Immunomodulatory potentials of the water-soluble yam (Dioscorea opposita Thunb) polysaccharides for the normal and cyclophosphamide-suppressed mice

ABSTRACT

Immunomodulatory potentials of water-soluble yam (Dioscorea opposita Thunb) polysaccharides (WYPs) for the normal and immuno-suppressed mice were described in this study. For the normal mice, the WYPs at 500 mg kg⁻¹ elevated spleen and thymus indices by 22–42%; promoted macrophages' phagocytosis; lymphocytes' proliferation and natural killer (NK) cell activity by 18–53%; enhanced IL-2 and IFN-γ levels in the splenocytes by 42–45%; raised IL-1β, IL-6, TNF-α, iNOS and lysozyme levels in the macrophages by 40–219%; and increased serum IgM, IgA and IgG levels by 44–51%. The WYPs could restore and improve immune status of the cyclophosphamide-treated mice, as they at 500 mg kg⁻¹ elevated spleen and thymus indices by 85–172%; promoted macrophages' phagocytosis, lymphocytes' proliferation and NK cell activity by 24–98%; enhanced IL-2 and IFN-γ levels in the splenocytes by 44–109% or IL-1β, IL-6, TNF-α, iNOS and lysozyme levels in the macrophages by 53–287%; and increased the three immunoglobulins by 24–69%.

KEYWORDS:

• Yam polysaccharides
• immune activity
• mice model
• cyclophosphamide

1. Introduction

Cancer is the second leading cause of death in the developing countries (Thun, DeLancey, Center, Jemal, & Ward, 2010). Chemotherapeutic agents used for cancer treatment can cause some adverse problems such as nausea, fatigue and immuno-suppression (Sitzia & Huggins, 1998). Cyclophosphamide (CY) as a mainstay cancer chemotherapy agent has been used extensively in clinics to treat various types of cancer (Ehrke, 2003). However, CY has cytotoxic side (Emadi, Jones, & Brodsky, 2009; Pass et al., 2005). Some clinic indices such as body weight, relative weights of spleen and thymus, differential leukocytes, total leukocyte count, B-cell and T-cell proliferation, and the activity of natural killer (NK) cells might be decreased by high doses of CY (Hussain, Shadma, Maksood, & Ansari, 2013). At the same time, CY is also capable of damaging the structure of DNA, killing immune cells, interfering with the proliferation and differentiation of macrophages, T cells as well as B cells, and restraining the humoral and cellular immune responses (Atsamo, Nguelefack, Datté, & Kamanyi, 2011; Wang et al., 2011). If immune system of the host is once impaired by the chemotherapy, the incidence of infections and immuno-deficiency will be increased (Ramioul & Zutterman, 1961). However, immuno-modulating agents such as these native polysaccharides of different resources can reduce these side effects, and thus enhance the curative effects of the chemotherapy.

Plant polysaccharides are one kind of the most important components in traditional Chinese medicine (Sun et al., 2015). These polysaccharides not only elicit antiviral effects, but also have immune-enhancing effects, via regulating cytokines, lymphocytes and antibody levels. Polysaccharides can influence specific, non-specific, humoral, and cellular immunity (Li et al., 2011; Liu et al., 2015). Many studies show that plant polysaccharides are effective biological response modifiers with low toxicity, and can be served as immuno-modulating agents (Meng et al., 2014; Yang, Wang, Li, & Yu, 2015). Immune modulation is the most important function of the plant polysaccharides, and has become a hot topic in the recent years (Abula et al., 2011; Dobreva, Popov, Georgieva, & Stanilova, 2015; Guo, Zhang, Yan, & Tong, 2008; Sultan, Buttxs, Qayyum, & Suleria, 2014; Zhang, Li, Smith, & Musa, 2015). Among these studied plant polysaccharides are the polysaccharides from yam (Dioscorea opposite Thunb) (Zava, Dollbaum, & Blen, 1998), because yam is one of the well-known edible and traditional medicinal plants in China. Yam contains flavonoids, allantoin and other bioactive components (Wang, Yu, Gao, Liu, & Xiao, 2006) and especially water-soluble polysaccharides (important biological macromolecules) in yam mucilage (Zhao, Kan, Li, & Chen, 2005). Yam has been thus well-studied for its antioxidant, anticancer, and immune activities (Jeon et al., 2006; Zhao et al., 2005). The soluble yam polysaccharides have molecular weights of 17–42 kDa, and contain glucose, mannose, and galactose units in their structure (Yang et al., 2015; Zhao et al., 2005). The structure of water-soluble yam polysaccharides (WYPs) is resistant to the milder digestion (e.g. by a diluted HCl solution, proteases, or amylases) and long-time thermal treatments (e.g. at 100°C for 30 or 60  min), but very sensitive to the severe digestion (e.g. by a HCl solution of 3 mol L⁻¹) (Hao & Zhao, 2015), which verify that the WYPs will keep their structure and immune activities during thermal processing and body digestion. However, in vivo immune activities of the WYPs are not well-assessed so far.

