International Journal of Biological Macromolecules 65 (2014) 229–233
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Anti-tumour and immunomodulatory activities of oligosaccharides isolated from Panax ginseng C.A. Meyer Lili Jiao a , Xiaoyu Zhang a , Bo Li a , Zhen Liu a , Mingzhu Wang a , Shuying Liu a,b,∗ a b
Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun 130117, China Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
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Article history: Received 6 December 2013 Received in revised form 8 January 2014 Accepted 16 January 2014 Available online 24 January 2014 Keywords: Panax ginseng Oligosaccharides Anti-tumour Immunomodulatory
a b s t r a c t Water-soluble ginseng oligosaccharides (WGOS) composed of d-glucose with a degree of polymerisation ranging from 2 to 14 were obtained from Panax ginseng C.A. Meyer. In this study, the anti-tumour and immunoregulatory effects of WGOS were evaluated in Hepatoma-22 (H22)-bearing mice. Treatment with WGOS inhibited tumour growth in vivo and significantly increased relative spleen and thymus weight, serum tumour necrosis factor-␣ level, spleen lymphocyte proliferation, natural killer cell activity, phagocytic function and nitric oxide production secreted by macrophage in H22-bearing mice. However, no direct cytotoxicity was detected. Therefore, the anti-tumour activity of WGOS may be related to their immunomodulatory effects. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cancer is a serious medical condition; however, the current medical treatments for cancer, including chemotherapy and radiotherapy, do not guarantee complete tumour remission and metastasis prevention [1]. In addition, many anti-carcinogens used for cancer patients are immunosuppressive agents. They repress tumour growth but have adverse effects on the immune system of the organism. Immunotherapy has been attempted in various animal models and human patients to address these problems [2]. The host response to cancer therapies could be stimulated by the treatment of immunoadjuvants, which non-specifically activate the immune system [3]. Plant-derived compounds have potent immunotherapeutic properties for the prevention and treatment of cancer [4]. Thus, numerous researchers have shifted their focus into natural compounds to discover and identify new anti-tumour drugs that can potentially improve immune function [5,6]. Panax ginseng C.A. Meyer of the family Araliaceae has been traditionally used as a tonic and prophylactic agent in Asia. Numerous active ingredients in P. ginseng, including ginsenosides, peptides, polysaccharides, oligosaccharides, polypeptides, fatty acid, amino acids, and aetherolea, have been reported [7]. However, reports on
∗ Corresponding author at: Changchun University of Chinese Medicine, Jingyue Economic Development Zone, 1035, Boshuo Road, Changchun 130117, Jilin Province, China. Tel.: +86 43186045155; fax: +86 43186045258. E-mail address: [email protected] (S. Liu). 0141-8130/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2014.01.039
the bioactive property of valuable oligosaccharides in ginseng are limited. Our previous study demonstrated that oligosaccharides in ginseng can promote lymphocyte proliferation [8], augment phagocytosis, and stimulate an increase in nitric oxide (NO) and tumour necrosis factor (TNF)-␣ production in macrophages [9]. However, the anti-tumour properties of ginseng oligosaccharides remain unclear. Therefore, the present study aims to evaluate the anti-tumour and immunoadjuvant potentials of ginseng oligosaccharides to restrict cancer development in Hepatoma-22 (H22)-bearing mice.
2. Materials and methods 2.1. Materials and reagents Roots of P. ginseng, a cultivated ginseng, were collected from Changbai Mountain, Jinlin Province, China in August 2010. Lipopolysaccharide (LPS), concanavalin A (ConA) and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. RPMI-1640 medium and foetal bovine serum (FBS) were purchased from Gibio Invitrogen Co. The complete RPMI-1640 medium that was used for immunological tests was supplemented with 10% (v/v) heat-inactivated, endotoxin-free FBS, penicillin (100 IU/ml) and streptomycin (100 g/ml, pH 7.4). Greiss reagent was purchased from Beyotime Institute of Biotechnology (Shanghai, China). All other reagents were of analytical grade.
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2.2. Preparation of oligosaccharides Water-soluble ginseng oligosaccharides (WGOS) were extracted as previously described [9]. Air-dried P. ginseng root was crushed and extracted with 95% ethanol under reflux extraction at 40 ◦ C to remove pigments and small lipophilic molecules. The residue was further extracted thrice (90 min each) with distilled water at 70 ◦ C. All aqueous extracts were combined, concentrated under reduced pressure and then lyophilised to yield crude oligosaccharides. Then, the crude oligosaccharides were re-dissolved in distilled water and dialysed against distilled water for 72 h in a dialysis sack (molecular weight cut-off of 3000 Da). The fraction out of dialysis sack (Mw < 3000 Da) was collected and concentrated at 50 ◦ C in vacuum and then lyophilised. Subsequently, the obtained oligosaccharides were applied to Sephadex G-25 (2.5 cm × 90 cm) and equilibrated with degassed distilled water. The eluate was detected using phenol–sulphuric acid [10]. The appropriate fractions were combined, concentrated and then lyophilised to obtain WGOS. The sugar content in WGOS was determined to be 95.87% by the phenol–sulphuric acid method. The ginsenoside content in WGOS was determined to be 1.43% following the method described by Chen et al. [11]. The protein and uronic acid contents in WGOS were undetectable using the Bradford method and m-hydroxydiphenyl analysis, respectively [12,13].
