Preliminary characterization and immunomodulatory activity of polysaccharide fractions from litchi pulp

Abstract

Two purified fractions were prepared by the sequential purification of litchi pulp polysaccharide (LP) through ion-exchange chromatography and gel-filtration chromatography. Their preliminary structures and immunomodulatory activities were investigated. The two fractions, LPI and LPII, were homogeneous heteropolysaccharides mainly composed of arabinose, glucose, galactose and mannose with average molecular weights (M_ws) of 213 and 36.9 kDa, respectively. LPII was quite different from LPI; it had a triple helix structure and a much higher content of neutral sugar, uronic acid, arabinose, glucose and mannose (p < 0.05). Nuclear magnetic resonance (NMR) spectroscopy data showed LPI contained α-D-Galp and α-L-Araf-(1→ and LPII was consisted of →3)-α-L-Araf-(1→, β-(1 → 2)-Galp and →4)-α-D-Glcp-(1→. The results from in vitro immunomodulatory activities indicate that LPII was a better stimulator than LPI on splenocyte proliferation, cytokine secretion and natural killer (NK) cell cytotoxicity from 100–300 μg mL⁻¹ (p < 0.05). LPII exhibited stronger immunostimulatory activity, which may be attributed to its unique structure.

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1. Introduction

Litchi (Litchi chinensis Sonn.) is a subtropical fruit that is native to Southern China and is now cultivated widely in subtropical areas around the world. As a result of its attractive appearance and delicious flavour, litchi has been widely accepted by consumers and has gained significant popularity in the international market. Litchi pulp has been used in Traditional Chinese Medicine as a remedy for cough, diarrhoea, stomach ulcers, diabetes, dyspepsia and obesity, as well as to kill intestinal worms.¹ Early studies suggested that the numerous health benefits of litchi pulp might be related to its polysaccharides, which are its main bioactive ingredients.^2–4

There are many litchi cultivars, and they mature during different time periods. Feizixiao, Guiwei, Nuomici, Heiye and Huaizhi are representative commercial litchi cultivars that are broadly cultivated in China, and they mature from early to middle June, late June to early July and middle to late July, respectively.⁵ The differences in the maturation time and cultivation environment have endowed litchi cultivars with varied volatile profiles⁶ and phenolic compositions and activities.⁷ Similarly, the polysaccharide structures are different in various litchi cultivars. For example, Yang found the average molecular weights (M_w) of a polysaccharide from Huaizhi litchi pulp to be 2.40 × 10⁶ Da,⁸ compared to 2.19 × 10⁵ Da for a polysaccharide from Nuomici⁹ and 105.88–986.47 × 10³ Da for the polysaccharides from Guiwei.¹⁰ Furthermore, the monosaccharide composition and glycosidic linkage features of polysaccharides were significantly different in Huaizhi, Nuomici and Guiwei.^8–10 Although researchers have purified litchi polysaccharides and analysed their structures from Huaizhi, Nuomici and Guiwei, there is little information about the structure of polysaccharides from Feizixiao, which is the second largest cultivar planted in China.¹¹ Thus, two fractions were purified from a polysaccharide extract of litchi pulp (cv. Feizixiao) using diethylaminoethyl (DEAE)-52 cellulose and Sephadex G-100 columns. The chemical composition, spectroscopic characteristics and helical structures of two polysaccharide fractions were analysed in the present study.

The biological activities of polysaccharides are closely related to their structure. Glucans from Lentinus edodes exhibited significant anti-tumour and immunomodulatory activities with a triple-helix structure, whereas their bioactivity almost disappeared with a single flexible chain.¹² Polysaccharides from litchi pulp (cv. Guiwei) exhibited excellent anti-tumour and/or immunomodulatory activities.^4,10,13 It is unclear whether litchi polysaccharides from Feizixiao have immunomodulatory activities. Therefore, we further evaluated the immunomodulatory activities of the polysaccharide fractions from litchi pulp (cv. Feizixiao) on splenocyte proliferation, cytokine secretion and natural killer (NK) cell cytotoxicity in vitro.