In the immune system of the body, both lymphocytes and macrophages are important immune cells. Macrophages can remove foreign substances, and produce various effector molecules such as nitric oxide synthase (iNOS), lysozyme (LZM), and cytokines, which all can protect the host (Sideras et al., 2014). Lymphocytes are the major cellular components of the adaptive immune response (Sideras et al., 2014). Several immune molecules such as IL-2, IL-4, IFN-γ, IL-1β, IL-6, TNF-α, iONS, LZM, IgM, IgA, and IgG are also very important to the immune system (Audibert & Lise, 1993; Zhao et al., 2014). These cells and factors have been used to reflect in vitro and in vivo immune activities of the plant polysaccharides from Ganoderma atrum, Cyrtomium macrophyllum, and Radix Cyathulae officinalis Kuan (Feng et al., 2014; Ren et al., 2014; Yu et al., 2015). In vivo immune activities of the WYPs toward the mice model should be studied via evaluating corresponding indices of these cells and factors.

In this study, the WYPs were assessed for in vivo immunomodulatory potentials for the normal and CY-treated mice. Some immune indices of the mice model – including spleen and thymus indices, macrophages phagocytosis, lymphocytes proliferation, NK cell activity, levels of IL-2, IL-4, IFN-γ, IL-1β, IL-6, TNF-α, iONS, LZM and three immunoglobulins (IgM, IgA and IgG) – were evaluated and compared, to reflect in vivo immune responses of the mice model toward the WYPs. This study aimed to verify if the WYPs are beneficial to the immune status of the normal mice, or especially if they can restore and improve the immune status of the immuno-suppressed mice.

2. Materials and methods

2.1. Materials and reagents

Fresh yam (Dioscorea opposite Thunb) was purchased from Henan Province, China. Fetal bovine serum (FBS) and RPMI-1640 were obtained from Thermo Fisher Scientific Inc. (Cleveland, OH, USA) and HyClone Co. (Logan, Utah, USA), respectively. Concanavalin A (ConA) was obtained from Sigma Chemical Co. (St. Louis, MO, USA). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kyushu, Japan). The mouse iNOS, LZM and immunoglobulins test kits were bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All cytokines assaying kits were bought from Wuhan Boster Biological Technology Co. Ltd. (Wuhan, China). The water used in this study was redistilled or ultrapure water, while other chemicals used were analytical grade.

Female BALB/c mice (7 weeks old, 18–21 g) were provided by Beijing Vital River Experimental Animal Technical Co. Ltd (Beijing, China). The YAC-1 cells used in the assay of NK cells activity were purchased from Chinese Academy of Sciences (Shanghai, China).