where A and B are the average tumour weights of the model control and experimental groups, respectively [14]. At 24 h after last drug administration, the blood were collected and centrifuged at 4000 × g at 4 ◦ C for 10 min to separate the serums required. The concentration of TNF-␣ was measured using an enzyme-linked immunosorbent assay according to the manufacturer’s instructions. 2.7. Splenocyte proliferation assay Spleens were aseptically removed from the sacrificed mice, gently homogenised with scissors and forceps in chilled RPMI-1640 medium and then filtered through a sterilised mesh of 200 to obtain single cell suspensions. Erythrocytes in the splenic lymphocytes were lysed by hypo-osmostic haemolysis. After centrifugation at 1000 rpm for 10 min, the cells were suspended to a final density of 5 × 106 cells/ml in a solution of complete RPMI-1640 medium. The purity and viability of splenocytes (tested by trypan blue dye exclusion) were consistently >90%. Spleen cells (100 l/well) were plated in 96-well plates and then incubated with or without ConA (5 g/ml) or LPS (20 g/ml). Cell concentration was determined by MTT assay [15]. The absorbance at 570 nm of the culture was measured in an ELISA reader (TECAN GENios, Switzerland). 2.8. Assay for natural killer (NK) cell cytotoxicity
2.3. Animals ICR male mice weighing 20.0 ± 2.0 g were obtained at 6–8 weeks of age. The mice were purchased from the Changchun Institute of Biological Products Co., Ltd. The mice were randomly housed by tens into cages and received standard mouse chow and water. The room conditions were maintained at room temperature with 12/12-h light–dark cycle. Rodent laboratory mouse chow and water were provided ad libitum. All animals were maintained and used in strict accordance with the PR China legislation on the use and care of laboratory animals and with the guidelines issued by Experimental Animal Centre of Changchun University of Chinese Medicine and approved by the university committee for animal experiments. 2.4. Cell lines and culture H22 cell lines and Yac-1 selected for resistance to NK cell lines were purchased from the Tumour Hospital of Jilin Province, Changchun, Jilin Province, China. The cells were suspended in complete RPMI-1640 medium with 100 IU/ml penicillin, 100 g/ml streptomycin and 10% foetal bovine serum at pH 7.4. 2.5. Treatment Seven-day-old H22 ascites (0.2 ml, 1 × 107 cells/ml) were inoculated into the right armpits of ICR male mice on Day 0; another 10 mice served as normal control. The inoculated mice were divided into five groups (10 mice in each group): three treatment groups, positive control group and model control group. In addition, 1, 12.5 and 25 mg/kg body weight of the WGOS were intragastrically administered to each group from Day 1 to Day 10. The positivecontrol mice received the same volume of cyclophosphamide (CTX, 25 mg/kg body weight), whereas the model control mice and normal control received saline (0.2 ml). 2.6. In vivo anti-tumour activity At 24 h after last drug administration, all mice were sacrificed, and the spleen, thymus and tumour were dissected and weighed. The anti-tumour activity of the oligosaccharides in vivo was expressed as an inhibitory rate calculated as [(A − B)/A] × 100,
Spleens obtained as described above were used as effector cells and seeded into 96-well plates at 2.5 × 105 cells/well in RPMI-1640 complete medium. YAC-1 cells were used as target cells, and 1 × 104 cells/well were added to achieve an E/T (effector/target) ratio of 25:1 [16]. After 20 h of incubation at 37 ◦ C in 5% CO2 atmosphere, each well was added with 20 l of MTT solution (5 mg/ml) and then incubated for another 4 h. The absorbance was measured at 490 nm. Three types of control measurements were performed: target cell control, blank control, and effect cell control. NK cell activity was calculated using the following formula: NK activity (%) =
[ODT − (ODS − ODE )] × 100 ODT
where ODT is the optical absorption value of the target cell control, ODS is the optical absorption value of test samples and ODE is the optical absorption value of the effect cell control. 2.9. Phagocytosis of macrophage assay Peritoneal macrophages were harvested from the H22-bearing mice by peritoneal lavage and centrifugation [17]. Briefly, peritoneal macrophages were harvested by peritoneal washing with 10 ml serum-free RPMI-1640 medium and centrifugation at 1500 rpm for 5 min at 4 ◦ C. Then, the peritoneal macrophages were cultured in complete RPMI-1640 medium in 96-well plates for 2 h at 37 ◦ C in a 5% CO2 cell incubator. Non-adherent cells were removed by washing the plate with warm PBS (0.01 M, pH 7.4). In the aforementioned method, the purity and viability of the peritoneal macrophages were consistently >90%. All cells were cultured at 37 ◦ C in a humidified atmosphere containing 5% CO2 . The number and viability of macrophages were assessed microscopically using trypan blue exclusion [18]. The amount of neutral red dye adsorbed by the macrophages was measured as previously described [19]. The macrophages were plated at a density of 2 × 105 cells/well to a final volume of 100 l in a 96-well plate and then incubated in a medium alone or in a medium with various concentrations of ginseng oligosaccharide. The cells were incubated at 37 ◦ C in humidified 5% CO2 for 24 h, added with 100 l of 0.075% aseptic neutral red solution and then incubated again for 1 h. Subsequently, the plate was washed thrice
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Table 1 Effects of WGOS on thymus, spleen indexes and tumour weights in H22-bearing mice. Group
Dose (mg/kg)
Normal control Model control CTX WGOS
– – 25 1 12.5 25
Tumour weight (g) – 1.31 0.26 0.84 0.55 0.76
± ± ± ± ±
0.18 0.11 0.091 0.07** 0.10**
Thymus index (mg/g) 3.46 2.18 1.40 2.78 3.34 2.88
± ± ± ± ± ±
0. 47 0.0.64 0.35 0.57* 0. 74** 0.53**
Spleen index (mg/g) 13.54 7.82 7.35 9.23 11.82 9.81
± ± ± ± ± ±
1.82 0.53 0.91 0.99 1.93* 0.96*
Inhibitory rate (%) – – 80.29 36.27 58.19** 41.90*
Drug treatment started to H22-bearing mice on Day 1 by oral administration. Normal control and model control group received saline, positive group received cyclophosphamide (CTX, 25 mg/kg), and oligosaccharide groups received the oligosaccharide WGOS (1, 12.5, 25 mg/kg). The dose volume was 0.2 ml. Values were shown as mean ± SD of 10 mice. Significance was determined using the Student’s t-test: * P < 0.05 vs. model control. ** P < 0.01 vs. model control.
with PBS, and 150 l cell lysate (a mixture of 100% ethanol and 99.9% acetic acid (1:1, v/v)) was added. The mixture was mixed fully and evaluated at 550 nm in an ELISA reader (TECAN GENios, Switzerland). The absorbance represented the phagocytic ability of the macrophages. All determinations were conducted in triplicate.
excellent inhibitory activity against solid tumour formation of H22, with inhibitory ratios of 36.27%, 58.19% and 41.90%. Furthermore, the relative spleen and thymus weights of the mice treated with 12.5 and 25 mg/kg WGOS significantly increased (Table 1) compared with that of the model control group (P < 0.05). In addition, the optimum feeding rate of oral administration was 12.5 mg/kg/d.
2.10. NO assay 3.2. Effect of WGOS on TNF-˛ section Peritoneal macrophages obtained by the abovementioned methods were cultured in complete RPMI-1640 medium at 1 × 106 cells per well in a 48-well plate [3]. The cells were cultured for 24 h at 37 ◦ C in humidified 5% CO2 incubator. Then, 100 l of the cell supernatants was removed and mixed with an equal volume of Greiss reagent (1% sulphanilamide in 2.5% phosphoric acid and 0.1% napthyl ethyl diamine dihydrochloride in 2.5% phosphoric acid). Absorbance was read at 540 nm. Nitrite products were calculated by equation from a NaNO2 standard curve. 2.11. Direct cytotoxicity assay The cytotoxicity of WGOS was investigated in H22 tumour cells. Proliferation was determined using the colourimetric MTT assay as previously described [20]. The cells were seeded at a density of 1 × 104 cells/well in 100 l volume of the medium in 96-well plates and then pre-incubated for 24 h. The medium was replaced with fresh medium containing different dosages of WGOS (0.5–100 g/ml), whereas the negative control was treated with the complete RPMI-1640 medium only. Approximately 20 l of MTT (5 mg/ml) was added 48 h later. After 4 h of incubation at 37 ◦ C, the supernatant was aspirated, and 150 l dimethyl sulphoxide was added to each well. Absorbance was measured at 570 nm. All samples were tested in triplicate.