2. Materials and methods

2.1. Materials and chemicals

2.1.1. Chemicals and reagents. Standard dextrans (including T-4: molecular mass 4 × 10³ Da, T-10: 1 × 10⁴ Da, T-40: 4 × 10⁴ Da, T-70: 7 × 10⁴ Da, T-500: 5 × 10⁵ Da, and T-2000: 2 × 10⁶ Da), rhamnose, arabinose, glucose, ribose, galactose, mannose, penicillin–streptomycin solution, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Sephadex G-100 and anion-exchange DEAE-52 cellulose were purchased from Sigma Chemical Co. (St. Louis, MO, USA). RPMI-1640 medium and fetal calf serums were purchased from Gibco Life Technologies (Grand Island, NY, USA). Erythrocyte lysis buffer and enzyme-linked immunosorbent assay (ELISA) kits were purchased from Neobioscience (Shenzhen, China). All other reagents were of analytical grade.

2.1.2. Animals and cells. BALB/c mice (male, 20.0 ± 2.0 g) aged 6–8 weeks were approved by the Animal Ethical and Welfare Committee of Sun Yat-Sen University and followed the Guiding Principles in the Care and Use of Animals. The mice were acclimatized for 1 week before being studied.

2.2. Preparation of crude polysaccharides from litchi pulp

The crude litchi pulp polysaccharides were prepared according to the previous report.¹⁴ Fresh litchi (cv. Feizixiao) fruits were dried with hot air, and dried pulps were ground into a fine powder using a high-speed disintegrator (YB-1000A, Yunbang Instrument Co., Ltd., Zhejiang, China). The powder was soaked three times in 80% ethanol at room temperature, each for 12 h, to remove the pigments, monosaccharides and oligosaccharides. Then, the purified sample powder was filtered and extracted by microwave-assisted extraction which was the optimized extraction method of polysaccharides from litchi pulp for maximum yield by our lab.¹⁴ The microwave power, extraction time, ratio of water to material and pH were 559 W, 11 min, 23 mL g⁻¹ and 8.2, respectively. The obtained aqueous was adjusted to neutral pH and then concentrated in a vacuum evaporator (Eyela, Tokyo, Japan) at 50 °C to one fifth of the initial volume. Proteins in the extract were removed using Sevag reagent,¹⁵ and polysaccharides were precipitated with absolute ethanol (1 : 4, v/v) for 24 h at 4 °C. The prepared precipitates were collected, washed successively with acetone and petroleum ether, and lyophilized to obtain crude litchi pulp polysaccharides (LP).

2.3. Purification of crude LP

The crude LP was dissolved in distilled water at a concentration of 10.0 mg mL⁻¹ and loaded onto a DEAE-52 cellulose column (60 × 2.6 cm). The column was stepwise eluted with 0, 0.1, 0.2, 0.3 and 0.5 M NaCl at a flow rate of 1.0 mL min⁻¹. The eluate was collected by a fraction collector (6 mL/tube) and analysed by the phenol–sulphuric acid method to determine the carbohydrate content.¹⁶ The protein absorbance was measured on a UV-vis spectrophotometer (UV 1800, Shimadzu, Japan) at 280 nm. As a result, two fractions (Fig. 1a) eluted by 0.1 and 0.3 M NaCl were concentrated, dialysed and further purified by gel filtration chromatography. The Sephadex G-100 column (40 × 2.0 cm) was eluted with distilled water at a flow rate of 0.1 mL min⁻¹, and the elute was collected (3 mL/tube) and analysed as mentioned above. The corresponding fractions were combined, concentrated and freeze-dried, yielding two purified fractions: LPI and LPII. The lyophilized samples were stored in a desiccator at room temperature until further use.

Fig. 1 Anion-exchange chromatograms of crude litchi polysaccharides on DEAE52-cellulose (60 cm × 2.6 cm) column, eluted with 0, 0.1, 0.2, 0.3 and 0.5 M NaCl (a). Sephadex G-100 column chromatogram of LPI and LPII from distilled water stepwise elution (c and d). GPC elution profiles of the LPI and LPII with refractive index detector (b).

