Immunological activities of polysaccharide extracted from Elaeagnus angustifolia L.

ABSTRACT The study was to investigate the immunological activities of polysaccharide isolated from Elaeagnus angustifolia L. The extracted Elaeagnus angustifolia L. polysaccharide (EAP) was purified by DEAE cellulose-52 chromatography (DEAE-52) and Sephadex G-100 size-exclusion chromatography (G-100). Then the average molecular weight (Mw) of purified polysaccharide was determined by high-performance gel permeation chromatography (HPGPC). One main fraction (EAP-1a) was obtained through the isolation and purification steps. The results showed that EAP-1a had strong immunological activities. EAP-1a could significantly increase spleen and thymus index, enhance the NK cell activity, stimulate the phagocytosis of peritoneal phagocyte and increase the level of IL-2, IFN-γ and IgG in serum. Thus, EAP-1a isolated from Elaeagnus angustifolia L. could enhance the immunological activities of immunosuppressed mice induced by cyclophosphamide (CY).

At present, the research of Elaeagnus angustifolia L. mainly stays on the phytochemical constituents, nutritional and medicinal value, exploitation and application, while the study of immunological activities of Elaeagnus angustifolia L. polysaccharide (EAP) is still rare. Therefore, the aim of this study is to investigate the biological activities of EAP which can provide a certain theoretical basis for further research.

Materials and chemicals
Elaeagnus angustifolia L. was collected from Kashi Prefecture, Xinjiang Province, China. The fruit pulp was dried and pulverized into powder for the next experiment. It was authenticated by professor Hui-ping Liu (Tianjin University of Science & Technology, Department of Food Engineering and Biotechnology, China). The plant materials were deposited at Tianjin University of Science & Technology.

Extraction, isolation and purification of the polysaccharide
The dry powder (20 g) of Elaeagnus angustifolia L. pulp was used to extract polysaccharide with distilled water (1:20, w/v) for 3 h at 80℃. We repeated this process twice. The extract solution was merged and removed protein by Sevage (chloroform-butanol 4:1, v/v) method. The above solution was concentrated by rotary vacuum evaporator and precipitated by adding 3-fold volume of 95% ethanol for 24 h at 4℃. After centrifugation, the precipitate was dialyzed and lyophilized to get crude polysaccharide (EAP). The polysaccharide content was quantified by the phenol-sulfuric acid method (Masuko et al., 2005).
The crude polysaccharide dissolved with distilled water (5 mg/mL) was filtered through 0.45 μm filters and fractionated with a DEAE-52 cellulose column (1.2 × 50 cm) equilibrated with distilled water. The column was eluted with sodium chloride solution (0-0.3 M) at a flow rate of 1.0 mL/min. We obtained three fractions (EAP-1, EAP-2 and EAP-3). The relevant fractions were merged, concentrated, dialyzed and lyophilized. The EAP-1 (5 mg/mL) was further purified by size-exclusion chromatography by a Sephadex G-100 column (2.6 × 50 cm) and eluted with distilled water at a flow rate of 1.0 mL/min. We obtained two fractions (EAP-1a and EAP-1b). The major fraction EAP-1a was merged, concentrated, dialyzed and lyophilized for further experiment.

Immune organ indexes
After 24 h of last gavage, we weighted the body weight of mice and collected blood from retroocular venous plexus. The serum was collected after centrifuged at 4°C, 1000 × g for 15 min. Then, the mice were dislocated to death; then, thymus and spleen were quickly gathered from each mouse. The immune organ index was calculated using the following equation:

Lymphocytes proliferation assay
Spleen of each mouse was washed, ground, collected with 5 mL RPMI-1640 medium and passed through 200 nesh sieve in clean bench. The cell suspension was precipitated for five min in order to remove large tissue. Then, the upper suspension was collected and centrifuged at 4°C, 300 × g for five min. After abandoning the supernatant, the precipitation was added 3 mL erythrocyte lysis buffer for five min, then 3 mL RPMI-1640 medium was added to finish lysis. According to the effect of lysis, repeat above process one or two times. After centrifugation, the precipitation was washed with RPMI-1640 medium for twice. Then, the precipitation was re-suspended in RPMI-1640 medium containing 10% fetal bovine serum (FBS) to obtain splenocyte suspension. The cells were stained with trypan blue and adjusted the cells (survival rate more than 95%) concentration to 2 × 10 7 cells· mL −1 . The splenocyte suspension (100 μL· well −1 ) were added to 96-well plates with Con A (final concentration 5 μg· mL −1 , 100 μL· well −1 ) or LPS (final concentration 10 μg· mL −1 , 100 μL· well −1 ). The control wells were added RPMI-1640 solution (100 μL· well −1 ). After incubating at 5% CO 2 incubatorat for 44 h, MTT (5 mg· mL −1 , 20 μL· well −1 ) was added to each well and the plates were incubated for another four hours. After that, DMSO (150 μL· well −1 ) was added to each well, and the 96-well plates were shaken for 20 min. At last, the absorbance was measured at 570 nm by microplate reader (Bio-Rad, Hercules, CA). The enhancement for lymphocyte proliferation was expressed as a stimulation index (SI).