TNF-␣ has an important function in tumouricidal and immune response; it also party mediates tumour cell elimination. Thus, we measured the serum TNF-␣ level. As shown in Fig. 1, the production of TNF-␣ was significantly decreased in the CTX-treated mice compared with the model control (P < 0.05). Thus, CTX could suppress the secretion of the cytokine. WGOS significantly augmented TNF-␣ production and stimulated TNF-␣ level to normal control in the serum of H22-bearing mice at high doses. 3.3. Effect of WGOS on splenocyte proliferation in tumour-bearing mice We evaluated the effects of WGOS on the proliferation of spleen lymphocytes from tumour-bearing mice to confirm the effect of WGOS on the cellular immune response (Table 2). All tested dosages of WGOS (1, 12.5 and 25 mg/kg) significantly increased splenocyte proliferation and Con A-/LPS-induced splenocyte proliferation compared with the model control mice. The results generated a bell-shaped dose–response curve. The splenocyte proliferation
2.12. Statistical analysis Results were expressed as mean ± SD. The statistical significance of the differences between the groups was evaluated by variance analysis, followed by Student’s t-test. The levels of significance were as follows: P < 0.05 is signified by *, P < 0.01 is signified by **. 3. Results 3.1. In vivo anti-tumour activity The anti-tumour activity of WGOS is shown in Table 1. The ICR mice were administered with WGOS orally and continuously with the same dose for 10 d. The tumour weights of the groups treated with 1, 12.5 and 25 mg/kg body weight of WGOS were 0.84, 0.55 and 0.76 g, respectively, whereas that of the negative control group was 1.31 g on Day 10. Therefore, the fraction of WGOS showed
Fig. 1. Concentration of TNF-␣ in serum of H22 tumour-bearing mice. Values are mean ± S.D. from 3 independents (n = 10). WGOS1, P. ginseng oligosaccharide (1 mg/kg) treated group; WGOS12.5, P. ginseng oligosaccharide (12.5 mg/kg) treated group; WGOS25, P. ginseng oligosaccharide (25 mg/kg) treated group; CTX, cyclophosphamide (25 mg/kg) treated group (positive control). *P < 0.05, **P < 0.01 vs. model group.
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Table 2 Effects of WGOS on spleen lymphocyte proliferation in H22-bearing mice. Group
Dose (mg/kg)
Normal control Model control CTX WGOS
– – 25 1 12.5 25
A 570 nm Lymphocyte 0.285 0.148 0.127 0.166 0.211 0.201
± ± ± ± ± ±
0.025** 0.037 0.034 0.043 0.027** 0.044*
ConA 0.303 0.134 0.130 0.207 0.244 0.237
LPS ± ± ± ± ± ±
0.038** 0.009 0.011* 0.073* 0.059** 0.063**
0.351 0.157 0.124 0.193 0.243 0.211
± ± ± ± ± ±
0.072** 0.033 0.014* 0.055 0.053** 0.040*
Drug treatment started to H22-bearing mice on Day 1 by oral administration. Normal control and model control group received saline, positive group received cyclophosphamide (CTX, 25 mg/kg), and oligosaccharide groups received the oligosaccharide WGOS (1, 12.5, 25 mg/kg). The dose volume was 0.2 ml. The lymphocyte proliferation activities were expressed as the absorption at 570 nm. Values are shown as mean ± SD of 10 mice. Significance was determined using the Student’s t-test: * P < 0.05 vs. model control. ** P < 0.01 vs. model control.
index with and without mitogenic stimuli reached the peak at 12.5 mg/kg.
3.4. Effect of WGOS on NK cell activities in tumour-bearing mice Tumour cell elimination can be partly mediated by the cytotoxic activity of NK cells. As shown in Fig. 2, NK cell activity was significantly decreased in both H22-bearing mice and CTX-treated mice. Each concentration of WGOS enhanced NK cell activity in YAC-1 cell lysis at the E:T cell ratio used. In addition, the cytotoxic activity of NK cells from both groups (12.5 and 25 mg/kg) of WGOS-treated mice was significantly different from that of the model control mice (P < 0.01).
3.6. Direct cytotoxicity assay H22 tumour cells were incubated with different concentrations (0.5, 1, 12.5, 25, 50, 100, 200, 400 and 800 g/ml) of WGOS for 72 h and measured by the MTT method. The highest inhibition on H22 cells was 13.6%, suggesting that WGOS had no significant cytotoxicity to H22 cells in vitro, even at the highest concentration of 800 g/ml. By contrast, CTX showed strong cytotoxicity (data not shown).
3.5. Effect of WGOS on peritoneal macrophage activation in tumour-bearing mice Macrophages have essential functions in innate immunity and serve as an important bridge between innate and adaptive immunity. In this study, oral administration of 1, 12.5 and 25 mg/kg WGOS to H22-bearing mice not only improved macrophage phagocytosis but also promoted NO generation, especially at 12.5 and 25 mg/kg (P < 0.01). The highest stimulation index was reached at 12.5 mg/ml (Fig. 3A and B).
Fig. 2. Effect of WGOS on cytotoxicity of NK cells from H22 tumour-bearing mice. Values are means ± S.D. from 3 independents (n = 10). WGOS1, P. ginseng oligosaccharide (1 mg/kg) treated group; WGOS12.5, P. ginseng oligosaccharide (12.5 mg/kg) treated group; WGOS25, P. ginseng oligosaccharide (25 mg/kg) treated group; CTX, cyclophosphamide (25 mg/kg) treated group (positive control). *P < 0.05, **P < 0.01 vs. model group.
Fig. 3. (A) Effects of different doses of WGOS on phagocytosis of murine peritoneal macrophages from H22 tumour-bearing mice. Phagocytosis was evaluated as OD 550 nm. Values are mean ± S.D. from 3 independents (n = 10). WGOS1, P. ginseng oligosaccharide (1 mg/kg) treated group; WGOS12.5, P. ginseng oligosaccharide (12.5 mg/kg) treated group; WGOS25, P. ginseng oligosaccharide (25 mg/kg) treated group; CTX, cyclophosphamide (25 mg/kg) treated group (positive control). *P < 0.05, **P < 0.01 vs. model group. (B) Effects of different doses of WGOS on NO level of murine peritoneal macrophages from H22 tumour-bearing mice. Values are mean ± S.D. from 3 independents (n = 10). WGOS1, P. ginseng oligosaccharide (1 mg/kg) treated group; WGOS12.5, P. ginseng oligosaccharide (12.5 mg/kg) treated group; WGOS25, P. ginseng oligosaccharide (25 mg/kg) treated group; CTX, cyclophosphamide (25 mg/kg) treated group (positive control). *P < 0.05, **P < 0.01 vs. model group.