2.4. Characterization analysis

2.4.1. Analysis of chemical characteristics. The neutral polysaccharide content was determined using the phenol–sulfuric acid method¹⁶ and expressed as glucose equivalents. The protein concentration was determined using the Bradford assay with a bovine serum albumin (BSA) standard curve,¹⁷ and the uronic acid content was determined using the modified m-hydroxydiphenyl method with galacturonic acid standards.¹⁸

The monosaccharide compositions of LPI and LPII were determined by gas chromatography-mass spectrometry (GC-MS) according to our previous study.³ Briefly, polysaccharide samples (40 mg) were dissolved in 2 mol L⁻¹ H₂SO₄ (10 mL) and hydrolysed at 100 °C for 6 h. After neutralizing the residual acid with 2 mol L⁻¹ BaCO₃, the hydrolysate was filtered through 0.2 μm syringe filters (Whatman, Sanford, ME, UK) and dried under a stream of N₂. The dried hydrolysate was then dissolved in 5 mL of pyridine containing 14 mg mL⁻¹ hydroxylamine at 90 °C for 30 min. The samples were cooled to room temperature, 1 mL of acetic anhydride was added, and the mixtures were incubated at 90 °C for 30 min. The acetylated hydrolysate was extracted with trichloromethane, following by evaporation under a stream of N₂. The final product was analysed by GC-MS using an Agilent 6890 GC instrument (Agilent Technologies Co., Ltd., Colorado Springs, CO, USA) equipped with a DB-1 column and an Agilent 5973 MS detector. The temperature programme was as follows: the temperature of the column was initially set to 190 °C, increased to 230 °C at 2 °C min⁻¹, held for 2 min, increased to 240 °C at 5 °C min⁻¹, and held for 2 min. The detector temperature was 290 °C, and the vaporizing chamber temperature was 260 °C. The GC/MSD ChemStation software was used. Six monosaccharides (arabinose, mannose, rhamnose, galactose, xylose, and glucose) were used as external standards to determine the composition of the polysaccharides. Each sample was analysed three times.

2.4.2. Analysis of molecular weight. The average M_ws of LPI and LPII were determined using an Agilent 1100 HPLC system equipped with a refractive index detector (RID). The samples were separated on a TSK-GEL G3000SWxl column (7.5 × 300 mm, Tosoh Corp., Japan). The column oven temperature was 25 °C, and the column was eluted with 0.1 M Na₂SO₄ solution in PBS buffer (0.01 M, pH 6.8) at a flow rate of 1.0 mL min⁻¹. Standard dextrans, including T-4 (molecular mass 4 × 10³ Da), T-10 (1 × 10⁴ Da), T-40 (4 × 10⁴ Da), T-70 (7 × 10⁴ Da), T-500 (5 × 10⁵ Da), and T-2000 (2 × 10⁶ Da), were used as molecular mass markers.

2.4.3. Analysis of Fourier transform infrared (FTIR) spectroscopy. According to our previous study,⁴ 2 mg polysaccharide samples were mixed with 100 mg of potassium bromide (KBr) powder and pressed into 1 mm thick pellets for FTIR measurements. FTIR spectra were recorded on a Nexus 5DXC FTIR spectrophotometer (Thermo Nicolet, Austin, TX, USA) at frequencies from 4000–400 cm⁻¹.

2.4.4. Analysis of nuclear magnetic resonance (NMR) spectroscopy. The polysaccharide samples were kept over P₂O₅ in vacuum for several days. The deuterium-exchanged polysaccharides (50 mg) were dissolved in 0.7 mL of 99.96% D₂O and placed in 5 mm NMR tubes. ¹H and ¹³C NMR spectra were recorded with a Bruker AM 600 MHz spectrometer (Bruker, Rheinstetten, Germany, operating frequencies 600 MHz for ¹H NMR and 151 MHz for ¹³C NMR) at 30 °C. The chemical shift was expressed in ppm. Tetramethylsilane was used as an internal standard.¹⁰

2.4.5. Analysis of the helix coil transition. The helical structure of the polysaccharides was identified by characterizing the Congo red–polysaccharide complex.¹⁹ Briefly, a polysaccharide solution (2 mL, 0.5 mg mL⁻¹) was mixed with Congo red solution (2 mL, 50 μmol L⁻¹) in a tube, and NaOH solution (1 mL, final concentration of 0, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 or 0.50 mol L⁻¹) was added. Distilled water (2 mL), Congo red solution (2 mL) and NaOH solution (1 mL) were mixed as a control. After 10 min at room temperature, the maximum absorption wavelength (λ_max) of the mixture was scanned at 400–600 nm.

2.5. Immunomodulatory activity analysis

2.5.1. Splenic lymphocyte proliferation. Spleens were removed from sacrificed BALB/c mice and minced in sterile PBS. Splenic cells were harvested using a sterilized stainless steel mesh (200 meshes) at room temperature. Red blood cells were lysed with erythrocyte lysis buffer, and the remaining cells were washed twice and resuspended in RPMI 1640 complete medium containing 10% fetal calf serum, 100 U mL⁻¹ penicillin and 100 μg mL⁻¹ streptomycin. The trypan-blue dye exclusion test showed greater than 95% cell viability.

