Sulfated modification promotes the immunomodulatory bioactivities of Lyciumbarbarum polysaccharides in vitro

Abstract

Three kinds of purified Lyciumbarbarum polysaccharides (LBPSs), LBPS₃₀, LBPS₇₀ and total LBPS (LBPS_t), were modified using chlorosulfonic acid-pyridine method based on the previous experiment, forming three sulfated LBPS (sLBPS), sLBPS_t, sLBPS₃₀ and sLBPS₇₀ respectively. They were characterized by ultrasonic-acidic barium chromate spectrophotometry, infrared (FT-IR) and high performance gel permeation chromatography (HPGPC). The immunomodulatory activity of each kind of LBPSs and sLBPSs was further examined to determine the relationship between the structure and bioactivity, and the sLBPS with the highest activity was selected. The results showed that sulfate contents were 390.67, 542.75 and 291.71 mg/g respectively, with different molecular masses. The appearance of two new characteristic absorption bands at near 1230 and 855, 853 or 808 cm^-1 in FT-IR spectra revealed the success of sulfation. sLBPS_t with high molecular weight and moderate sulfate content exhibited the best immunomodulatory activity by promoting lymphopoiesis and T lymphocyte differentiation as well as increasing IL-2, IL-6, IFN-γ and TFN-α production in vitro compared with the inartificial polysaccharides. These results indicated that sulfated modification could be considered as an effective way to enhance immune activity of LBPSs. Furthermore, sLBPS_t showed the best performances and would be expected as a new source of immunopotentiator.

Introduction

Lyciumbarbarum (also called Wolfberry, Fructus Lycii or Gouqizi) of the plant family Solanaceae has been widely used in traditional Chinese Medicine for more than 2000 years [1]. The major active ingredients are thought to be the L. barbarum polysaccharides (LBPSs), scopoletin and 2-O-β-D-glucopyranosyl-L-ascorbic acid (AA-2βG) [2]. LBPSs have been reported to exert a large variety of biological functions including immunomodulation, antitumor activity, antioxidant properties, neuroprotection, radioprotection, anti-diabetes, hepatoprotection, anti-osteoporosis and antifatigue [3-9].

L. barbarum polysaccharide fractions generally were composed of two kinds of monosaccharides, namely glucose and fructose in molar ratios of 1:2.1 according to Fourier Transform Infrared Spectroscopy (FT-IR) and high-performance liquid chromatography (HPLC) [10]. In addition, LBPS also contains a small amount of galactose, rhamnose, arabinose, mannose [11], and 18 kinds of amino acids [12].

Chemical modification of polysaccharides provided an opportunity to obtain new pharmacological agents with possible therapeutic uses. Polysaccharides have been chemically modified in various ways to improve their physical or biological properties, thus allowing a broader range of applications [13]. Most studies have demonstrated that biological activities of polysaccharides are greatly increased by molecular modification [14]. Sulfated polysaccharides are also called polysaccharides sulfate, referring to a series of polysaccharide derivatives with complex chemical structure, varied bioactivity and distinct structure-activity relationship, which are produced by the replacement of one or several hydroxides of the monosaccharides molecules in the polysaccharide macromolecular chain by sulfate. Both naturally occurring and artificially synthesized sulfated polysaccharides exerted some biological activities, such as antitumor, anticoagulation, antivirus, antioxidation and immunoregulation [15].

Our previous research found that sulfated modification of LBPS could significantly enhance its antiviral activity [16]. In this study, we prepared sulfate derivatives of LBPS₃₀, LBPS₇₀, LBPS_t by chlorosulfonic acid-pyridine method respectively. The objective of this research was to investigate the possibility of improving the immune-enhancing activities of LBPSs through sulfated modification and to compare their immunoregulatory properties for seeking new-type functional food and pharmaceutical products.

Materials and methods

Material and chemical

Three purified Lycium barbarum polysaccharides (LBPSs), total LBPS (LBPS_t) and two fractions LBPS₃₀ and LBPS₇₀ were prepared in our laboratory. Chlorosulfonic acid was the production of Shanghai Experimental Reagent Ltd.; pyridine and dimethylformamide (DMF) were purchased from Chemical Reagent Ltd. of National Drug Group. Lymphocyte separation medium was obtained from Tianjin Hanyang Biologicals Technology Co. Ltd. Penicillin, streptomycin, RPMI-1640 and fetal bovine serum (FBS) were obtained from Hyclone (USA). Calcium and magnesium-free phosphate-buffered saline (CMF-PBS, pH 7.4) was used to prepare 0.25% trypsin (Amresco). Concanavalin A (ConA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were obtained from sigma, Germany. All the other chemicals used were of analytical grade.