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4. Discussion Oligosaccharides have long been considered as a healthy food source whose biological functions are limited to antigenic properties of various blood groups [21]. However, oligosaccharides have recently been extensively studied by experts because of their biological and physiological activities, such as anti-tumour [22], antioxidant [23] and immunoregulatory [24]. According to various investigations, oligosaccharides from various sources exhibit potent immunostimulating activities [25]. An aqueous extract containing mainly oligosaccharides and polysaccharides from North American ginseng (Panax quinquefolium), a species that belongs to the same family as P. ginseng, can stimulate the proliferation of normal mouse spleen in vitro [26]. Murosaki et al. also demonstrated that nigero-oligosaccharides exert immunopotentiating activity by activating an IL-12-dependent T-helper-1-like immune response [27]. The present study showed that ginseng oligosaccharides can reliably inhibit the growth of mouse transplantable H22 cells and improve the weight of immune organs of tumour-bearing mice. The relative spleen and thymus weights in the WGOS test groups were significantly higher than those of the control groups. Thymus and spleen are important immune organs; the thymus and spleen indices reflect the immune function of the organism. Immunopotentiation can increase the weights of thymus and spleen. Furthermore, the TNF-␣ level in the serum of WGOS-treated H22 murine serum was significantly increased. These results suggest that TNF-␣ is involved in the immune response. In addition, the appetite, activity and coat lustre of each animal in the WGOS groups were better than those of the mice treated with saline and CTX. Furthermore, WGOS did not directly kill the tumour cells. Thus, the anti-tumour activity of WGOS can be ascribed to their capacity to stimulate the immune system. Thus, WGOS can have great significance in tumour growth therapy. The specific immune responses include humoural immunity and cellular immunity. The humoural immune response by B cells is an antigen-specific antibody reaction. Cellular immunity can be mediated specifically by T cells, including NK cells. Lymphocyte proliferation induced by antigen or mitogens is an indicator of immunoenhancement. Cellular multiplication induced by Con A can be used as a method to detect T lymphocyte activity in vitro. Moreover, the LPS-induced activation of B cells and subsequent immunoglobulin synthesis reflect B lymphocyte immunity. Extensive in vivo and in vitro evidence demonstrated that oligosaccharides display immunomodulating function by stimulating cellular and humoural immune responses [28,29]. The present results showed that ginseng oligosaccharides can augment splenocyte proliferation and NK cell activity in H22-bearing mice. Therefore, ginseng oligosaccharides can enhance the humoural immunity and cell-mediated immunity in H22-bearing mice. However, significant signs of immunosuppressive effect were observed in the mice treated with the positive control CTX based on the activities of splenocyte proliferation and NK cells. Macrophages are fundamental in maintaining homeostasis and perform essential and pivotal functions in host defence. The innate defence response can be activated through the interaction of sugar moieties with innate receptors in the plasma membrane of macrophages; this interaction was assumed to be the immunoregulatory mechanism of several oligosaccharides [30]. Zhang et al. described that synthetic -(1 → 6)-branched -(1 → 3) glucohexaose can stimulate innate immune reactions by binding to selective receptors (such as dectin-1) mainly expressed on M2 macrophages [31]. Murosaki et al. also found that an
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oligosaccharide containing ␣-(1 → 3) linked-glucose increases the production of endogenous IL-12 that is locally produced by activated resident macrophages [27]. The present study showed that WGOS, a mixture of glucose units, has a positive effect on macrophage activation in H22-transplanted mice. WGOS enhanced the phagocytic effect of macrophages. In addition, NO production increased in the WGOS-treated (12.5–25 mg/kg) groups. Thus, the oligosaccharides from P. ginseng could indirectly exhibit antitumour activity through the activation of macrophages. In summary, our findings clearly showed that WGOS significantly inhibited tumour growth in vivo by enhancing the immune system instead of eliciting direct cytotoxicity. Acknowledgements We are grateful for the financial support of this research from Scientific Research Foundation of Jilin Provincial Science & Technology Department of China (Grant No. 20130202059YY) and Program for Innovative Research Team (in Science and Technology) in University of Jilin Province (No. JTD201213). References [1] H. Mushiake, T. Tsunoda, M. Nukatsuka, K. Shimao, M. Fukushima, H. Tahara, Cancer Immunol. Immunother. 54 (2005) 120–128. [2] S.B. Han, C.W. Lee, M.R. Kang, Y.D. Yoon, J.S. Kang, K.H. Lee, W.K. Yoon, K. Lee, S.K. Park, H.M. Kim, Cancer Lett. 243 (2006) 264–273. [3] D. Ghosh, T.K. Maiti, Immunobiology 212 (2007) 589–599. [4] B.Z. Zaidman, M. Yassin, J. Mahajna, S.P. Wasser, Appl. Microbiol. Biotechnol. 67 (2005) 453–468. [5] B. Faanes, V.J. Merluzzi, P. Ralph, in: B. Serrou, C. Rosenfield (Eds.), International Symposium on New Trends in Human Immunology and Cancer Immunotherapy, Doin, Paris, 1980, pp. 953–964. [6] T.K. Yun, S.Y. Choi, Int. J. Epidemiol. 19 (1990) 871–876. [7] A.S. Attele, J.A. Wu, C.S. Yuan, Biochem. Pharmacol. 58 (1999) 1685–1693. [8] D.B. Wan, L.L. Jiao, H.M. Yang, Y.L. Shu, Planta 235 (2011) 1289–1297. [9] L.L. Jiao, D.B. Wan, X.Y. Zhang, B. Li, H.X. Zhao, S.Y. Liu, J. Ethnopharmacol. 144 (2012) 490–496. [10] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Anal. Chem. 28 (1956) 350–356. [11] R.Z. Chen, F.L. Meng, S.Q. Zhang, Z.Q. Liu, Sep. Purif. Technol. 66 (2009) 340–346. [12] J.J. Sedmark, S.E. Grossberg, Anal. Biochem. 79 (1979) 544–552. [13] M.C.C Tullia, C. Filisetti, Anal. Biochem. 197 (1991) 157–162. [14] K.C. Mousinho, C.C. Oliveira, J.R. Ferreira, A.A. Carvalho, H.I. Magalhães, D.P. Bezerra, A.P.N.N. Alevs, L.V. Costa-lotufo, C. Pessoa, M.P.V. Matos, M.V. Ramos, M.O. Moraes, J. Ethnopharmacol. 137 (2011) 421–426. [15] C.M. Chien, J.L. Cheng, W.T. Chang, M.H. Tien, C.M. Tsao, Y.H. Chang, Bioorg. Med. Chem. 12 (2004) 5603–5609. [16] L. Cao, X.Z. Liu, T.X. Qian, G.B. Sun, Y. Guo, F. Chang, S. Zhou, X. Sun, Int. J. Biol. Macromol. 48 (2011) 160–164. [17] W.C.S. Cho, K.N. Leung, Cancer Lett. 252 (2007) 43–54. [18] D.L. Felice, J. Sun, R.H. Liu, J. Funct. Food 1 (2009) 109–118. [19] B.A. Weeks, A.S. Keisler, Q.N. Myrvik, Dev. Comp. Immunol. 11 (1987) 117–124. [20] A. Akrout, L.A. Gonzalez, H.E.P. Jani, C. Madrid, Food Chem. Toxicol. 