The splenocyte proliferation assay was performed as previously described.²⁰ In the mitogenic test, 5 × 10⁵ cells per well in 0.1 mL of medium were seeded onto a 96 well plate. Cells were stimulated with LPI and LPII at a final concentration of 0, 100, 200 or 300 μg mL⁻¹ for 68 h. Next, 20 μL of MTT (5 mg mL⁻¹) was added to each well, and the cells were cultured for 4 h. Acidified isopropyl alcohol (100 μL) was added, and the cells were incubated for 12 h at 37 °C to dissolve the formazan crystals. The absorbance at 570 nm was recorded using a microplate reader (Tecan Infinite Pro 200, Männedorf, Switzerland).

2.5.2. Cytokine production by splenic lymphocytes. Splenocyte cells (5 × 10⁶ cells/well) in 90 μL of medium were pretreated in 96 well culture plates. Then, 100 μL of LPI and LPII (0, 100, 200 or 300 μg mL⁻¹ final concentration) and 10 μL of ConA (5 μg mL⁻¹ final concentration) were added and incubated for 48 h. The culture media were centrifuged at 3000 × g for 10 min at 4 °C, and supernatant IL-6 and INF-γ levels were quantified using a double-antibody sandwich ELISA according to the manufacturer's instructions. Three replicates were performed for each treatment.

2.5.3. Cytotoxicity assay of natural killer cells. Splenocytes were prepared as the effector cells for a splenic NK activity assay as described in our previous study.⁴ The splenocytes were plated onto 96-well plates at a density of 1 × 10⁷ cells/mL per well in a 50 μL volume and stimulated with 40 μL of LPI and LPII at different concentrations (final concentration of 0, 100, 200 or 300 μg mL⁻¹) for 24 h, using twelve replicate wells for each concentration. Then, 10 μL of YAC-1 cells (1 × 10⁶ cells/mL) was added to six wells as the experimental group, and complete medium was placed in the other six wells as the effector control. At the same time, 100 μL of complete medium containing 10 μL of YAC-1 cells (1 × 10⁶ cells/mL) was added to the empty wells as the target control. The plates were incubated for 4 h, followed by another 4 h incubation with 20 μL of MTT (5 mg mL⁻¹). Then, 100 μL of acidified isopropyl alcohol was added to each well, followed by a 12 h incubation. The absorbance was measured at 570 nm using a microplate reader. The cytotoxicity of the NK cells was expressed as the percent lysis of target cells: [OD_T − (OD_exp − OD_E)]/OD_T × 100, where OD_T, OD_exp and OD_E represent the OD value of the target control group, experimental group and effector control group, respectively.

2.6. Statistical analysis

The data are presented as the mean ± standard deviation (SD). The significance was evaluated by one-way ANOVA followed by Student–Newman–Keuls test using SPSS 19.0 software. A p-value of 0.05 was chosen as the threshold for significance.

3. Results

3.1. Preparation of polysaccharide fractions

The crude LP was prepared from litchi pulp by ultrasound-assisted extraction and ethanol precipitation. The overall yield of crude LP from the dried litchi pulp was 24.02%. Then, the crude LP was fractionated by anion-exchange chromatography on a DEAE-52 cellulose column to yield two fractions (Fig. 1a), which were concentrated, dialysed and further purified on a Sephadex G-100 column. Each fraction generated one single elution peak: LPI and LPII (Fig. 1c and d).

The yields of LPI and LPII were 22.54% and 38.91% of the crude polysaccharide, respectively. The homogeneity of LPI and LPII was evaluated by gel filtration chromatography (Fig. 1c and d) and GPC (Fig. 1b). The figures show that LPI and LPII had single symmetrical and concentrated sharp peaks, indicating that they were homogeneous polysaccharides.