2.2. WYPs preparation

The fresh yam was washed by drinking water to remove the dust in the surface, and then rewashed by the redistilled water. The washed yam was crushed into paste, and then suspended in redistilled water with a mass proportion of 1:10. The mixture was adjusted into pH 4.5 using a HCl solution of 0.2 mol L⁻¹, kept at 100°C for 3 h, cooled and then centrifuged at 12,000 × g for 15 min to collect supernatant. The proteins in the supernatant were removed using the Sevage agent (chloroform: n-butyl alcohol, 5:1, v/v) and an alkaline protease Alcalase. The treated supernatant was then concentrated at 100°C into one fifth of its original volume. Three volumes of 95% ethanol was added into the concentrated supernatant and kept at 4°C for 24 h. The precipitates were collected, dissolved in the water and retreated by the ethanol again. The final precipitates were lyophilized to obtain the WYPs. Saccharide content of the WYPs was evaluated as per the method of Dubosi, Gilles, and Hamilton (1951).

2.3. Animal experiments

The mice were maintained under constant conditions (facility temperature of 22 ± 1°C, relative humidity of 50–60%, light and dark cycles of 12 h), and allowed free access to the routine feed and water during the experiment. All animal procedures were performed in accordance with the Ethical Guidelines of the Animal Care and Use Committee of Northeast Agricultural University (Harbin, China).

The mice were randomly divided into seven groups (10 mice for each group) after being adapted to facility environment for one week. The four groups of the normal mice were control, low, medium and high WYPs dose groups (0, 100, 300 and 500 mg kg⁻¹ body weight), while other three groups of the CY-treated mice were control, low and high WYPs dose groups (0, 100 and 500 mg kg⁻¹ body weight), respectively. The normal mice in the control group were treated every day with physiological saline solution by gavage for 18 d, but those mice of the other groups were given the WYPs of one of the three dose levels. For the CY-treated mice, from days 1 to 3, all mice were given CY at 80 mg kg⁻¹ d⁻¹ via gavage. From days 4 to 18, the CY-treated mice in the control group were given physiological saline solution by gavage while those in the other two groups were given the WYPs at one of the two dose levels. Twenty-four hours after the last administration, the mice were weighed and used for the following evaluations.

2.4. Assays of thymus and spleen indices

The mice were weighed and killed by cervical dislocation. Spleens and thymuses were removed and weighed immediately. Thymus and spleen indices were calculated as per the method of Ren et al. (2014).

2.5. Assays of lymphocytes proliferation, phagocytosis of peritoneal macrophages, and the activity of NK cells

Following the reference method (Yuan, Song, Li, Li, & Dai, 2006), spleens were aseptically removed from the mice, put in the Hank's balanced salt solution of 5 mL, cut into pieces, and passed through a 200-mesh sieve to prepare single cell suspension. Lymphocyte proliferation was detected as per the method of Yuan et al. (2010). At the same time, Hank's balanced salt solution of 10 mL was used for peritoneal lavage. The collected cells were centrifuged, and re-suspended at a final cell density of 2 × 10⁵ cells mL⁻¹ in the RPMI 1640-FBS medium. Macrophages phagocytosis was measured as per the method of Wu, Lu, Huang, Li, and Jiang (2014). NK cell activity was determined using the CCK-8 as per the method of Yuan et al. (2010).

2.6. Assays of cytokines, enzymes, and immunoglobulins

Splenocytes or macrophages (2 × 10⁵ cells mL⁻¹) were seeded onto 6-well plates. The cells were cultured at 37°C in 5% CO₂ for 48 h, and centrifuged to collect cellular supernatants to assay the levels of cytokines and enzymes using the ELISA kits, following the protocols provided by the kit manufacturers.

The serum was collected by retro-orbital bleeding 24 h after the last administration. The concentrations of cytokines, enzymes and immunoglobulins in the sera were determined using the ELISA kits according to the instruction of the kit manufacturers.

2.7. Statistical analysis

All reported data were collected from at least three independent evaluations, and expressed as means ± standard deviations. The statistical analysis was performed using SPSS software (SPSS Inc., Chicago, IL, USA). The differences (P < .05) between the mean values of multiple groups were analyzed by one-way analysis of variance (ANOVA) with Duncan's multiple range tests.