49 (2011) 342–347. [21] A.C. Weymouth-Wilson, Nat. Prod. Rep. 14 (1997) 99–110. [22] H.M. Yuan, J.M. Song, X.G. Li, N. Li, J.C. Dai, Cancer Lett. 243 (2006) 228–234. [23] B.R. Veenashri, G. Muralikrishna, Food Chem. 126 (2011) 1475–1481. [24] S.M. Zhou, X.Z. Liu, Y. Guo, Q. Wang, D.Y. Peng, L. Cao, Carbohydr. Polym. 81 (2010) 784–789. [25] X. Qiang, Y.L. Chao, Q.B. Wan, Carbohydr. Polym. 77 (2009) 435–441. [26] M.Q. Wang, L.J. Guilbert, L. Ling, J. Li, Y.Q. Wu, S. Xu, P. Pang, J.J. Shan, J. Pharm. Pharmacol. 53 (2001) 1515–1523. [27] S. Murosaki, K. Muroyama, Y. Yamamoto, H. Kusaka, T. Liu, Y. Yoshikai, Biosci. Biotechnol. Biochem. 63 (1999) 373–378. [28] J. Yan, H.L. Zong, A.G. Shen, S. Chen, X.L. Yin, X.Y. Shen, W.C. Liu, X.S. Gu, J.X. Gu, Int. Immunopharmacol. 3 (2003) 1861–1871. [29] H.J. Chen, B.Q. Yuan, Z.C. Zheng, Z. Liu, S.S. Wang, X. Lewis, Cell Immunol. 269 (2011) 144–148. [30] J.N. Arnold, R.A. Dwek, P.M. Rudd, R.B. Sim, Immunol. Lett. 106 (2006) 103–110. [31] M. Zhang, P.C.K. Cheung, L.C.M. Chiu, E.Y.L. Wong, V.E.C. Ooi, Carbohydr. Polym. 66 (2006) 455–462.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Anti-tumour and immunomodulatory activities of oligosaccharides isolated from Panax ginseng C.A. Meyer Lili Jiao a , Xiaoyu Zhang a , Bo Li a , Zhen Liu a , Mingzhu Wang a , Shuying Liu a,b,∗ a b
Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun 130117, China Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
a r t i c l e
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Article history: Received 6 December 2013 Received in revised form 8 January 2014 Accepted 16 January 2014 Available online 24 January 2014 Keywords: Panax ginseng Oligosaccharides Anti-tumour Immunomodulatory
a b s t r a c t Water-soluble ginseng oligosaccharides (WGOS) composed of d-glucose with a degree of polymerisation ranging from 2 to 14 were obtained from Panax ginseng C.A. Meyer. In this study, the anti-tumour and immunoregulatory effects of WGOS were evaluated in Hepatoma-22 (H22)-bearing mice. Treatment with WGOS inhibited tumour growth in vivo and significantly increased relative spleen and thymus weight, serum tumour necrosis factor-␣ level, spleen lymphocyte proliferation, natural killer cell activity, phagocytic function and nitric oxide production secreted by macrophage in H22-bearing mice. However, no direct cytotoxicity was detected. Therefore, the anti-tumour activity of WGOS may be related to their immunomodulatory effects. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cancer is a serious medical condition; however, the current medical treatments for cancer, including chemotherapy and radiotherapy, do not guarantee complete tumour remission and metastasis prevention [1]. In addition, many anti-carcinogens used for cancer patients are immunosuppressive agents. They repress tumour growth but have adverse effects on the immune system of the organism. Immunotherapy has been attempted in various animal models and human patients to address these problems [2]. The host response to cancer therapies could be stimulated by the treatment of immunoadjuvants, which non-specifically activate the immune system [3]. Plant-derived compounds have potent immunotherapeutic properties for the prevention and treatment of cancer [4]. Thus, numerous researchers have shifted their focus into natural compounds to discover and identify new anti-tumour drugs that can potentially improve immune function [5,6]. Panax ginseng C.A. Meyer of the family Araliaceae has been traditionally used as a tonic and prophylactic agent in Asia. Numerous active ingredients in P. ginseng, including ginsenosides, peptides, polysaccharides, oligosaccharides, polypeptides, fatty acid, amino acids, and aetherolea, have been reported [7]. However, reports on
∗ Corresponding author at: Changchun University of Chinese Medicine, Jingyue Economic Development Zone, 1035, Boshuo Road, Changchun 130117, Jilin Province, China. Tel.: +86 43186045155; fax: +86 43186045258. E-mail address: [email protected] (S. Liu). 0141-8130/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2014.01.039
the bioactive property of valuable oligosaccharides in ginseng are limited. Our previous study demonstrated that oligosaccharides in ginseng can promote lymphocyte proliferation [8], augment phagocytosis, and stimulate an increase in nitric oxide (NO) and tumour necrosis factor (TNF)-␣ production in macrophages [9]. However, the anti-tumour properties of ginseng oligosaccharides remain unclear. Therefore, the present study aims to evaluate the anti-tumour and immunoadjuvant potentials of ginseng oligosaccharides to restrict cancer development in Hepatoma-22 (H22)-bearing mice.
2. Materials and methods 2.1. Materials and reagents Roots of P. ginseng, a cultivated ginseng, were collected from Changbai Mountain, Jinlin Province, China in August 2010. Lipopolysaccharide (LPS), concanavalin A (ConA) and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. RPMI-1640 medium and foetal bovine serum (FBS) were purchased from Gibio Invitrogen Co. The complete RPMI-1640 medium that was used for immunological tests was supplemented with 10% (v/v) heat-inactivated, endotoxin-free FBS, penicillin (100 IU/ml) and streptomycin (100 g/ml, pH 7.4). Greiss reagent was purchased from Beyotime Institute of Biotechnology (Shanghai, China). All other reagents were of analytical grade.
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2.2. Preparation of oligosaccharides Water-soluble ginseng oligosaccharides (WGOS) were extracted as previously described [9]. Air-dried P. ginseng root was crushed and extracted with 95% ethanol under reflux extraction at 40 ◦ C to remove pigments and small lipophilic molecules. The residue was further extracted thrice (90 min each) with distilled water at 70 ◦ C. All aqueous extracts were combined, concentrated under reduced pressure and then lyophilised to yield crude oligosaccharides. Then, the crude oligosaccharides were re-dissolved in distilled water and dialysed against distilled water for 72 h in a dialysis sack (molecular weight cut-off of 3000 Da). The fraction out of dialysis sack (Mw < 3000 Da) was collected and concentrated at 50 ◦ C in vacuum and then lyophilised. Subsequently, the obtained oligosaccharides were applied to Sephadex G-25 (2.5 cm × 90 cm) and equilibrated with degassed distilled water. The eluate was detected using phenol–sulphuric acid [10]. The appropriate fractions were combined, concentrated and then lyophilised to obtain WGOS. The sugar content in WGOS was determined to be 95.87% by the phenol–sulphuric acid method. The ginsenoside content in WGOS was determined to be 1.43% following the method described by Chen et al. [11]. The protein and uronic acid contents in WGOS were undetectable using the Bradford method and m-hydroxydiphenyl analysis, respectively [12,13].