Sulfated modification of LBPSs

LBPS_t, LBPS₃₀ and LBPS₇₀ were sulfated by the chlorosulfonic acid-pyridine (CSA-PD) method and the optimum modification conditions were chosen based on our preparative experiment through orthogonal design. In brief, the chlorosulfonic acid-pyridine complex (1:8) was prepared in ice bath. LBPSs of 400 mg was added in the complex, cooled to room temperature after reaction at 80°C for 2 h, dissolved in 100 mL ice-cold water, neutralized with saturated NaOH solution, precipitated with 95% ethanol (EtOH) following by standing for 24 h. The precipitation was dialyzed in dialysis sack in flowing tap water for 48 h and in distilled water for 24 h and lyophilized to obtain three sulfated LBPSs: sLBPS_t, sLBPS₃₀ and sLBPS₇₀. The sulfate contents of sulfated polysaccharides was calculated by ultrasonic-acidic barium chromate spectrophotometry using sodium sulphate (Na₂SO₄) as a standard as described [17,18].

Based on our previous experiments, all the polysaccharides were diluted to 3.907, 1.953, 0.977, 0.488 and 0.244 µg/Ml (net content) in turn with RPMI-1640 basic solution, sterilized and stored at 4°C.

Infrared (IR) spectroscopy analysis

IR spectra of the polysaccharides were characterized by IR spectroscopy. Fourier transform IR spectra were recorded with a Nicolet 6700 IR spectrophotometer between 400 and 4000 cm^-1. The samples were analyzed as potassium bromide (KBr) pellets.

Determination of the molecular weight

The molecular weight of six polysaccharides were evaluated and determined by high performance gel permeation chromatography (HPGPC) (Wyatt technology Corp., USA) coupled with HSPgelTM AQ 4.0 column (6.0×150 mm) (Waters, Japan) and a refractive index detector. The mobile phase was 0.1 M NaNO₃ at a flow rate of 0.5 mL/min at 40°C. The polysaccharide samples were dissolved in the mobile phase (10 mg/mL). The column was calibrated with T-series dextran (T-10, 40, 70, 110, 400) as standards, and the molecular weight of polysaccharide fractions was estimated by reference to the calibration curve. The weight-average molecular weight (Mw), the number-average molecular weight (Mn), and the polydispersity were calculated using molecular weight calculation software connected to the HPLC integration system.

Animals

Female Balb/C mice aged from 6 to 8 week were purchased from Laboratory Animal Center of Zhengzhou University. Given free access to granulated feed and water, the mice were housed for one week and maintained under standard conditions prior to experiment. All management and experimental contact with the mice was carried out under specific pathogen free facilities. All the procedures for animal use were performed in accordance with internationally accepted principles and the PR China legislation on the use and care of laboratory animals and approved by the Bioethics Committee of Zhengzhou University.

The spleen lymphocytes proliferation assay for in vitro

Balb/C mice were sacrificed. The spleens were aseptically removed, placed in Petri dishes containing Hanks’ solution and crushed gently with a syringe through a sterile nylon strainer to prepare single cell suspension. After centrifugation at 1000 r/min for 10 min, supernatant was removed and erythrocytes lysed with Tris-NH₄Cl lysis buffer (0.144 M NH₄Cl, 0.017 M Tris-HCl) at room temperature. The lymphocytes were collected by centrifugation with lymphocyte separation medium and washed twice with Hanks’ solution by centrifugation and resuspended in RPMI-1640 medium supplemented with fetal bovine serum. The cell concentration was adjusted to 1×10⁷ cell/mL. Splenocyte suspension was plated at 100 µL/well in a 96-well culture plate containing ConA (final concentration was 5 mg/L), then polysaccharides at series concentrations were added at 100 μL/well in five wells for each concentration, taking RPMI-1640 medium as cell control (CC), RPMI-1640 medium with ConA as ConA control. The plates were cultured at 37°C in a 5% CO₂ humidified incubator for 48 h, 20 µL of MTT solution (5 mg/mL) was added to each well and re-incubated for another 4 h. Cell proliferation was measured by MTT assay [19]. The proliferation ratio was calculated according to the formula:

Proliferation ratio (%) = (Ā (experimental group) - Ā (ConA control group))/Ā (cell control group) × 100% [20]

Enzyme-linked immunosorbent assay (ELISA)

Peripheral blood of 5 ml was sampled from each mouse and transferred immediately into aseptic centrifuge tubes with heparin, and lymphocytes were isolated from the blood sample and washed by centrifugation with the same method as above. The resulting pellet was re-suspended to 1×10⁵ mL^-1 with RPMI-1640 medium, and incubated in 12-well culture plates at 1 mL per well. The supernatants were removed after 24 h of culture in 5% CO₂ at 37°C. In polysaccharide groups the six polysaccharides each at the concentrations of 3.907, 0.977 and 0.244 µg/mL were added respectively, taking RPMI-1640 as CC group, 500 µL per well, four wells for each concentration. The plates were incubated at 37°C in a humid atmosphere of 5% CO₂. After incubation for 24 h, cytokine levels were determined by collecting the supernatants from cell cultures through centrifugation at 1000 rpm for 20 min. The concentrations of IL-2, IL-6, interferon-γ (IFN-γ) and TFN-α in the culture media were determined using ELISA kit (Elabscience Biotechnology Co. Ltd, Wuhan) according to the manufacturer’s instructions.

Determination of T lymphocyte subsets

The preparation of lymphocytes was similar as 2.6. The adjusted cells were incubated in 6-well culture plates with 1×10⁵ mL^-1 at 2 mL per well. In polysaccharide groups six polysaccharides each at 3.907 µg/mL were added in plates at 1 mL per well, four wells each concentration, while RPMI-1640 medium added as CC group. After 24-h culture at 37°C in a humid atmosphere of 5% CO₂, cells were suspended and centrifuged at 1000 rpm for 3 min, washed twice with phosphate-buffered saline (PBS) at 4°C and fixed 40 min with 4% paraformaldehyde at room temperature, and then the supernatants were removed after centrifugation at 1000 rpm for 5 min. The cells were suspended with PBS and stained with surface antibodies (CD3-FITC, CD56-PE) for 40 min at 4°C. Cells were then washed, fixed with 1% paraformaldehydein PBS, and analyzed with flow cytometer BD Accuri^TM C6. All surface antibodies were purchased from Abcam (USA), data acquisition and analysis was performed using CELL Quest software.

Statistics

The results were depicted as the means ± standard deviations (SD) and the difference between groups was analyzed by one-way ANOVA. All statistical analysis was conducted using SPSS 16.0 software. A value of P<0.05 and P<0.01 were considered to be statistically significant.

Results

Characterizations of LBPSs

sLBPS_t, sLBPS₃₀ and sLBPS₇₀ were obtained under the same modification condition. Each sample showed a single and symmetrically sharp peak, indicating its homogeneity on HPGPC (data not shown). According to estimation in reference to standard dextrans, the average molecular weights (Mw) of sLBPSs significantly increased compared with those of non-sulfated LBPSs, which proved that CSA-PD method is an effective means of sulfated modification. Mw/Mn values of sLBPS_t and sLBPS₃₀ increased, demonstrating that molecular weight distribution of polysaccharide derivatives is more dispersive. However, Mw/Mn values of sLBPS₇₀ decreased, showing that after the introduction of sulfate groups, molecular weight became larger, as well as molecular weight distribution more narrow and molecular chain distribution more uniform. The sulfate contents of sLBPS₃₀, sLBPS₇₀ and sLBPS_t were 542.75, 291.71 and 390.67 mg/g respectively based on ultrasonic-acidic barium chromate spectrophotometry (see Table 1).

Table 1.