3. Results and discussion

3.1. Effects of the WYPs on spleen, thymus as well as three immune cells

The prepared WYPs had a saccharide content of 818.2 g kg⁻¹ on a dry weight basis. The results obtained when the WYPs were orally administered to the normal and CY-treated mice are listed in Table 1. For the normal mice, high WYPs dose (500 mg kg⁻¹) resulted in the mice with 22% and 42% increases in spleen and thymus indices (P < .05), respectively; however, low and medium WYPs doses (100 and 300 mg kg−1) showed insignificant impact on the two indices (P > .05). For the CY-treated mice, CY clearly impaired the immune status of the mice via lowering spleen and thymus indices (P < .05), in comparison with the respective values of the normal mice in the control group; however, WYPs administration in a dose-dependent manner conferred the CY-treated mice with 37–85% and 90–172% increases in spleen and thymus indices (P < .05), respectively. That is, the WYPs had immune effects on the normal and CY-treated mice via restoring and increasing spleen and thymus indices. At the same time, the WYPs were also capable of activating the three immune cells (Table 1). For the normal mice, WYPs administration at 500 mg kg⁻¹ increased lymphocyte proliferation, macrophage phagocytosis and NK cells activity by 46%, 53% and 18%, respectively, in comparison with the respective values of the normal mice in the control group (P < .05). For the CY-treated mice, CY showed immune suppression on the mice in the control group via decreasing the three cell indices by 15–35% (P < .05). However, WYPs administration at 500 mg kg⁻¹ showed activation on these immune cells, as the three cell indices were enhanced by 80%, 98% and 24% (P < .05), respectively. The WYPs were thus proved capable of improving and restoring immune status of the normal and immuno-suppressed mice, showing their immunomodulatory potentials for the body.

Table 1. Effects of WYPs administration on two immune organs and three immune cells of the mice.

Groups	WYPs doses	Spleen index	Thymus index	Lymphocyte proliferation	Macrophage phagocytosis	NK cell activity
				(A450)	(A540)	(%)
Normal mice	No	5.11 ± 0.13^bc	2.07 ± 0.11^bc	0.553 ± 0.019^c	0. 116 ± 0.014^b	60.7 ± 1.7^bc
	Low	5.42 ± 0.32^bcd	2.41 ± 0.23^cd	0.570 ± 0.032^c	0.134 ± 0.013^bc	62.4 ± 5.7^c
	Medium	5.87 ± 0.91^cd	2.51 ± 0.17^de	0.676 ± 0.038^d	0.154 ± 0.013^cd	67.4 ± 3.7^cd
	High	6.22 ± 0.48^d	2.93 ± 0.14^e	0.809 ± 0.018^e	0.177 ± 0.008^d	71.8 ± 4.1^d
CY-treated mice	No	3.32 ± 0.25^a	0.96 ± 0.15^a	0.358 ± 0.030^a	0.085 ± 0.011^a	52.5 ± 3.6^a
	Low	4.56 ± 0.40^b	1.82 ± 0.10^b	0.481 ± 0.010^b	0.155 ± 0.011^cd	54.6 ± 2.8^ab
	High	6.15 ± 0.38^d	2.61 ± 0.43^de	0.643 ± 0.010^d	0.168 ± 0.007^d	65.1 ± 2.5^cd

Note: Different lowercase letters after the mean values as the superscripts in same column indicate that one-way ANOVA of the mean values is significantly different (P < .05).

The indices of immune organs reflect the development of immune organs as well as the respective immune function (Cui, Chen, Wang, Kai, & Fang, 2011). Lymphocyte proliferation reflects the situation of immune responses, while phagocyte phagocytosis is the first and pivotal step in the immune responses (Mishra et al., 2006). NK cells are also important in the defense against tumors and viruses (Cho et al., 2015). Some past studies have assessed the effects of polysaccharides on immune organs and cells, and they are used here for data comparison. Schisandra polysaccharides have been found to enhance immune organ indices of the mice (Zhao et al., 2014). Cheonggukjang polysaccharides at 200 mg kg⁻¹ could increase spleen and thymus indices by 13% and 23%, respectively (Cho et al., 2015). Radix Cyathulae officinalis Kuan polysaccharides are able to enhance phagocytic capacity of peritoneal macrophage, splenocyte proliferation, and the activity of NK cells (Feng et al., 2014). Cheonggukjang polysaccharides are capable of increasing T-lymphocyte proliferation and NK cell activity of the CY-treated mice by 67% (Cho et al., 2015). Umbilicaria esculenta polysaccharides could enhance phagocytic capacity of macrophage by 175% (Du, Liu, & Wang, 2015). These studies support that the WYPs had immunomodulatory potentials via improving or restoring the status of two immune organs and three immune cells for the mice model.