where A and B are the average tumour weights of the model control and experimental groups, respectively [14]. At 24 h after last drug administration, the blood were collected and centrifuged at 4000 × g at 4 ◦ C for 10 min to separate the serums required. The concentration of TNF-␣ was measured using an enzyme-linked immunosorbent assay according to the manufacturer’s instructions. 2.7. Splenocyte proliferation assay Spleens were aseptically removed from the sacrificed mice, gently homogenised with scissors and forceps in chilled RPMI-1640 medium and then filtered through a sterilised mesh of 200 to obtain single cell suspensions. Erythrocytes in the splenic lymphocytes were lysed by hypo-osmostic haemolysis. After centrifugation at 1000 rpm for 10 min, the cells were suspended to a final density of 5 × 106 cells/ml in a solution of complete RPMI-1640 medium. The purity and viability of splenocytes (tested by trypan blue dye exclusion) were consistently >90%. Spleen cells (100 l/well) were plated in 96-well plates and then incubated with or without ConA (5 g/ml) or LPS (20 g/ml). Cell concentration was determined by MTT assay [15]. The absorbance at 570 nm of the culture was measured in an ELISA reader (TECAN GENios, Switzerland). 2.8. Assay for natural killer (NK) cell cytotoxicity
2.3. Animals ICR male mice weighing 20.0 ± 2.0 g were obtained at 6–8 weeks of age. The mice were purchased from the Changchun Institute of Biological Products Co., Ltd. The mice were randomly housed by tens into cages and received standard mouse chow and water. The room conditions were maintained at room temperature with 12/12-h light–dark cycle. Rodent laboratory mouse chow and water were provided ad libitum. All animals were maintained and used in strict accordance with the PR China legislation on the use and care of laboratory animals and with the guidelines issued by Experimental Animal Centre of Changchun University of Chinese Medicine and approved by the university committee for animal experiments. 2.4. Cell lines and culture H22 cell lines and Yac-1 selected for resistance to NK cell lines were purchased from the Tumour Hospital of Jilin Province, Changchun, Jilin Province, China. The cells were suspended in complete RPMI-1640 medium with 100 IU/ml penicillin, 100 g/ml streptomycin and 10% foetal bovine serum at pH 7.4. 2.5. Treatment Seven-day-old H22 ascites (0.2 ml, 1 × 107 cells/ml) were inoculated into the right armpits of ICR male mice on Day 0; another 10 mice served as normal control. The inoculated mice were divided into five groups (10 mice in each group): three treatment groups, positive control group and model control group. In addition, 1, 12.5 and 25 mg/kg body weight of the WGOS were intragastrically administered to each group from Day 1 to Day 10. The positivecontrol mice received the same volume of cyclophosphamide (CTX, 25 mg/kg body weight), whereas the model control mice and normal control received saline (0.2 ml). 2.6. In vivo anti-tumour activity At 24 h after last drug administration, all mice were sacrificed, and the spleen, thymus and tumour were dissected and weighed. The anti-tumour activity of the oligosaccharides in vivo was expressed as an inhibitory rate calculated as [(A − B)/A] × 100,
Spleens obtained as described above were used as effector cells and seeded into 96-well plates at 2.5 × 105 cells/well in RPMI-1640 complete medium. YAC-1 cells were used as target cells, and 1 × 104 cells/well were added to achieve an E/T (effector/target) ratio of 25:1 [16]. After 20 h of incubation at 37 ◦ C in 5% CO2 atmosphere, each well was added with 20 l of MTT solution (5 mg/ml) and then incubated for another 4 h. The absorbance was measured at 490 nm. Three types of control measurements were performed: target cell control, blank control, and effect cell control. NK cell activity was calculated using the following formula: NK activity (%) =
[ODT − (ODS − ODE )] × 100 ODT
where ODT is the optical absorption value of the target cell control, ODS is the optical absorption value of test samples and ODE is the optical absorption value of the effect cell control. 2.9. Phagocytosis of macrophage assay Peritoneal macrophages were harvested from the H22-bearing mice by peritoneal lavage and centrifugation [17]. Briefly, peritoneal macrophages were harvested by peritoneal washing with 10 ml serum-free RPMI-1640 medium and centrifugation at 1500 rpm for 5 min at 4 ◦ C. Then, the peritoneal macrophages were cultured in complete RPMI-1640 medium in 96-well plates for 2 h at 37 ◦ C in a 5% CO2 cell incubator. Non-adherent cells were removed by washing the plate with warm PBS (0.01 M, pH 7.4). In the aforementioned method, the purity and viability of the peritoneal macrophages were consistently >90%. All cells were cultured at 37 ◦ C in a humidified atmosphere containing 5% CO2 . The number and viability of macrophages were assessed microscopically using trypan blue exclusion [18]. The amount of neutral red dye adsorbed by the macrophages was measured as previously described [19]. The macrophages were plated at a density of 2 × 105 cells/well to a final volume of 100 l in a 96-well plate and then incubated in a medium alone or in a medium with various concentrations of ginseng oligosaccharide. The cells were incubated at 37 ◦ C in humidified 5% CO2 for 24 h, added with 100 l of 0.075% aseptic neutral red solution and then incubated again for 1 h. Subsequently, the plate was washed thrice
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Table 1 Effects of WGOS on thymus, spleen indexes and tumour weights in H22-bearing mice. Group
Dose (mg/kg)
Normal control Model control CTX WGOS
– – 25 1 12.5 25
Tumour weight (g) – 1.31 0.26 0.84 0.55 0.76
± ± ± ± ±
0.18 0.11 0.091 0.07** 0.10**
Thymus index (mg/g) 3.46 2.18 1.40 2.78 3.34 2.88
± ± ± ± ± ±
0. 47 0.0.64 0.35 0.57* 0. 74** 0.53**
Spleen index (mg/g) 13.54 7.82 7.35 9.23 11.82 9.81
± ± ± ± ± ±
1.82 0.53 0.91 0.99 1.93* 0.96*
Inhibitory rate (%) – – 80.29 36.27 58.19** 41.90*
Drug treatment started to H22-bearing mice on Day 1 by oral administration. Normal control and model control group received saline, positive group received cyclophosphamide (CTX, 25 mg/kg), and oligosaccharide groups received the oligosaccharide WGOS (1, 12.5, 25 mg/kg). The dose volume was 0.2 ml. Values were shown as mean ± SD of 10 mice. Significance was determined using the Student’s t-test: * P < 0.05 vs. model control. ** P < 0.01 vs. model control.
with PBS, and 150 l cell lysate (a mixture of 100% ethanol and 99.9% acetic acid (1:1, v/v)) was added. The mixture was mixed fully and evaluated at 550 nm in an ELISA reader (TECAN GENios, Switzerland). The absorbance represented the phagocytic ability of the macrophages. All determinations were conducted in triplicate.
excellent inhibitory activity against solid tumour formation of H22, with inhibitory ratios of 36.27%, 58.19% and 41.90%. Furthermore, the relative spleen and thymus weights of the mice treated with 12.5 and 25 mg/kg WGOS significantly increased (Table 1) compared with that of the model control group (P < 0.05). In addition, the optimum feeding rate of oral administration was 12.5 mg/kg/d.
2.10. NO assay 3.2. Effect of WGOS on TNF-˛ section Peritoneal macrophages obtained by the abovementioned methods were cultured in complete RPMI-1640 medium at 1 × 106 cells per well in a 48-well plate [3]. The cells were cultured for 24 h at 37 ◦ C in humidified 5% CO2 incubator. Then, 100 l of the cell supernatants was removed and mixed with an equal volume of Greiss reagent (1% sulphanilamide in 2.5% phosphoric acid and 0.1% napthyl ethyl diamine dihydrochloride in 2.5% phosphoric acid). Absorbance was read at 540 nm. Nitrite products were calculated by equation from a NaNO2 standard curve. 2.11. Direct cytotoxicity assay The cytotoxicity of WGOS was investigated in H22 tumour cells. Proliferation was determined using the colourimetric MTT assay as previously described [20]. The cells were seeded at a density of 1 × 104 cells/well in 100 l volume of the medium in 96-well plates and then pre-incubated for 24 h. The medium was replaced with fresh medium containing different dosages of WGOS (0.5–100 g/ml), whereas the negative control was treated with the complete RPMI-1640 medium only. Approximately 20 l of MTT (5 mg/ml) was added 48 h later. After 4 h of incubation at 37 ◦ C, the supernatant was aspirated, and 150 l dimethyl sulphoxide was added to each well. Absorbance was measured at 570 nm. All samples were tested in triplicate.