3.2. Preliminary characterization of LPI and LPII

3.2.1. Chemical composition. The neutral sugar, uronic acid and protein content and M_w of each fraction are summarized in Table 1. LPII contained a greater content of neutral sugar and uronic acid than LPI (p < 0.05), while the M_w of LPII was significant lower than that of LPI (p < 0.05). The difference in uronic acid content between LPI and LPII agreed with the elution order of crude LP on the DEAE-52 cellulose column. As the NaCl concentration increased from 0.1 to 0.3 M, the uronic acid content of the extracted polysaccharides increased, which is consistent with polysaccharides extracted from the peduncles of Hovenia dulcis²¹ and Prunella vulgaris Linn.²²

Table 1 The chemical compositions of LPI and LPII^a

	LPI	LPII
a Each value is expressed as the mean ± standard deviation (n = 3).b p < 0.05.
Neutral sugar (%)	55.34 ± 2.01	61.75 ± 2.18^b
Protein (%)	3.26 ± 0.15	2.95 ± 0.218
Uronic acid (%)	12.14 ± 0.24	16.88 ± 0.79^b
Molecular weight (Da)	2.13 × 10^5	3.69 × 10^4
Monosaccharide composition (%)
Ribose	3.28 ± 0.11	7.49 ± 0.42^b
Rhamnose	5.18 ± 0.36	5.61 ± 0.24
Arabinose	30.00 ± 1.02	36.09 ± 0.57^b
Mannose	8.72 ± 0.47	11.15 ± 0.26^b
Glucose	16.29 ± 1.09	23.99 ± 1.46^b
Galactose	36.53 ± 2.17	15.67 ± 1.38^b

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A monosaccharide analysis revealed that LPI and LPII were heteropolysaccharides (Table 1). The major monosaccharides in LPI were arabinose and galactose, whereas those of LPII were arabinose and glucose. The percentage contributions of ribose, arabinose, mannose and glucose in LPII were higher than those of LPI while the galactose content was an opposite trend.

3.2.2. FT-IR spectrum characterisation. The FTIR spectra, functional groups and structural characteristics of LPI and LPII are summarized in Table 2. Both LPI and LPII showed characteristic polysaccharide bands, including hydroxyl group bands at 3408.9 and 3421.9 cm⁻¹, alkyl group bands at approximately 2929.5 cm⁻¹ and carboxyl group bands at approximately 1655.2, 1560.6 and 1419 cm⁻¹. The absorption bands between 1150 and 1000 cm⁻¹ were attributed to characteristic C–O–C glycosidic bond vibrations and ring vibrations overlapping with stretching vibrations of the side group C–O–H link bonds. The absorption peaks at approximately 3410 and 1655 cm⁻¹ are typical of protein infrared peaks.²³ Absorption peaks at approximately 840 and 768 cm⁻¹, which indicate α-type glycosidic linkage and D-glucopyranose ring, respectively, were present in LPI and LPII. However, the β-type glycosidic linkage at 894.0 cm⁻¹ only existed in LPII.

Table 2 FTIR spectra of LPI and LPII

Absorption^a (cm^−1)		Functional group^b	Structural characteristics
LPI	LPII
a The FTIR spectra of litchi polysaccharides LPI and LPII were determined using a Fourier-transform infrared spectrophotometer over the frequency range of 4000–400 cm^−1.b The functional group and structural characteristics were obtained from Zhang (1994).
3408.9	3421.9	Hydroxyl group (–OH)	O–H stretching vibration
		Amino group (–NH2)	N–H stretching vibration
2929.5	2929.9	Alkyl group (–CH2–)	C–H stretching vibration
	2346.3	Amino group (–NH2)	N–H stretching vibration
1655.2	1657.8	Carbonyl group (–C=O or –CHO)	C=O stretching vibration
		Amide group (–NH2 or –COR)	N–H bending vibration or C=O stretching vibration
		Amino group (–NH2)	N–H bending vibration
	1560.6	Carbonyl group (–C=O)	C=O stretching vibration
1546.6		Amino group (–NH2) or amide group (–NH2)	N–H bending vibration
1412.6	1419.7	Carboxyl group (–COOH)	C–O stretching vibration
	1330.7	Alkyl group (–CH2)	C–H deformation vibration
	1241.7
1077.5	1143.3	Ether (–C–O–C–)	C–O stretching vibration
	1100.6
	1014.6
	954.6	Alkyl group (–CH)	C–H bending vibration
917.9	894.0	D-Glucopyranose ring	Asymmetric stretching vibration
		β-Type glycosidic linkage	C–H bending vibration
848.9	836.4	α-Type glycosidic linkage	C–H bending vibration
764.8	768.4	D-Glucopyranose ring	Symmetrical ring vibration