3.2. Effects of the WYPs on cytokines and enzymes secretion

As seen from the data in Table 2, we can see WYPs could enhance the secretion of IL-2 and IFN-γ from the lymphocytes. If the normal mice were given the WYPs of 500 mg kg⁻¹, IL-2 and IFN-γ secretion were increased by 42% and 45%, respectively. Although CY treatment resulted in the mice with decreased secretion of IL-2 and IFN-γ clearly, the CY-treated mice with low and high WYPs administration (100 and 500 mg kg⁻¹) showed enhanced secretion of IL-2 and IFN-γ (P < .05) (Table 2). High WYPs dose led to the CY-treated mice with greater secretion of IL-2 and IFN-γ, as respective IL-2 and IFN-γ levels were increased from 33.9 and 58.1 to 48.9 and 121.5 pg mL⁻¹. WYPs administration also had helpful effects on the mice in the secretion of IL-1β, IL-6, TNF-α, iNOS and LZM from the macrophages (Table 2). For the normal mice with WYPs administration of 500 mg kg⁻¹, IL-1β and IL-6, and TNF-α secretion as well as LZM and iNOS levels were increased by 90%, 40%, 40%, 59%, and 219%, respectively. For the CY-treated mice, WYPs administration of 500 mg kg⁻¹ increased IL-1β, IL-6, and TNF-α secretion by 165%, 161%, and 64%, and enhanced iNOS and LZM levels by 287% and 53%, respectively. It was in a dose-dependent manner that the WYPs enhanced the secretion of the five cytokines and two enzymes from the lymphocytes and macrophages. These results verify again that the WYPs had immunomodulatory potentials via enhancing and restoring the secretion of these cytokines and enzymes.

Table 2. Effects of WYPs administration on the levels of cytokines and enzymes secreted from the lymphocytes and macrophages of the mice.

Groups	WYPs doses	Lymphocytes			Macrophages
		IL-2 (pg mL^−1)	IFN-γ (pg mL^−1)	IL-1β (pg mL^−1)	IL-6 (pg mL^−1)	TNF-α (pg mL^−1)	iNOS (pg mL^−1)	LZM (ng mL^−1)
Normal mice	No	38.7 ± 1.2^b	85.2 ± 3.1^b	50.7 ± 1.3^b	59.9 ± 1.3^c	124.9 ± 8.7^b	73.8 ± 5.5^b	22.5 ± 1.3^b
	Low	44.5 ± 1.6^c	106.7 ± 3.1^c	64.5 ± 5.0^c	77.2 ± 1.7^d	138.3 ± 9.7^bc	88.9 ± 10.7^b	24.1 ± 1.2^b
	Medium	49.4 ± 1.5^d	112.2 ± 9.9^cd	78.8 ± 2.6^d	80.2 ± 2.3^d	148.6 ± 5.2^c	168.9 ± 6.7^c	29.7 ± 2.0^c
	High	55.1 ± 2.1^e	123.9 ± 9.8^d	96.2 ± 5.9^e	100.6 ± 4.6^f	174.4 ± 6.4^d	235.6 ± 8.0^e	35.8 ± 1.3^d
CY-treated mice	No	33.9 ± 2.4^a	58.1 ± 3.1^a	38.0 ± 1.2^a	35.1 ± 1.0^a	102.4 ± 3.9^a	51.1 ± 6.7^a	18.2 ± 0.9^a
	Low	42.8 ± 1.1^c	92.5 ± 4.2^b	58.2 ± 5.0^bc	54.5 ± 1.8^b	125.0 ± 5.0^b	84.6 ± 10.0^b	23.9 ± 1.2^b
	High	48.9 ± 1.8^d	121.5 ± 6.8^d	100.8 ± 10.7^e	91.7 ± 1.4^e	167.5 ± 9.4^d	197.9 ± 10.2^d	27.9 ± 1.3^c

Note: Different lowercase letters after the mean values as the superscripts in same column indicate that one-way ANOVA of the mean values is significantly different (P < .05).