TNF-␣ has an important function in tumouricidal and immune response; it also party mediates tumour cell elimination. Thus, we measured the serum TNF-␣ level. As shown in Fig. 1, the production of TNF-␣ was significantly decreased in the CTX-treated mice compared with the model control (P < 0.05). Thus, CTX could suppress the secretion of the cytokine. WGOS significantly augmented TNF-␣ production and stimulated TNF-␣ level to normal control in the serum of H22-bearing mice at high doses. 3.3. Effect of WGOS on splenocyte proliferation in tumour-bearing mice We evaluated the effects of WGOS on the proliferation of spleen lymphocytes from tumour-bearing mice to confirm the effect of WGOS on the cellular immune response (Table 2). All tested dosages of WGOS (1, 12.5 and 25 mg/kg) significantly increased splenocyte proliferation and Con A-/LPS-induced splenocyte proliferation compared with the model control mice. The results generated a bell-shaped dose–response curve. The splenocyte proliferation
2.12. Statistical analysis Results were expressed as mean ± SD. The statistical significance of the differences between the groups was evaluated by variance analysis, followed by Student’s t-test. The levels of significance were as follows: P < 0.05 is signified by *, P < 0.01 is signified by **. 3. Results 3.1. In vivo anti-tumour activity The anti-tumour activity of WGOS is shown in Table 1. The ICR mice were administered with WGOS orally and continuously with the same dose for 10 d. The tumour weights of the groups treated with 1, 12.5 and 25 mg/kg body weight of WGOS were 0.84, 0.55 and 0.76 g, respectively, whereas that of the negative control group was 1.31 g on Day 10. Therefore, the fraction of WGOS showed
Fig. 1. Concentration of TNF-␣ in serum of H22 tumour-bearing mice. Values are mean ± S.D. from 3 independents (n = 10). WGOS1, P. ginseng oligosaccharide (1 mg/kg) treated group; WGOS12.5, P. ginseng oligosaccharide (12.5 mg/kg) treated group; WGOS25, P. ginseng oligosaccharide (25 mg/kg) treated group; CTX, cyclophosphamide (25 mg/kg) treated group (positive control). *P < 0.05, **P < 0.01 vs. model group.
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Table 2 Effects of WGOS on spleen lymphocyte proliferation in H22-bearing mice. Group
Dose (mg/kg)
Normal control Model control CTX WGOS
– – 25 1 12.5 25
A 570 nm Lymphocyte 0.285 0.148 0.127 0.166 0.211 0.201
± ± ± ± ± ±
0.025** 0.037 0.034 0.043 0.027** 0.044*
ConA 0.303 0.134 0.130 0.207 0.244 0.237
LPS ± ± ± ± ± ±
0.038** 0.009 0.011* 0.073* 0.059** 0.063**
0.351 0.157 0.124 0.193 0.243 0.211
± ± ± ± ± ±
0.072** 0.033 0.014* 0.055 0.053** 0.040*
Drug treatment started to H22-bearing mice on Day 1 by oral administration. Normal control and model control group received saline, positive group received cyclophosphamide (CTX, 25 mg/kg), and oligosaccharide groups received the oligosaccharide WGOS (1, 12.5, 25 mg/kg). The dose volume was 0.2 ml. The lymphocyte proliferation activities were expressed as the absorption at 570 nm. Values are shown as mean ± SD of 10 mice. Significance was determined using the Student’s t-test: * P < 0.05 vs. model control. ** P < 0.01 vs. model control.
index with and without mitogenic stimuli reached the peak at 12.5 mg/kg.
3.4. Effect of WGOS on NK cell activities in tumour-bearing mice Tumour cell elimination can be partly mediated by the cytotoxic activity of NK cells. As shown in Fig. 2, NK cell activity was significantly decreased in both H22-bearing mice and CTX-treated mice. Each concentration of WGOS enhanced NK cell activity in YAC-1 cell lysis at the E:T cell ratio used. In addition, the cytotoxic activity of NK cells from both groups (12.5 and 25 mg/kg) of WGOS-treated mice was significantly different from that of the model control mice (P < 0.01).
3.6. Direct cytotoxicity assay H22 tumour cells were incubated with different concentrations (0.5, 1, 12.5, 25, 50, 100, 200, 400 and 800 g/ml) of WGOS for 72 h and measured by the MTT method. The highest inhibition on H22 cells was 13.6%, suggesting that WGOS had no significant cytotoxicity to H22 cells in vitro, even at the highest concentration of 800 g/ml. By contrast, CTX showed strong cytotoxicity (data not shown).
3.5. Effect of WGOS on peritoneal macrophage activation in tumour-bearing mice Macrophages have essential functions in innate immunity and serve as an important bridge between innate and adaptive immunity. In this study, oral administration of 1, 12.5 and 25 mg/kg WGOS to H22-bearing mice not only improved macrophage phagocytosis but also promoted NO generation, especially at 12.5 and 25 mg/kg (P < 0.01). The highest stimulation index was reached at 12.5 mg/ml (Fig. 3A and B).
Fig. 2. Effect of WGOS on cytotoxicity of NK cells from H22 tumour-bearing mice. Values are means ± S.D. from 3 independents (n = 10). WGOS1, P. ginseng oligosaccharide (1 mg/kg) treated group; WGOS12.5, P. ginseng oligosaccharide (12.5 mg/kg) treated group; WGOS25, P. ginseng oligosaccharide (25 mg/kg) treated group; CTX, cyclophosphamide (25 mg/kg) treated group (positive control). *P < 0.05, **P < 0.01 vs. model group.
Fig. 3. (A) Effects of different doses of WGOS on phagocytosis of murine peritoneal macrophages from H22 tumour-bearing mice. Phagocytosis was evaluated as OD 550 nm. Values are mean ± S.D. from 3 independents (n = 10). WGOS1, P. ginseng oligosaccharide (1 mg/kg) treated group; WGOS12.5, P. ginseng oligosaccharide (12.5 mg/kg) treated group; WGOS25, P. ginseng oligosaccharide (25 mg/kg) treated group; CTX, cyclophosphamide (25 mg/kg) treated group (positive control). *P < 0.05, **P < 0.01 vs. model group. (B) Effects of different doses of WGOS on NO level of murine peritoneal macrophages from H22 tumour-bearing mice. Values are mean ± S.D. from 3 independents (n = 10). WGOS1, P. ginseng oligosaccharide (1 mg/kg) treated group; WGOS12.5, P. ginseng oligosaccharide (12.5 mg/kg) treated group; WGOS25, P. ginseng oligosaccharide (25 mg/kg) treated group; CTX, cyclophosphamide (25 mg/kg) treated group (positive control). *P < 0.05, **P < 0.01 vs. model group.