Sulfation of LBPSs and molecular characterization of sulfated LBPSs (sLBPSs)

Group	Sulfating complexa (CSA: PD)	Time (h)	Temperature (°C)b	Carbohydrate (%)	MW×10-4 c	Mw/Mn	Sulfate content (mg/g)d
sLBPS30	1:8	2	80	63.21	18.58	1.37	542.75
sLBPS70				44.25	20.35	1.21	291.71
sLBPSt				35.37	29.06	3.90	390.67
LBPS30	-	-	-	68.08	11.87	1.27	-
LBPS70	-	-	-	73.50	11.94	2.08	-
LBPSt	-	-	-	80.59	13.12	1.55	-

^aThe ratio of chlorosulfonic acid to pyridine in sulfated reagent.

^bSetting temperature.

^cDetermined by gel permeation chromatography.

^dDetermined by ultrasonic-acidic barium chromate spectrophotometry.

IR spectroscopy

The FT-IR spectra of three non-sulfated polysaccharides were quite similar (Figure 1A), and they both revealed typical carbohydrate absorption peaks characteristics. The spectra showed a typical major broad stretching peak at 3396 and 3419 cm^-1 for the -OH group. The weak bands at 2928 and 2932 cm^-1 showed the C-H stretching vibration of -CH₂. The relatively strong absorption peak at 1625, 1619 and 1601 cm^-1 were attributed to N-H (-CONH-) variable angle vibration, proving the presence of polypeptide or protein combined with the polysaccharides. The bands at 1414, 1406 and 1409 cm^-1 were due to C-O (-COOH) stretching vibration, the bands at 1251 and 1241 cm^-1 in LBPS₃₀ and LBPS₇₀ were ascribed to O-H (-COOH) variable angle vibration. The strong absorptions at 1078 and 1061 cm^-1 were assigned asymmetric vibration of C-O-C glycosidic rings, indicating the presence of pyranose. While the weak absorbance at 920-869 cm^-1 suggested the characteristics of β-D-pyranoid glucose in the polysaccharides, and the almost imperceptible shoulders at 778 and 779 cm^-1 may suggest the presence of α-isomers of pyranose.

Figure 1.

FT-IR spectra of LBPSs and sLBPSs. A. LBPSs; B. sLBPSs.

In the FT-IR spectrum of sLBPSs (Figure 1B), the carbohydrate characteristic peak of sulfated polysaccharides due to O-H stretching vibration shifts to around 3430 cm^-1 and peak shape becomes narrow compared with that of non-sulfated polysaccharides. The signal around 2930 cm^-1 also corresponds to the bending vibration of C-H bonds. In addition, The IR spectrum of sulfated polysaccharides presented a characteristic band around 1230 cm^-1, describing an asymmetrical ester sulphate groups (S=O) stretching vibration. Moreover, a discernible shoulder at 855 and 853 cm^-1 in the spectrum of sLBPS₃₀ and sLBPS₇₀, as well as 808 cm^-1 of sLBPS_t were due to symmetrical C-O-S stretching vibrations. These bands were affected by the anomeric structure and the position of substitution. The sulfated polysaccharides were obtained successfully without degradation of the polysaccharides.

Splenocyte proliferation activity of sLBPSs in vitro

In this study, polysaccharides were subjected to immune test to evaluate their effects on lymphocyte proliferation. As shown in Figure 2, in the presence or absence of ConA as mitogens for lymphocytes, the A ₅₇₀ values of LBPS₃₀ at 1.953-0.244 µg/mL, LBPS₇₀ and LBPS_t at 1.953-0.488 µg/mL, sLBPS₃₀ and sLBPS_t at 3.907-0.244 µg/mL, sLBPS₇₀ at 0.488 µg/mL groups were all significantly higher than those of corresponding cell control and ConA control groups (all at P<0.01); the A ₅₇₀ values of LBPS₃₀ at 3.907 µg/mL, LBPS_t at 3.907 and 0.244 µg/mL groups were all significantly higher than those of corresponding cell control (all at P<0.01) and of corresponding ConA control group (all at P<0.05). sLBPS₇₀ at 0.977 and 0.244 µg/mL groups were significantly higher than that of corresponding cell control group (P<0.01) while there were no significant differences compared with that of corresponding ConA control group (P>0.05).

Figure 2.

