Article pubs.acs.org/JAFC
Structural Characterization and Immunostimulatory Activity of a Homogeneous Polysaccharide from Sinonovacula constricta Qingxia Yuan,† Longyan Zhao,§ Qianqian Cha,† Yi Sun,† Hong Ye,† and Xiaoxiong Zeng*,† †
College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650201, People’s Republic of China
Downloaded by TEXAS A&M INTL UNIV on September 6, 2015 | http://pubs.acs.org Publication Date (Web): September 4, 2015 | doi: 10.1021/acs.jafc.5b03306
§
ABSTRACT: Sinonovacula constricta has been widely used as a health food and medicine in China, Japan, and Korea. In the present study, a water-soluble polysaccharide fraction (SCP-1) was prepared from S. constricta by enzyme-assisted extraction and purification of chromatography with DEAE-52 cellulose anion-exchange column and Sephadex G-100 size exclusion column. On the basis of the analytical results of high-performance liquid chromatography, Fourier transform-infrared spectroscopy, methylation analysis, and NMR spectroscopy, SCP-1 was found to have an average molecular weight of 15.63 kDa and a linear backbone of (1→4)-linked α-D-Glcp residue with one branch, α-D-Glcp, attached to the main chain by a (1→6) glycosidic bond at every five α-D-Glcp units. Furthermore, it was found that SCP-1 could significantly increase the viability of macrophages, enhance the capability of macrophage phagocytosis, increase the activity of acid phosphatase, and promote the production of nitric oxide, mouse tumor necrosis factor (TNF)-α, mouse interferon (IFN)-γ, and mouse interleukin (IL)-1β. The results suggest that SCP-1 possesses potent immunomodulating effect and may be explored as a potential biological response modifier. KEYWORDS: Sinonovacula constricta, polysaccharide, structure, immunomodulatory activity
■
INTRODUCTION Sinonovacula constricta, a bivalve mollusk belonging to the Solcnidoe family, is widely distributed in intertidal zones and estuarine regions of China, Japan, and Korea.1 As an ecologically and commercially important mollusk food in China, it contains rich nutrients, such as protein, fat, amino acids, betaine, and polysaccharides.2 According to traditional Chinese medicine, S. constricta can rectify deficiency, improve disease-resistant ability, and treat dysenteric diseases. Recent research has mainly focused on the genome,3−5 physiology,6 metabolism,7 and reproductive cycle8 of S. constricta, whereas there are not so many studies on its bioactive compounds.1,9−12 It has been reported that the hot water extract and protein of S. constricta exhibited anticancer and immunological activities.1,9−11 In addition, the polysaccharides from S. constricta (SCP) showed a potent virus−cell fusion inhibitory activity.12 However, research on its biological activities and potential mechanisms is still in its infancy. Polysaccharides and oligosaccharides have intrigued biologists for decades as therapeutic agents and mediators of complex cellular events.13,14 There is increasing evidence that a great number of polysaccharides isolated from plants, fungi, and bacteria are associated with immunostimulatory effects and do not cause significant side effects.15 For example, glucans from edible mushrooms have been used as a nonspecific immune modulatory supplement,16−18 and the polysaccharides from animals, mainly common bivalve mollusk, also possess immune activity.19−21 It is well-known that the structure features of polysaccharides including their molecular weights, monosaccharide compositions, glycosidic bonds, branched degrees, and conformation are related to their various biological functions.18,22,23 Moreover, the exact structure of a polysaccharide is a prerequisite to probe its potential mechanisms and structure−activity relationship. However, information about © XXXX American Chemical Society
the purification, structural feature and immunostimulatory activity of SCP is not available. In our present study, therefore, a purified polysaccharide fraction (SCP-1) was first prepared from S. constricta by extraction and purification of crude SCP through chromatography of DEAE cellulose anion-exchange column and Sephadex G-100 size exclusion column. Afterward, the structure of SCP-1 was characterized by a series of analytical techniques including Fourier transform-infrared spectroscopy (FT-IR), high-performance liquid chromatography (HPLC), methylation analysis, and 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. Finally, the immunomodulatory activity in vitro of SCP-1 was evaluated by using RAW264.7 murine macrophages to investigate the effects on cell viability, phagocytosis, acid phosphatase, and production of nitric oxide (NO) and cykotines.