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4. Discussion Oligosaccharides have long been considered as a healthy food source whose biological functions are limited to antigenic properties of various blood groups [21]. However, oligosaccharides have recently been extensively studied by experts because of their biological and physiological activities, such as anti-tumour [22], antioxidant [23] and immunoregulatory [24]. According to various investigations, oligosaccharides from various sources exhibit potent immunostimulating activities [25]. An aqueous extract containing mainly oligosaccharides and polysaccharides from North American ginseng (Panax quinquefolium), a species that belongs to the same family as P. ginseng, can stimulate the proliferation of normal mouse spleen in vitro [26]. Murosaki et al. also demonstrated that nigero-oligosaccharides exert immunopotentiating activity by activating an IL-12-dependent T-helper-1-like immune response [27]. The present study showed that ginseng oligosaccharides can reliably inhibit the growth of mouse transplantable H22 cells and improve the weight of immune organs of tumour-bearing mice. The relative spleen and thymus weights in the WGOS test groups were significantly higher than those of the control groups. Thymus and spleen are important immune organs; the thymus and spleen indices reflect the immune function of the organism. Immunopotentiation can increase the weights of thymus and spleen. Furthermore, the TNF-␣ level in the serum of WGOS-treated H22 murine serum was significantly increased. These results suggest that TNF-␣ is involved in the immune response. In addition, the appetite, activity and coat lustre of each animal in the WGOS groups were better than those of the mice treated with saline and CTX. Furthermore, WGOS did not directly kill the tumour cells. Thus, the anti-tumour activity of WGOS can be ascribed to their capacity to stimulate the immune system. Thus, WGOS can have great significance in tumour growth therapy. The specific immune responses include humoural immunity and cellular immunity. The humoural immune response by B cells is an antigen-specific antibody reaction. Cellular immunity can be mediated specifically by T cells, including NK cells. Lymphocyte proliferation induced by antigen or mitogens is an indicator of immunoenhancement. Cellular multiplication induced by Con A can be used as a method to detect T lymphocyte activity in vitro. Moreover, the LPS-induced activation of B cells and subsequent immunoglobulin synthesis reflect B lymphocyte immunity. Extensive in vivo and in vitro evidence demonstrated that oligosaccharides display immunomodulating function by stimulating cellular and humoural immune responses [28,29]. The present results showed that ginseng oligosaccharides can augment splenocyte proliferation and NK cell activity in H22-bearing mice. Therefore, ginseng oligosaccharides can enhance the humoural immunity and cell-mediated immunity in H22-bearing mice. However, significant signs of immunosuppressive effect were observed in the mice treated with the positive control CTX based on the activities of splenocyte proliferation and NK cells. Macrophages are fundamental in maintaining homeostasis and perform essential and pivotal functions in host defence. The innate defence response can be activated through the interaction of sugar moieties with innate receptors in the plasma membrane of macrophages; this interaction was assumed to be the immunoregulatory mechanism of several oligosaccharides [30]. Zhang et al. described that synthetic -(1 → 6)-branched -(1 → 3) glucohexaose can stimulate innate immune reactions by binding to selective receptors (such as dectin-1) mainly expressed on M2 macrophages [31]. Murosaki et al. also found that an
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oligosaccharide containing ␣-(1 → 3) linked-glucose increases the production of endogenous IL-12 that is locally produced by activated resident macrophages [27]. The present study showed that WGOS, a mixture of glucose units, has a positive effect on macrophage activation in H22-transplanted mice. WGOS enhanced the phagocytic effect of macrophages. In addition, NO production increased in the WGOS-treated (12.5–25 mg/kg) groups. Thus, the oligosaccharides from P. ginseng could indirectly exhibit antitumour activity through the activation of macrophages. In summary, our findings clearly showed that WGOS significantly inhibited tumour growth in vivo by enhancing the immune system instead of eliciting direct cytotoxicity. Acknowledgements We are grateful for the financial support of this research from Scientific Research Foundation of Jilin Provincial Science & Technology Department of China (Grant No. 20130202059YY) and Program for Innovative Research Team (in Science and Technology) in University of Jilin Province (No. JTD201213). References [1] H. Mushiake, T. Tsunoda, M. Nukatsuka, K. Shimao, M. Fukushima, H. Tahara, Cancer Immunol. Immunother. 54 (2005) 120–128. [2] S.B. Han, C.W. Lee, M.R. Kang, Y.D. Yoon, J.S. Kang, K.H. Lee, W.K. Yoon, K. Lee, S.K. Park, H.M. Kim, Cancer Lett. 243 (2006) 264–273. [3] D. Ghosh, T.K. Maiti, Immunobiology 212 (2007) 589–599. [4] B.Z. Zaidman, M. Yassin, J. Mahajna, S.P. Wasser, Appl. Microbiol. Biotechnol. 67 (2005) 453–468. [5] B. Faanes, V.J. Merluzzi, P. Ralph, in: B. Serrou, C. Rosenfield (Eds.), International Symposium on New Trends in Human Immunology and Cancer Immunotherapy, Doin, Paris, 1980, pp. 953–964. [6] T.K. Yun, S.Y. Choi, Int. J. Epidemiol. 19 (1990) 871–876. [7] A.S. Attele, J.A. Wu, C.S. Yuan, Biochem. Pharmacol. 58 (1999) 1685–1693. [8] D.B. Wan, L.L. Jiao, H.M. Yang, Y.L. Shu, Planta 235 (2011) 1289–1297. [9] L.L. Jiao, D.B. Wan, X.Y. Zhang, B. Li, H.X. Zhao, S.Y. Liu, J. Ethnopharmacol. 144 (2012) 490–496. [10] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Anal. Chem. 28 (1956) 350–356. [11] R.Z. Chen, F.L. Meng, S.Q. Zhang, Z.Q. Liu, Sep. Purif. Technol. 66 (2009) 340–346. [12] J.J. Sedmark, S.E. Grossberg, Anal. Biochem. 79 (1979) 544–552. [13] M.C.C Tullia, C. Filisetti, Anal. Biochem. 197 (1991) 157–162. [14] K.C. Mousinho, C.C. Oliveira, J.R. Ferreira, A.A. Carvalho, H.I. Magalhães, D.P. Bezerra, A.P.N.N. Alevs, L.V. Costa-lotufo, C. Pessoa, M.P.V. Matos, M.V. Ramos, M.O. Moraes, J. Ethnopharmacol. 137 (2011) 421–426. [15] C.M. Chien, J.L. Cheng, W.T. Chang, M.H. Tien, C.M. Tsao, Y.H. Chang, Bioorg. Med. Chem. 12 (2004) 5603–5609. [16] L. Cao, X.Z. Liu, T.X. Qian, G.B. Sun, Y. Guo, F. Chang, S. Zhou, X. Sun, Int. J. Biol. Macromol. 48 (2011) 160–164. [17] W.C.S. Cho, K.N. Leung, Cancer Lett. 252 (2007) 43–54. [18] D.L. Felice, J. Sun, R.H. Liu, J. Funct. Food 1 (2009) 109–118. [19] B.A. Weeks, A.S. Keisler, Q.N. Myrvik, Dev. Comp. Immunol. 11 (1987) 117–124. [20] A. Akrout, L.A. Gonzalez, H.E.P. Jani, C. Madrid, Food Chem. Toxicol. 49 (2011) 342–347. [21] A.C. Weymouth-Wilson, Nat. Prod. Rep. 14 (1997) 99–110. [22] H.M. Yuan, J.M. Song, X.G. Li, N. Li, J.C. Dai, Cancer Lett. 243 (2006) 228–234. [23] B.R. Veenashri, G. Muralikrishna, Food Chem. 126 (2011) 1475–1481. [24] S.M. Zhou, X.Z. Liu, Y. Guo, Q. Wang, D.Y. Peng, L. Cao, Carbohydr. Polym. 81 (2010) 784–789. [25] X. Qiang, Y.L. Chao, Q.B. Wan, Carbohydr. Polym. 77 (2009) 435–441. [26] M.Q. Wang, L.J. Guilbert, L. Ling, J. Li, Y.Q. Wu, S. Xu, P. Pang, J.J. Shan, J. Pharm. Pharmacol. 53 (2001) 1515–1523. [27] S. Murosaki, K. Muroyama, Y. Yamamoto, H. Kusaka, T. Liu, Y. Yoshikai, Biosci. Biotechnol. Biochem. 63 (1999) 373–378. [28] J. Yan, H.L. Zong, A.G. Shen, S. Chen, X.L. Yin, X.Y. Shen, W.C. Liu, X.S. Gu, J.X. Gu, Int. Immunopharmacol. 3 (2003) 1861–1871. [29] H.J. Chen, B.Q. Yuan, Z.C. Zheng, Z. Liu, S.S. Wang, X. Lewis, Cell Immunol. 269 (2011) 144–148. [30] J.N. Arnold, R.A. Dwek, P.M. Rudd, R.B. Sim, Immunol. Lett. 106 (2006) 103–110. [31] M. Zhang, P.C.K. Cheung, L.C.M. Chiu, E.Y.L. Wong, V.E.C. Ooi, Carbohydr. Polym. 66 (2006) 455–462.