Effects of different concentrations of polysaccharides on spleen cells proliferation. A. Effect of three non-sulfated polysaccharides on splenocyte proliferation. B. Effect of three sulfated polysaccharides on splenocyte proliferation. C. Proliferation effect of six polysaccharides on splenocyte proliferation, respectively. Bars represent the mean ± SD of three independent experiments as compared to the control. Line charts represent the proliferation ratio of the changes of six polysaccharides of five different concentrations, respectively. Viability was quantified by the MTT assay and expressed as the absorption at 570 nm. The statistically significant differences among the groups were evaluated with ANOVA. *or#p<0.05 represents significant differences, **or ##p<0.01 represents extremely significant differences compared with cell control (CC) or Con A control group, respectively.

The proliferation ratio of lymphocytes treated in all polysaccharides groups showed first rising then declining trend with the increase of polysaccharide concentrations (Figure 2C). The overall average proliferation ratio of sLBPS_t was the highest (50.62%), followed by sLBPS₃₀ (45.61%) and LBPS_t (39.33%).

The changes of cytokine levels

The levels of four cytokines in all groups were presented in Figure 3. The results showed that IL-2 levels of sLBPS_t at all the concentrations, sLBPS₃₀ and LBPS_t at 3.907 and 0.977 µg/mL, LBPS₇₀ at 3.907 µg/mL, sLBPS₇₀ at 0.977 µg/mL, were significantly higher than that of control group (CC) (P<0.01) (Figure 3A). IL-6 levels of sLBPS₃₀ and sLBPS_t at all the concentrations, LBPS₇₀, LBPS_t and sLBPS₇₀ at 3.907 and 0.977 µg/mL, LBPS₃₀ at 0.977 µg/mL, were significantly higher than that of control group (P<0.01) (Figure 3B). TFN-α contents of sLBPS_t at all concentrations, sLBPS₃₀, sLBPS₇₀ and LBPS_t at 3.907 and 0.977 µg/mL while others at 3.907 µg/mL were significantly higher than those of control groups (P<0.01) respectively (Figure 3C). IFN-γ content of three sulfated polysaccharide at 3.907 and 0.977 µg/mL, LBPS₃₀ and LBPS₇₀ at 0.977 and 0.244 µg/mL, LBPS_t at 3.907 µg/mL were significantly higher than those of control groups (P<0.01) respectively while LBPS₃₀ at 0.977 and 0.244 µg/mL and LBPS_t at 0.244 µg/mL were higher than that of control group (P<0.05) (Figure 3D).

Figure 3.

IL-2, IL-6, TFN-α and IFN-γ contents of all groups with three concentrations. The four cytokine levels were measured by ELISA method. Results are expressed as mean±SEM. A. IL-2 levels. B. IL-6 levels. C. TFN-α levels. D. IFN-γlevels. “**” extremely significantly different (p<0.01) or “*” significantly different (p<0.05) from CC group, respectively by one-way ANOVA.

Flow cytometric analysis of T-Cell subsets of peripheral blood lymphocytes

The results in Figure 4 showed that after 24-h culture of peripheral blood lymphocytes with polysaccharides, the frequency of CD3⁺ T cells in all groups was increased compared with that in cell control group, among sLBPS_t was the highest. Moreover, the ratios of CD3⁺CD56⁺ in LBPS₇₀, LBPS_t, sLBPS₃₀ and sLBPS_t were higher than that of cell control group and that of LBPS₇₀ was the highest, followed by that of sLBPS_t.

Figure 4.

Flow Cytometry of CD3 and CD56 molecules expression on lymphocyte. Lymphocytes were double stained for CD3-FITC, CD56-PE after cultured for 24 h.

Discussion

The result of FT-IR spectra showed that the sulfate group has been introduced into polysaccharide chain of sLBPSs. The characterizations of natural polysaccharides and their derivatives with various chemical components, molar mass and chain conformation were important because of their critical effects on end-use structure-property relations [21]. Compared with nature polysaccharides, the molecular weights of three sulfated polysaccharides were obviously increased, which was mainly due to the introduction of sulfate group. The carbohydrate characteristic absorption peaks of the sulfated polysaccharides had little variation change, but the characteristic absorption peaks of the sulfate group enhanced obviously, showing that other groups of sLBPSs were not affected after modification except the sulfate content with evident changes. Total and fractions of LBPSs reacted successfully with the chlorosulfonic acid-pyridine complex to have obtained sulfated derivatives forming different Mw values with sulfate contents ranging from 291 to 543 mg/g.