Cytokines play key role in the regulating functions of the Th cells, which can induce IFN-γ and IL-2 secretion (Yuan et al., 2010). Some studies have found that plant polysaccharides can enhance cytokines and LZM secretion. Radix Cyathulae officinalis Kuan polysaccharides can increase respective IL-2 and IFN-γ secretion in CD4⁺ by 60% and 57% (Feng et al., 2014). Kadsura polysaccharide can enhance respective IL-2 IFN-γ and TNF-α secretion in lymphocytes and macrophages by 31%, 8% and 9% (Wang et al., 2013). Pleurotus nebrodensis polysaccharide is capable of enhancing TNF-α and IL-6 secretion by 62% and 19%, respectively (Cui, Wang, Wang, Li, & Zhang, 2015). Acemannan, a polysaccharide from Aloe vera, can increase TNF-α and IL-1 levels in the irradiated mice (Kumar & Tiku, 2016). Pleurotus tuber-regium (Fr.) polysaccharide can increase LZM activity in the macrophages (Yuan et al., 2010). These five studies above show the same conclusion to support the present results. That is, the WYPs could display immunomodulatory potentials for the mice model, via stimulating the secretion of IL-2 and IFN-γ from the lymphocytes as well as the secretion of the five immune-related molecules from the macrophages.

3.3. Effects of the WYPs on serum levels of cytokines, enzyme, and immunoglobulins

As shown in Table 3, the WYPs were in a dose-dependent manner to enhance serum levels of IL-2, IL-1β, IL-6, and LZM of the mice. For the normal mice, WYPs dose at 500 mg kg⁻¹ enhanced IL-1β, IL-6, IL-2, and LZM levels in the sera by 67%, 39%, 18%, and 67%, respectively. For the CY-treated mice, the WYPs at low and high dose levels (100 and 500 mg  kg⁻¹) enhanced L-1β, IL-6, and IL-2 levels (by 9–68%) and LZM level (by 25–106%) (P < .05). The levels of three immunoglobulins IgM, IgA and IgG in the sera were also detected and compared (Table 3). WYPs dose of 500 mg kg⁻¹ increased respective serum IgM, IgA and IgG levels by 44%, 49% and 51% for the normal mice. However, this WYPs dose could enhance these respective levels by 24%, 49% and 69% for the CY-treated mice. It was also in a dose-dependent manner that the WYPs increased three immunoglobulin levels in the sera. All these results proved again that the WYPs had immunomodulatory potentials, via enhancing the serum levels of IL-2, IL-1β, IL-6, LZM, IgM, IgA, and IgG.

Table 3. Effects of WYPs administration on serum levels of cytokines, lysozyme (LZM) and immunoglobulins.

Groups	WYPs doses	IL-2 (pg mL^−1)	IL-1β (pg mL^−1)	IL-6 (pg mL^−1)	LZM (ng mL^−1)	IgM (μg mL^−1)	IgA (μg mL^−1)	IgG (mg mL^−1)
Normal mice	No	104.2 ± 3.9^b	86.8 ± 1.2^b	87.7 ± 1.7^b	22.4 ± 1.7^b	813.3 ± 9.0^b	745.2 ± 7.0^b	12.3 ± 0.7^b
	Low	108.8 ± 2.8b^c	107.9 ± 1.1^c	96.0 ± 1.7^c	27.8 ± 1.0^cd	856.7 ± 12.8^c	868.7 ± 6.8^d	15.6 ± 0.3d
	Medium	112.5 ± 3.2^cd	122.7 ± 8.2^d	105.0 ± 1.7^d	30.3 ± 1.2^d	1002.3 ± 9.7^e	916.5 ± 13.8^e	16.4 ± 0.4^de
	High	123.1 ± 4.1^e	145.0 ± 3.3^e	121.9 ± 2.4^f	37.3 ± 1.7^e	1171.8 ± 9.3^f	1113.0 ± 5.2^g	18.6 ± 0.5^f
CY-treated mice	No	94.8 ± 2.7^a	67.2 ± 7.2^a	67.3 ± 3.0^a	17.4 ± 1.0^a	754.0 ± 8.4^a	628.3 ± 12.4^a	10.0 ± 0.1^a
	Low	103.7 ± 3.9^b	113.1 ± 3.8^cd	83.1 ± 1.6^b	24.3 ± 1.3^bc	825.7 ± 13.3^b	800.3 ± 6.5^c	14.4 ± 0.1^c
	High	118.7 ± 3.3^de	138.8 ± 5.3^e	111.6 ± 5.0^e	34.5 ± 2.9^e	936.7 ± 6.2^d	938.2 ± 5.8^f	16.9 ± 0.2^e