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3.2.3. NMR spectrum characterisation. The NMR spectra of LPI and LPII were showed in Fig. 2. The ¹H signal at 4.70 ppm belonged to D₂O. The ¹H and ¹³C NMR of LPI and LPII were crowded in a narrow region ranging from 3.0 to 5.2 ppm and 60 to 112 ppm which were typical signal of polysaccharides.²³ The ¹H NMR spectrum at 5.16, 4.22, 4.14, 3.95 and 3.7 ppm were assigned to the H-1, H-2, H-4, H-3 and H-5 while the ¹³C signal at 109.88, 77.6 and 84.67 ppm were assigned to the C-1, C-3 and C-4 of α-L-Araf-(1→ in LPI⁸ while the ¹H NMR spectrum at 5.18, 3.34, 4.15, 3.42 and 3.15 ppm and the ¹³C signal at 110.22, 84.04, 84.8, 79.43 and 73.43 ppm were assigned to the H1/C1, H2/C2, H3/C3, H4/C4 and H5/C5 of →3)-α-L-Araf-(1→ in LPII.²⁴ In addition, the ¹H NMR spectrum at 4.45, 3.57, 3.64 and 3.95 ppm assigned to H-1, H-2, H-3 and H-4 while the ¹³C signal at 104.11, 71.34, 73.43 and 69.39 were assigned to the C-1, C-2, C-3 and C-4 of α-D-Galp in LPI.^8,25 Moreover, the ¹H NMR spectrum at 5.02, 4.06, 4.10, 3.89 ppm were assigned to the H-1, H-2, H-3 and H-4 of β-(1 → 2)-Galp in LPII.²⁶ The ¹H signals at 3.65, 3.95, 3.82, 3.72 and 3.42 ppm were assigned to H-2, H-3, H-4, H-5 and H-6 and the ¹³C signals at 76.1, 74.1, 75.6 and 72.2 ppm were assigned to the C-3, C-4, C-5 and C-6 of →4)-α-D-Glcp-(1→ in LPII.²⁷ Comprehensive analysis of the results of the monosaccharide composition, FTIR and NMR characterization, LPI mainly consisted of α-D-Galp and α-L-Araf-(1→ and LPII contained →3)-α-L-Araf-(1→, β-(1 → 2)-Galp and →4)-α-D-Glcp-(1→.

Fig. 2 NMR spectra of litchi polysaccharide: (A) ¹H spectra of LPI; (B) ¹H spectra of LPII; (C) ¹³C spectra of LPI; (D) ¹³C spectra of LPII. The ¹H and ¹³C NMR spectra of LPI and LPII in D₂O were recorded using a Bruker AM 600 MHz spectrometer at 30 °C.

3.2.4. Helical structures. Congo red dye can combine with helical polysaccharides, particularly single-helical polysaccharides, to red shift the λ_max.^19,28 The λ_max values of the litchi polysaccharide–Congo red complexes within a NaOH concentration range of 0–0.5 mol L⁻¹ are shown in Fig. 3. The λ_max values of the complexes and Congo red alone gradually decreased with increasing NaOH concentration. The λ_max of the LPII–Congo red complex was nearly constant between 0 and 0.05 mol L⁻¹ NaOH, corresponding to depolymerization from a triple helix to a single helix, whereas the subsequent decreases in λ_max corresponded to the change from a single helix to a random coil.²⁹ In contrast, the λ_max values of the LPI–Congo red complex decreased continuously with increasing NaOH concentration, and they were clearly lower than those of the LPII–Congo red complex at the same NaOH concentration, indicating that LPI had a less organized conformation without a triple helix structure.³⁰

Fig. 3 The maximum absorption wavelengths of litchi polysaccharide–Congo red complexes at NaOH concentrations between 0 and 0.5 mol L⁻¹.

3.3. Immunomodulatory activities of LPI and LPII

3.3.1. Effects on splenocyte proliferation. The stimulatory effects of LPI and LPII on mouse splenocyte proliferation are shown in Fig. 4. LPI and LPII stimulated proliferation in a dose-dependent manner from 100–300 μg mL⁻¹ (p < 0.05). Both LPI and LPII exhibited the highest proliferation indexes of 58.28% and 71.65%, respectively, at 300 μg mL⁻¹. LPII showed stronger stimulatory effects than did LPI at the same concentration (p < 0.05).

Fig. 4 Effects of LPI and LPII on splenocytes proliferation index at different concentrations (100, 200 and 300 μg mL⁻¹). The proliferation indices were assessed using an MTT assay. The results were expressed as the means ± standard deviation (n = 6). Bars labeled with different letters represent a statistical difference at p < 0.05 among the different sample concentrations.