NK cells activity
The preparation of splenocyte suspension was same with lymphocytes proliferation assay. The spleen cells kept in Ficoll-Hypaque were centrifuged at 700 × g for 20 min to obtain the low-density cells. The low-density cells were centrifuged and washed twice with RPMI-1640 medium to obtain NK cells. Then, the cells concentration of NK (effector cells) and YAC-1 (target cells) were adjusted to 2 × 10 6 cells· mL −1 and 1 × 10 5 cells· mL −1 (effector-to-target rate was 20:1). The test wells were added NK cells and YAC-1 to 96-well plates. The effector control wells were added NK cells and RPMI-1640 medium. The target control wells were added YAC-1 and RPMI-1640 medium. The adding volume of cells was 100 μL·well-1. Then MTT (5 mg mL −1 , 10 μL well −1 ) was added and the 96-well plates were incubated in a incubatorat with 5% CO 2 for four hours at 37°C. After centrifugation, 150 µL DMSO was added to each well. The absorbance was measured at 570 nm by microplate reader. The activities of NK cells were calculated using the following equation:

Macrophage phagocytosis assay
Phagocytosis of peritoneal macrophages was measured referred to in references with minor modification (Chen, Zhang, Shen, & Wang, 2010;Liu et al., 2017). 5 mL saline was injected to abdominal cavity of each mouse. The peritoneal cells were collected and centrifuged at 37°C, 300 × g for five min. After supernatant was removed, the macrophages were washed twice, resuspended in RPMI-1640 medium (containing 10% FBS) and adjusted the cells concentration to 1 × 10 6 cells· mL −1 . The test wells were added macrophages (100 μL well −1 ) and the blank wells were added RPMI-1640 medium (100 μL well −1 ). The 96-well plates were cultured at 37°C in a 5% CO 2 incubator for four hours. Non-adherent cells were removed by washing twice with preheated RPMI-1640 medium. Then, 200 μL neutral red solution (0.075%, m/v) was added to each well and the cells were incubated for another four hours. After that, the cultures were washed by medium three times. Then, 100 μL of cell lysis solution (ethanol: acid = 1:1) was added to each well overnight at 4°C. The absorbance was measured at 540 nm using microplate reader. Macrophage phagocytosis activity was calculated according to the following equation: Macrophage phagocytosis activity ¼ OD test ÀOD blank

Determination of IL-2, IFN-γ and IgG in peripheral blood
After 24 h of last gavage, we collected blood from the retroocular venous plexus of mice. The serum was collected after centrifuged at 4°C, 1000 × g for 15 min. The IFN-γ, IL-2 and IgG concentration in serum were measured with an enzyme-linked immunosorbent assay (ELISA kit, Rapidbio) according to the indication of the manufacturer.

Statistical analysis
The results were expressed as mean ± standard deviation and were analyzed using t-test and one-way analysis of variance (ANOVA), using IBM SPSS Statistics 19.0 for meaning differences among the samples. p < 0.05 were considered to be statistically significant.

Extraction, purification and molecular weight
In the present study, the extracted yield of crude polysaccharide was 5.9% and the polysaccharide content of the crude polysaccharide was 79.25%. Furthermore, the crude polysaccharide was firstly separated through an anionexchange chromatography of DEAE-52. We obtained three independent elution peaks: EAP-1, EAP-2 and EAP-3 as detected by the phenol-sulfuric acid assay (Figure 1).
The EAP-1 fraction (relative content 44.56%, polysaccharide content 87.22%, no protein) was collected and purified by gel filtration chromatography Sephadex G-100. In Figure 2, EAP-1 generated two fractions (EAP-1a and EAP-1b). The big peak named as EAP-1a was collected for further study. The high-performance gel permeation chromatography (HPGPC) was applied to determine the average molecular weight (Mw) of EAP-1a (Figure 3). The average molecular weight of EAP-1a was calculated to be 10.5 kDa. The structural characterization of EAP-1a was published in our early work (Liu et al., 2015).

Immunostimulatory activity in vivo
The mammalian immune system is comprised of two branches: innate and acquired immunity. The innate immune system includes phagocytes (macrophages), NK cells, cytokines (IFN-γ and IL-2) and so on. Acquired immunity is involved in elimination of pathogens in the late phase of infection as well as the generation of immunological memory, which includes humoral immunity mediated by B cells and cell immunity mediated by T cells and NK cells (Akira, Uematsu, & Takeuchi, 2006). Exogenous substances, for instance, polysaccharides, could trigger the human immune system to play their biological roles (Bradford, 1976;Brown & Gordon, 2005;Yang, Zhao, Shi, Yang, & Jiang, 2008).