Note: Different lowercase letters after the mean values as the superscripts in same column indicate that one-way ANOVA of the mean values is significantly different (P < .05).

Many immune-related enzymes, cytokines, and immunoglobulins exist in serum, and some polysaccharides are proved to influence the production of these immune-related molecules. Cyrtomium macrophyllum polysaccharides can improve serum levels of IL-2 and IL-6 for the CY-treated mice (Ren et al., 2014), while Astragalus membranaceus polysaccharides are able to increase serum level of IL-2 (Yang, Xiao, & Sun, 2013). Two other studies have also observed that both Hyriopsis cumingii and Ficus carica polysaccharides can increase serum LZM activity (Cui et al., 2011; Wu et al., 2014). These results are consistent with the present one; that is, the WYPs were able to increase serum levels of the three cytokines and LZM. It has also been reported that Cyrtomium macrophyllum polysaccharides can improve respective IgG and IgM secretion by 96% and 180% in the sera of the CY-treated mice (Ren et al., 2014). Another study shows that Schisandra chinensis polysaccharides can increase serum IgG, IgM and IgA levels by 14%, 23% and 19%, respectively (Zhao et al., 2014). These reported findings are consistent with the present results, proving WYPs’ immunomodulatory potentials to increase serum levels of the three immunoglobulins.

4. Conclusions

This study showed that the prepared WYPs had clear immunomodulatory potentials for the normal and CY-treated mice, and could enhance immune status of the mice model in a dose-dependent manner. For the normal mice, the WYPs could elevate spleen and thymus indices, improve macrophages phagocytosis, lymphocytes proliferation and NK cell activity, and stimulate cytokines, immune-related enzymes and immunoglobulins production in the macrophages, lymphocytes and sera. At the same time, the WYPs could restore and improve the immune status of the immuno-suppressed (i.e. CY-treated) mice, via markedly enhancing spleen and thymus indices, improving macrophages phagocytosis, lymphocytes proliferation and NK cell activity, as well as stimulating cytokines, immune-related enzymes and immunoglobulins production. It is thus concluded that the WYPs are potential ingredients when developing functional foods with immune functions.

Acknowledgements

The authors also thank the anonymous reviewers and editors for their valuable advices.

Disclosure statement

The authors declare that they have no conflict of interest.

Notes on contributors

Li-Xin Hao is studying in Northeast Agricultural University. She will shortly complete her MD in Food Science and Technology under the direction of Prof. Zhao X.H. Current connection postal address for her is Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, Harbin 150030, PR China, while her E-mail is [email protected].

Xin-Huai Zhao completed his MD and PhD in Food Science and Chemistry from Northeast Agricultural University and Ocean University of China, respectively. He is working in Northeast Agricultural University for more than 30 years, and has more than 200 research publications on different domains of food chemistry. His work mostly focuses on modification and bioactivities of food proteins/peptides, health benefits of phytochemicals, and application of food microorganism for bioconversion and biodegradation.

ORCID

Xin-Huai Zhao http://orcid.org/0000-0001-9682-5426

Additional information

Funding

This study was funded by the Specialized Research Fund for the Doctoral Program of Higher Education [20132325130001].