3.3.2. Effects on splenocyte cytokine production. The effects of LPI and LPII on the mouse splenocyte secretion of IL-6 and IFN-γ are shown in Fig. 5a and b. As the dose of LPI and LPII increased from 100–300 μg mL⁻¹, the splenocytes produced more IFN-γ and IL-6 (p < 0.05). Splenocytes produced the highest IL-6 concentrations were 136.08 and 161.66 pg mL⁻¹ when they were stimulated with 300 μg mL⁻¹ LPI and LPII, respectively. Moreover, LPI and LPII stimulated the secretion of IFN-γ to the highest levels of 1370.80 and 1616.28 pg mL⁻¹, respectively. LPII exhibited a stronger stimulatory activity than LPI at the same concentration (p < 0.05).

Fig. 5 Effects of LPI and LPII on the production of IL-6 (a) and IFN-γ (b) by ConA-induced splenocytes at different concentrations (100, 200 and 300 μg mL⁻¹). The proliferation indices were assessed using an MTT assay, and the production of IL-6 (a) and IFN-γ (b) were measured using ELISA kits. The results were expressed as the means ± standard deviation (n = 6). Bars labeled with different letters represent a statistical difference at p < 0.05 among the different sample concentrations.

3.3.3. Effects on NK cell cytotoxicity. The effects of LPI and LPII on the cytotoxicity of NK cells against YAC-1 cells are shown in Fig. 6. The percent lysis of target cells was 40.46–49.84% when the splenocytes were stimulated with 100–300 μg mL⁻¹ LPI, and the percent lysis was the highest when stimulated with 300 μg mL⁻¹. The percent lysis of target cells ranged from 41.30–55.97% when the splenocytes were stimulated with 100–300 μg mL⁻¹ LPII. Furthermore, compared to LPI, LPII showed stronger stimulation on NK cell cytotoxicity at 200 and 300 μg mL⁻¹ (p < 0.05).

Fig. 6 Cytotoxicity of NK cells stimulated by LPI and LPII toward target cells. Cytotoxicity of NK cells was assessed by the MTT assay and expressed as the mean ± standard deviation (n = 6). Bars labeled with different letters represent a statistical difference at p < 0.05 among the different sample concentrations.

4. Discussion

Two polysaccharide fractions (LPI and LPII) were isolated and purified from litchi pulp (cv. Feizixiao) in the present study. Their preliminary chemical characterization, including chemical composition, M_w, glycosidic linkage and helical structure, and in vitro immunostimulatory activities were analysed. The results showed that the immunostimulatory effects of LPII at the evaluated concentrations were significantly greater than those of LPI on splenocyte proliferation, cytokine secretion and NK cell cytotoxicity (p < 0.05). The greater activities of LPII are likely attributable to its chemical composition, M_w, monosaccharide composition, glycosidic linkage and helical structure. Polysaccharides rich in uronic acid exhibit considerable biological activity as the uronic acid residues may alter the polysaccharide properties and modify their solubility.³¹ The immunological activity of polysaccharides from the peduncles of Hovenia dulcis²¹ and the roots of Actinidia eriantha³² were positively correlated with their uronic acid content. LPII containing more uronic acid, exhibited stronger immunostimulatory activity than LPI, which is consistent with previous observations.

Mw is also an important factor that influences the immunological activity of polysaccharides. For example, the higher M_w polysaccharide fraction from Opuntia polyacantha had a stronger effect on macrophage function,³³ while the lower M_w polysaccharide fractions from Abrus cantoniensis³⁴ and Lentinus edodes³⁵ exhibited better immunological activity than the other fractions. A moderate M_w polysaccharide fraction (5–400 kDa) prepared from Aloe showed relatively higher macrophage-activating activity in vivo and in vitro compared to that of fractions with a lower or higher M_w (>400 kDa or <5 kDa).³⁶ It can be concluded that the M_w of polysaccharides from various sources had different effects on their immunological activity. In this study, LPII, with a lower M_w, showed better immunomodulatory activity than LPI.

