCJ-induced IL-12 production in macrophages may result in the enhancement of NK cell activity. Excessive CY administration is known to decrease the numbers of leukocytes and neutrophils by immunosuppression [38]. The number of WBC in the CY-treated group significantly decreased compared with the vehicle-treated control group, and the PSCJ-treated groups presented significantly increased WBC counts, thereby suggesting that the PSCJ can prevent CY-induced leukopenia. Because the reduction in the WBC count upon CY administration can cause severe immunodepression, various drugs have been developed to prevent leukopenia [39]. As such, the PSCJ has the potential to restore the immune activity inhibited by CY administration because it simultaneously increases lymphocyte proliferation, NK cell activity, and number of WBC in CY-treated immunosuppressed mice.
C.-W. Cho et al. / International Journal of Biological Macromolecules 72 (2015) 519–525
5. Conclusion In conclusion, the PSCJ exhibited potent immunostimulatory properties in vitro, ex vivo, and in vivo. The PSCJ was able to stimulate the complement system (in vitro), and the production of NO and immunostimulatory cytokines (IL-6 and IL-12) in primary cultured mouse peritoneal macrophages (ex vivo). The immunostimulatory activity of the PSCJ was also measured in normal and immunosuppressed mice models (in vivo). In the normal mice model, the PSCJ treatment increased the weight of immune organs (spleen and thymus), and improved the phagocytic rates of peritoneal macrophages and lymphocytes proliferation. Similarly, the PSCJ markedly restored the decreased lymphocyte proliferation, NK cell activity, and WBC count in the CY-induced immunosuppressed mice model. Taken together, we speculate that the PSCJ could be utilized as an effective immunostimulatory agent and that the PSCJ may have therapeutic potential in patients with inadequate immune functions. Acknowledgement This work was supported by research grants from the Korea Food Research Institute (Project No. E0132101). References [1] K.P. Mishra, Y.S. Padwad, M. Jain, D. Karan, L. Ganju, R.C. Sawhney, Immunopharmacol. Immunotoxicol. 28 (2006) 201–212. [2] T. Zhao, Y. Feng, J. Li, R. Mao, Y. Zou, W. Feng, D. Zheng, W. Wang, Y. Chen, L. Yang, X. Wu, Int. J. Biol. Macromol. 65 (2014) 33–40. [3] M.Y. Leung, C. Liu, J.C. Koon, K.P. Fung, Immunol. Lett. 105 (2006) 101–114. [4] M.H. Jiang, L. Zhu, J.G. Jiang, Expert Opin. Ther. Targets 14 (2010) 1367–1402. [5] M.J. Bae, H.S. Shin, H.J. See, O.H. Chai, D.H. Shon, J. Med. Food 17 (2014) 142–149. [6] I.S. Hwang, J.E. Kim, Y.J. Lee, M.H. Kwak, J. Go, H.J. Son, D.S. Kim, D.Y. Hwang, Nutr. Res. 34 (2014) 355–367. [7] J. Kim, J.N. Choi, J.H. Choi, Y.S. Cha, M.J. Muthaiya, C.H. Lee, Mol. Nutr. Food Res. 57 (2013) 1886–1891. [8] Y.J. Lee, J.E. Kim, M.H. Kwak, J. Go, D.S. Kim, H.J. Son, D.Y. Hwang, Int. J. Mol. Med. 33 (2014) 1185–1194. [9] Y.C. Hwang, K.S. Shin, Korean J. Food Sci. Technol. 40 (2008) 220–227. [10] H.R. Park, M.S. Lee, S.Y. Jo, H.J. Won, H.S. Lee, H. Lee, K.S. Shin, Korean J. Food Sci. Technol. 44 (2012) 228–234.