Effect of immune organ index in mice
The spleen and thymus are major organs of the immune system, determination of thymus and spleen weight can reflect the effects of drugs on the immune organs, viscera index is one of the indicators to measure the immune function (Fu et al., 2017;Zalys, Zagon, Bonneau, Lang, & Mclaughlin, 2000).
In the study, we used cyclophosphamide (CY) to establish the model of immunosuppression in mice. No mortality and any adverse reactions happened. After 10 days of gavage treatment, the effects of EAP-1a on the spleen and thymus index were measured. Figure 4 shows that the spleen index and thymus index of model group decreased significantly when compared to control group (p < 0.05). The spleen index of EAP-1a-L, EAP-1a-M and EAP-1a-H had significant increased when compared to model group (p < 0.05). While the thymus index, only EAP-1a-M and EAP-1a-H had significant increased when compared to model group (p < 0.05). Besides, EAP-1a-H had the highest spleen index and thymus index compared with other groups except the control group.
As a cytotoxic drug, CY can damage the structure of DNA, cut off its copy and result in cell death in vivo (Deng et al., 2018;Martello, David, & Matuo et al., 2016). It is widely used in the treatment of autoimmune diseases and cancer (Manente, Quinello, & Ferreira et al., 2017;Yin, Zhou, & Wang et al., 2016). Previous research revealed that high doses of CY could lead to suppression of immune system (Sun, Yang, & Pan et al., 2018). Because of immunosuppression, the major immune organs (spleen and thymus) were destroyed (Cheng et al., 2017;Duggina, Kalla, Varikasuvu, Bukke, & Tartte, 2015), resulting in the decrease of spleen index and thymus index, while EAP-1a could effectively protect these immune organs.

Effect of splenic lymphocyte proliferation
Proliferation of splenic cells is an important part in the activation pathway of cellular or humoral immunity (Zhao, Li, & Luo et al., 2006). The ability of lymphocytes to respond to mitogen reflects the immune potential of the organism (Singh, Haldar, & Rai, 2006). The mitogen ConA and LPS were frequently used to stimulate T and B lymphocyte proliferation, respectively (Cerqueira, Cordeirodasilva, & Gasparmarques et al., 2004). Proliferative capacity of splenic cells has been widely used in screening novel immune stimulants (Lee, Lee, & Park et al., 2007). The effects of EAP-1a on the splenic lymphocyte proliferation were investigated by comparing the stimulation indexes of T lymphocytes and B lymphocytes stimulated by ConA and LPS. Table 1 shows that the lymphocyte proliferation of model group decreased significantly when compared with the control group (p < 0.05). The study demonstrated that cyclophosphamide (CY) could suppress splenic lymphocyte proliferation. This result was in accordance with previous reports (Cho et al., 2014;Moynihan & Cohen, 1989) that mitogen-stimulated T and B lymphocytes proliferation could be suppressed by CY administration. Moreover, treatment with EAP-1a could significantly increase ConA or LPS      stimulated splenic lymphocyte proliferation compared with the model group (p < 0.05). In the three EAP-1a groups, high-dose EAP-1a group presented the strongest promoting effect, while low-dose EAP-1a group showed the weakest stimulating activity whether stimulated by ConA or LPS. In the other words, as the dose of EAP-1a increase, the capacity of the proliferation of T and B lymphocytes was increased.
CY can inhibit T lymphocyte and B lymphocyte proliferation through alkylating the DNA of the splenic cell (Artym, Zimecki, & Kruzel, 2004), while the study indicated that EAP-1a could promote the mitogen-stimulated proliferation of splenic lymphocyte. Previous studies showed that polysaccharides had the ability of activating lymphocytes and promoting their proliferation. Chen, Tan & Chan (2008) indicated that polysaccharides isolated from lycium barbarum L. significantly promoted the proliferation of T lymphocytes, and the signaling pathways that activated T lymphocytes might be achieved by activating NFAT, ap-1, and CD25. Hu and Zheng (2004) showed that sophora subprosrate polysaccharides enhanced the activity of PKC on T lymphocyte membrane and the concentration of free Ca 2+ in cells, and Ca 2+ was a key factor to activate T lymphocytes. Han et al. (2001) indicated that polysaccharides isolated from the radix of platycodon grandiflorum significantly enhanced the proliferation of B lymphocytes in mice, and the pathway of activated B lymphocytes was related to CD19 and CD79 receptors. Shao et al. (2004) showed that astragalus membranaceus polysaccharides stimulated B lymphocyte proliferation and regulated B lymphocyte activity in BALB/c mice by binding to mIg receptor. Han et al. (2003) indicated that polysaccharides isolated from acanthopanax senticosus could bind to the TLR2/4 receptor on the surface of B lymphocytes to promote the proliferation of B lymphocytes.