In addition, polysaccharides mediate immunostimulatory activity by interacting with different receptors and/or modulating various post-receptor intracellular signalling pathways. The monosaccharide composition of polysaccharides were correlated with the recognition of cell surface receptors, such as the mannose receptor, which binds mannosyl/fucosyl ligands.³⁷ Lo et al. reported that arabinose, mannose, xylose and galactose of Lentinula edodes polysaccharides played important roles in the stimulation of macrophages.³⁸ In addition, mannose played a key role in the immunomodulatory activities of polysaccharides from longan pulp³⁹ and aloe vera.⁴⁰ Arabinose was a crucial factor in a Sambucus nigra polysaccharide, influencing macrophage stimulation, and the loss of arabinose resulted in reduced immunostimulatory activity.⁴¹ Furthermore, it was found that the effects of polysaccharides on lymphocytes were closely related to the branch of molecular chain mainly composed of D-galactopyranosyl residues and D-glucopyranosyl residues.^42,43 α-1,4 glucosidic linkages in Coriolus versicolour polysaccharides were the most important structures contributing to their anti-tumour and immunomodulatory activities.⁴⁴ In tested litchi polysaccharides fractions, LPII but not LPI possessed the above structural features and showed higher immunostimulating activity.

Besides the chemical structural features, polysaccharide conformations are also important factors affecting their bioactivities. Previous studies revealed that the triple-helical structure increased the potential for immunological and anti-tumour activity because of interactions with immunological cell surface receptors.⁴⁵ The immunological and anti-tumour activity of Lentinula edodes polysaccharides significantly decreased when the triple-helical conformation was disrupted into single chains.³⁵ LPII, which possesses a triple-helical structure, exhibited stronger immunostimulatory activity than LPI, which is consistent with previous observations.

There are two kinds of possible mechanisms of polysaccharides to exert their immunomodulation activities.¹³ One is some polysaccharides could activate the intracellular signaling cascades in immune cells by membrane receptors,^46,47 and the other is some polysaccharides could directly or indirectly act on intestinal mucosal and trigger the intestinal mucosal immunity, and then regulate systemic immune response by homing of lymphocytes and cytokines.⁴⁸ As for LPI and LPII, they exhibited immunostimulatory activity in term of inducing splenocyte proliferation, cytokine secretion and NK cell cytotoxicity in vitro (Fig. 4–6). The results suggest they may interact directly with immune cells and activate the intracellular signaling cascades to exert immunomodulatory activity, but the detailed mechanism need a further study.

The structural features of polysaccharides from litchi pulp (cv. Feizixiao), including the M_w of 36.9–213 × 10³ Da, the triple helical structure in one fraction, the glycosidic linkage of →3)-α-L-Araf-(1→, β-(1 → 2)-Galp, →4)-α-D-Glcp-(1→, etc., were obviously different from those of other cultivars.^8–10 In addition, litchi (cv. Feizixiao) polysaccharides more strongly stimulated splenocyte proliferation than that did litchi (Guiwei) polysaccharides at the same concentration.⁴ The ability of litchi (cv. Feizixiao) polysaccharides to secrete IFN-γ was greater than that of the litchi polysaccharides in Jing's report.² The differences in structure and immunomodulatory activity may be due to the differences in cultivation environment and variety, as well as the extraction method. These results suggest that Feizixiao, the second largest cultivar planted in China, is a good source for the development of immune-enhancing functional food.

5. Conclusions

In the present study, crude LP was prepared by microwave-assisted extraction and further fractionated by DEAE-cellulose and Sephadex G-100 column chromatography into two purified fractions: LPI and LPII. Compared to LPI, LPII possessed a triple-helical structure, the glycosidic linkage of →3)-α-L-Araf-(1→, β-(1 → 2)-Galp, →4)-α-D-Glcp-(1→, lower M_w and higher content of neutral sugar, uronic acid, arabinose, glucose and mannose and exhibited stronger immunostimulatory activity in term of inducing splenocyte proliferation, cytokine secretion and NK cell cytotoxicity. Based on the above results, it was presumed that the monosaccharide composition, particular glycosidic linkage, uronic acid content and M_w are crucial for the immunostimulatory activity of LP.

Acknowledgements

This work was supported by a Joint Fund from the NSFC and Guangdong Provincial Government (U1301211), the Guangdong Provincial Science and Technology Project (2016B070701012), the National Nature Science Foundation of China (31301459), the Guangdong Natural Science Foundation (2016A030310321) and the Applied Research and Development Project of Guangdong Province (2015B020230005).

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Footnote

† Electronic supplementary information (ESI) available See DOI: 10.1039/c6ra20505d

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