525
[11] C.W. Cho, C.J. Han, Y.K. Rhee, Y.C. Lee, K.S. Shin, H.D. Hong, Molecules 19 (2014) 5266–5277. [12] S.J. Lee, H.K. Rim, J.Y. Jung, H.J. An, J.S. Shin, C.W. Cho, Y.K. Rhee, H.D. Hong, K.T. Lee, Food Chem. Toxicol. 59 (2013) 476–484. [13] E.A. Kabat, M.M. Mayer, Experimental Immunochemistry, second ed., Charles C. Thomas, Springfield, IL, 1964. [14] H. Saito, H. Tomioka, K. Sato, J. Gen. Microbiol. 134 (1988) 1029–1035. [15] I. Saiki, S. Saito, C. Fujita, H. Ishida, J. Iida, J. Murata, A. Hasegawa, I. Azuma, Vaccine 6 (1988) 238–244. [16] M. Wang, L.J. Guilbert, L. Ling, J. Li, Y. Wu, S. Xu, P. Pang, J.J. Shan, J. Pharm. Pharmacol. 53 (2001) 1515–1523. [17] X.Y. Wu, G.H. Mao, Q.Y. Fan, T. Zhao, J.L. Zhao, F. Li, L.Q. Yang, Food Res. Int. 48 (2012) 935–939. ´ V. Juri´sic, ´ I. Spuzi ´ c, ´ J. Immunol. Methods 200 (1997) 199–201. [18] G. Konjevic, [19] S.H. Bae, J.W. Choi, K.S. Ra, K.W. Yu, K.S. Shin, S.S. Park, H.J. Suh, J. Sci. Food Agric. 92 (2012) 1765–1770. [20] V.B. Liju, K. Jeena, R. Kuttan, Food Chem. Toxicol. 53 (2013) 52–61. [21] F. Cerqueira, A. Cordeiro-Da-Silva, C. Gaspar-Marques, F. Simões, M.M. Pinto, M.S. Nascimento, Bioorg. Med. Chem. 12 (2004) 217–223. [22] Y.M. Ha, S.H. Chun, S.T. Hong, Y.C. Koo, H.D. Choi, K.W. Lee, Int. J. Biol. Macromol. 60 (2013) 399–404. [23] H.Y. Park, A.R. Yu, I.W. Choi, H.D. Hong, K.W. Lee, H.D. Choi, Int. Immunopharmacol. 17 (2013) 191–197. [24] A.O. Tzianabos, Clin. Microbiol. Rev. 13 (2000) 523–533. [25] V.M. Holers, Clin. Immunol. 107 (2003) 140–151. [26] S.H. Bae, E.Y. Jung, S.Y. Kim, K.S. Shin, H.J. Suh, J. Food Biochem. 34 (2010) 233–248. [27] V.E. Ooi, F. Liu, Curr. Med. Chem. 7 (2000) 715–729. [28] H. Wang, J.K. Actor, J. Indrigo, M. Olsen, A. Dasgupta, Clin. Chim. Acta 327 (2003) 123–128. [29] R.M. Sobota, P.J. Müller, C. Khouri, A. Ullrich, V. Poli, T. Noguchi, P.C. Heinrich, F. Schaper, Cell Signal. 20 (2008) 1385–1391. [30] S.J. Kim, J.S. Park, N.Y. Myung, P.D. Moon, I.Y. Choi, H.J. An, N.H. Kim, H.J. Na, D.H. Kim, M.C. Kim, N.H. An, I.K. Kim, J.Y. Lee, H.J. Jeong, J.Y. Um, H.M. Kim, S.H. Hong, Immunopharmacol. Immunotoxicol. 31 (2009) 246–252. [31] H.L. Cui, Y. Chen, S.S. Wang, G.Q. Kaia, Y.M. Fang, J. Sci. Food Agric. 91 (2011) 2180–2185. [32] X. Chen, W. Nie, S. Fan, J. Zhang, Y. Wang, J. Lu, L. Jin, Carbohydr. Polym. 90 (2012) 1114–1119. [33] T.T. Kawabata, K.L. White, Cancer Res. 47 (1987) 2317–2322. [34] C. Zhao, M. Li, Y. Luo, W. Wu, Carbohydr. Res. 341 (2006) 485–491. [35] H. Wang, M.Y. Wang, J. Chen, Y. Tang, J. Dou, J. Yu, Int. Immunopharmacol. 11 (2011) 1946–1953. [36] J.W. Hadden, Int. Immunopharmacol. 3 (2003) 1061–1071. [37] J. Moynihan, N. Cohen, Int. J. Immunopharmacol. 11 (1989) 517–527. [38] I. Shalit, Y. Kletter, D. Halperin, D. Waldman, E. Vasserman, A. Nagler, I. Fabian, Eur. J. Haematol. 66 (2001) 287–296. [39] H. Zhou, F. Sun, H. Li, S. Zhang, Z. Liu, J. Pei, C. Liang, Immunopharmacol. Immunutoxicol. 35 (2013) 426–433.