Effect of NK cells activity
Natural killer (NK) cells are non-specific immune killer cells which belong to the innate immune system . It can kill target cells directly without antibodies participation (Pellicci et al., 2002). NK cells have been recognized as the most ideal effect cells in immune surveillance cells that can regulate the function of T cells, B cells and bone marrow stem cells (Subleski, Wiltrout, & Weiss, 2009).
As shown in Figure 5, the NK cells activity of mice in model was significantly lower than that of control group (p < 0.05) and EAP-1a group (p < 0.05). In addition, the NK cells activity of EAP-1a-L, EAP-1a-M and EAP-1a-H groups were dose dependent. Besides, the NK cells activity of the high EAP-1a group was considerable compared with control group.
In our study, EAP-1a could enhance the immunological activity of mice by improving the activity of NK cells. The reason why NK cell activity increased might be that EAP-1a activated NK cells and promoted their proliferation. Tsai et al. (2012) drew the similar results that oligosaccharide and peptidoglycan of ganoderma lucidum could activate the immune response of NK cells and promote their proliferation (Tsai et al., 2012).

Effect of macrophage phagocytosis
Macrophage is the most important profession phagocyte and it plays an essential and pivotal role in host defense against any type of invading cells including tumor cells (Katsiari, Liossis, & Sfikakis, 2010). As shown in Figure 6, the activity of peritoneal macrophages in the model was significantly lower than that of the control group (p < 0.05). Besides, it indicated that EAP-1a group had the activity of promoting peritoneal macrophages to devour neutral red and caused a significant increase compared with the model group (p < 0.05). The administration of EAP-1a-H group presented the strongest phagocytic activity compared with the other two EAP-1a groups.
Recent studies have shown that the main function of polysaccharides is to activate the immune response of macrophages (Schepetkin et al., 2008). Lee et al. (2004) indicated that polysaccharides isolated from poria cocos activated macrophages through the TLR4-mediated p38 signaling pathway. Li and Xu (2011) showed that polyporus umbellatus (pers.) fries polysaccharides activated macrophages in mice through the NF-κB signaling pathway mediated by TLR4 receptor. Han et al. (2001) indicated that polysaccharides isolated from the radix of platycodon grandiflorum activated macrophages through CD14 and CR3. Nakamura, Suzuki, Wada, Kodama, and Doi (2006) showed that fucosan polysaccharides activated macrophages to release NO through SR receptors. Once macrophages are activated, they can release a significant amount of NO and IL-2, which contributes to devour pathogens (  Note: a p < 0.05 when compared with control; b p < 0.05 when compared with model. Nota: a p < 0.05 comparado con el control; b p < 0.05 comparado con el modelo. Figure 5. Effects of EAP-1a on NK cell activity. Note: a p < 0.05 when compared with control; b p < 0.05 when compared with model.

Levels of IL-2, IFN-γ and IgG in mice serum
Previous reports showed that immune cells (T cells, B cells and NK cells) can synthesize and secret cytokines and cytokines can regulate immunological activities (Schoenaker et al., 2018). IL-2 could promote the killing activity of T cells, stimulate differentiation and activation of B lymphocytes and NK cells. INF-γ is an efficient antiviral cytokine, as well as has broad-spectrum immune regulative effects (Zhou et al., 2018). As shown in Table 2, the result showed that cyclophosphamide (CY) could decrease the immunological activity of mice by reducing the serum cytokine levels. While the levels of IL-2, IFN-γ and IgG in serum treated with different doses of EAP-1a significantly increased compared with the model group (p < 0.05). And the high-dose EAP-1a group had the highest cytokines level and low-dose EAP-1a group had the lowest cytokines level.

Conclusions
In this study, the results showed that EAP-1a had beneficial effect on immunological activity of cyclophosphamideinduced immunosuppression mice. Therefore, EAP-1a may be explored as an immunomodulator in the field of pharmaceutical. The clinical significance of these findings should be taken more attention in further investigation.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the Tianjin Science and Technology Commission [17ZXYENC00010]. Figure 6. Effects of EAP-1a on phagocytosis of macrophage. Note: a p < 0.05 when compared with control; b p < 0.05 when compared with model. Figura 6. Efectos de la EAP-1a en la fagocitosis del macrófago. Nota: a p < 0.05 comparado con el control; b p < 0.05 comparado con el modelo. Note: a p < 0.05 when compared with control; b p < 0.05 when compared with model. Nota: a p < 0.05 comparado con el control; b p < 0.05 comparado con el modelo.