Splenic lymphocytes are the body’s immune active cells, containing T and B lymphocytes. Cellular immunity mediated by T-lymphocyte is an important immunologic reaction. The proliferation of lymphocytes was the direct indexes reflecting the body’s cellular immune [22]. ConA, as T cell mitogen, mainly promotes T lymphocyte proliferation [23]. The proliferation assay showed that both natural and sulfated polysaccharides in all concentrations could stimulate lymphocyte proliferation statistically compared with the proliferation by ConA (at least at P<0.05). sLBPSs improved the proliferation effect and there was an obvious concentration-dependent trend. The lymphocytes proliferation ratios of sLBPS_t and sLBPS₃₀ were also higher than those of other groups, suggesting that the sulphation modification of LBPSs could improve the immunity of T lymphocyte in vitro, sLBPS_t and sLBPS₃₀ possessed the best efficacies. Similarly, our previous experimental confirmed that sLBPS_t could significantly promote proliferation and enhance serum antibody titer of chicken peripheral lymphocytes [24].

Cytokines are peptides and low-molecular proteins, IL-2, IL-6, TNF-α and IFN-γ were used as indicators for the lymphocyte responses under induction. The direction of the acquired immunity is regulated by the Th₁/Th₂ balance. IL-2 and IFN-γ are secreted by Th₁ cells, mainly connect natural and adaptive immune response [25]. IL-6 is secreted by Th₂ cell and plays an important role in humoral immunity [26]. TNF-α, mainly secreted by mononuclear macrophages, has a critical role in various physiological and pathological processes, including immune and inflammatory responses [27]. Our results showed that the secretion of IL-2, IL-6, TNF-α and IFN-γ was strongly stimulated by all the sulfated polysaccharides. Moreover, the lymphocyte harvested from in vitro culture with sLBPS_t group at all the concentrations produced the highest levels of four cytokines and with the decrease of sLBPS concentration, the cytokine levels of lymphocytes decreased gradually, demonstrating that the cellular immunity may be stimulated by sLBPS_t in an dose-dependent manner.

Cellular immune mediated by T lymphocytes which are heterogeneous groups is important immune mechanism of the body [28]. CD3 is the specific surface molecules of T lymphocytes; the ratio of peripheral CD3⁺ T lymphocytes means the mature T cell portions and reflects the degree of proliferation and differentiation of T lymphocytes to a certain extent. The CD3 vs CD56 dot plot also gated on the lymphoid region which can be used to evaluate NK cells (CD3^-CD56⁺) and NK T cells (CD3⁺CD56⁺) [29,30]. The results of flow cytometric analysis identified that CD3⁺ percentage of all the groups and the CD3⁺/CD56⁺ ratio in peripheral blood of most of polysaccharide-treated groups were higher than that in CC group, and the general effect of sLBPS_t was the best, supporting the fact that activation of T lymphocytes was involved in the immune effect of LBPSs via stimulating the expression of CD3 and CD3⁺CD56⁺ subset. It was indicated that sLBPS_t could promote T cells differentiation as well as NK cells. Many investigators also have reported that the high percentage of CD3⁺ and ratio of CD3⁺/CD56⁺ would be favor of activation of body’s immune [31,32].

Our earlier studies reported molecular weight and DS of sulfated polysaccharides were important parameters influencing activities [33]. In this study, sLBPS_t with high molecular weight and moderate sulfate content showed the best performances while sLBPS₃₀ and sLBPS₇₀ could also significantly enhance immune function of immunocytes to a certain extent, whose effects were better than those of natural LBPSs. This indicated that sulfated modification could improve the immunocompetence of LBPS and there was a strong relationship between the activities and high molecular weight while the DS of sulfated polysaccharides with the best immune activity may lie within the optimum scope. Chen et al. [34] also reported that the S-PSG (sulfated polysaccharides)-2 from Ganodermaatrum with moderate DS and molecular weight exhibited the highest immunomodulatory activity in vitro. This needs to be further proved in lymphocyte in vitro.

In conclusion, sulfation modification could improve the immune bioactivities of LBPS, and sulfated LBPS (sLBPS_t) presented the higher immunomodulatory activities and could be expected as a new source of immunopotentiator.

Disclosure of conflict of interest

None.