■
MATERIALS AND METHODS
Materials and Reagents. S. constricta was obtained from a local market in Nanjing, China. RAW264.7 murine macrophage cell line was purchased from Nanjing University of Chinese Medicine (Nanjing, China). DEAE-52 cellulose, Sephadex G-100, mannose (Man), arabinose (Ara), galactose (Gal), galacturonic acid (GalA), glucose (Glc), glucuronic acid (GlcA), ribose (Rib), xylose (Xyl), lipopolysaccharide (LPS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 3-methyl-1-phenyl-2-pyrazolin-5-one (PMP), penicillin, and streptomycin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rhamnose (Rha) and fucose (Fuc) were purchased from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits for NO, mouse Received: July 6, 2015 Revised: August 25, 2015 Accepted: August 28, 2015
A
DOI: 10.1021/acs.jafc.5b03306 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Downloaded by TEXAS A&M INTL UNIV on September 6, 2015 | http://pubs.acs.org Publication Date (Web): September 4, 2015 | doi: 10.1021/acs.jafc.5b03306
Journal of Agricultural and Food Chemistry
4000 cm−1 was recorded with a Nicolet 6700 FT-IR spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Methylation and GC-MS Analysis. SCP-1 was methylated according to the reported method.28,29 Briefly, SCP-1 (dried) was dissolved in dimethyl sulfoxide (DMSO), and NaOH was then added. The mixture was treated for 20 min under ultrasonic condition. Then, SCP-1 was methylated under darkness by the addition of methyl iodide with ultrasonic for 2 h, and the remaining methyl iodide was decomposed by the addition of deionized water. The methylated sample was extracted with trichloromethane and evaporated to dryness by a stream of nitrogen. Then, the dried methylated sample was hydrolyzed by 2 M TFA at 120 °C for 2 h, reduced with sodium borodeuteride at 50 °C for 2 h, and acetylated with pyridine and acetic anhydride at 100 °C for 1 h. Deionized water and dichloromethane were added to the acetylated derivatives, and the organic phase was dried under nitrogen, dissolved in dichloromethane, and analyzed by GC-MS. The GC-MS analysis was performed on a HP6890GC/5973MS system equipped with an ion trap MS detector and fitted with a HP-5MS quartz capillary column (30 mm × 0.25 mm, 0.25 μm film thickness, 150−200 °C at 2 °C/min and then 200−280 °C at 5 °C/min). NMR Analysis. NMR analysis was performed at 298 K on a Bruker DRX Avance III 500 MHz spectrometer (Bruker Corp., Karlsruhe, Germany) equipped with a 13C/1H dual probe in FT mode. The freezedried SCP-1 was dissolved in deuterium (D2O, 99.9% D) and lyophilized three times to replace exchangeable protons with D2O. The lyophilized sample was then dissolved in D2O at a concentration of 100 mg/mL and analyzed by NMR spectrometer. All chemical shifts are relative to internal sodium 2,2-dimethyl-isotope 2-silapentane-5sulfonate (DSS). Assay of Immunomodulatory Activity in Vitro of SCP-1. Cell Culture. The murine macrophage RAW264.7 cells were cultured in DMEM supplemented with 10% (v/v) new bovine calf serum and 1% (v/v) penicillin−streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. The cells were cultivated in sterile tissue culture flasks and subcultured for experimentation. Assay of Cell Viability. The viability of RAW264.7 cells was assessed by using MTT-based colorimetric assay.30 Briefly, 100 μL/well of RAW264.7 cell suspension was plated in a 96-well culture plate and incubated (37 °C, 5% CO2) for 12 h. The adherent RAW264.7 cells were washed twice by PBS and then incubated with medium containing various concentrations of sample (12.5, 25.0, 50.0, 100.0, and 200.0 μg/ mL) as well as complete medium alone (blank control) or LPS (10.0 μg/ mL, positive control) for 48 h. The stimulated cells were washed twice by PBS, and 200 μL of MTT solution (0.5 mg/mL) was added to each well. After further incubation for 4 h, 150 μL of DMSO was added to each well to dissolve the formazen crystals. The absorbance (Abs) at 570 nm was determined by using a microplate reader. The cell viability was calculated by using the following equation:
tumor necrosis factor (TNF)-α, mouse interferon (IFN)-γ, and mouse interleukin (IL)-1β were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All other reagents used in this study were of analytical grade. Preparation of SCP-1. Extraction of SCP. Briefly, fresh S. constricta was collected and washed carefully with water. After removal of the shells and impurities, the flesh was crushed by a high-speed disintegrator, and the homogenate was kept in 85% of ethanol (v/v) for 1 week to defat and remove pigments and small molecule substances. The collected flesh was then air-dried at 50 °C and used for the extraction. The dried powder of S. constricta was dissolved in deionized water (20 mL/g (v/w) for ratio of water/material) containing 2.0% papain. After incubation at 50 °C for 6 h, the mixture was boiled for 10 min and centrifuged at 3040g for 20 min. The supernatant was precipitated with 3 volumes of dehydrated ethanol (ethanol final concentration, 75%) at 4 °C for 12 h, and the resulting precipitates were obtained by centrifugation at 3040g for 15 min. The precipitates were dissolved in deionized water, the pH was adjusted with 1.0 M HCl to 2.5, and the mixtures were kept at 4 °C for 4 h and then centrifuged at 3040g for 15 min to remove acidic albumen.24 The supernatant was adjusted to neutral and precipitated again with dehydrated ethanol. Finally, the precipitates were collected by centrifugation at 3040g for 15 min and vacuum freeze-dried, affording the crude SCP. Purification of Crude SCP. The crude SCP was applied to a DEAE-52 cellulose column (2.6 × 50 cm) and eluted stepwise with NaCl solutions from low to high concentration (0, 0.1, and 0.3 M) at a flow rate of 1.0 mL/min. The fractions were collected (10 mL/tube) and checked by tracking the absorbance at 490 nm using the phenol−sulfuric acid method.25 The major polysaccharide fraction (F-1) was collected, dialyzed, freeze-dried, and loaded onto a column (1.6 × 60 cm) of Sephadex G-100. The column was eluted with deionized water at a flow rate of 15 mL/h, and the elution was checked as described above. As a result, one purified fraction was collected and vacuum freeze-dried, affording SCP-1. Determination of Contents of Carbohydrate, Protein, Uronic Acid, and Sulfuric Radical. The contents of carbohydrate, protein, uronic acid, and sulfuric radical in SCP-1 were determined according to the reported methods.26 Determination of Homogeneity and Molecular Weight. The homogeneity and molecular weight of SCP-1 were determined by highperformance gel permeation chromatography (HPGPC) with an Agilent 1200 series apparatus (Agilent Technologies, Santa Clara, CA, USA) equipped with a Shodex OHpak SB-804 HQ column (8 × 300 mm). The column was eluted with 0.1 M NaCl at a flow rate of 0.5 mL/ min, and the column temperature was kept constant at 35 °C. The molecular weight and polydispersity index (Mw/Mn) were estimated by HPGPC, and the column was calibrated by standard D-series dextrans (D-2, D-3, D-4, D-5, D-6, D-7, and D-8) with known molecular weight. Analysis of Monosaccharide Composition. The monosaccharide composition of SCP-1 was analyzed by HPLC as described previously.27 The polysaccharide solution (100 μL, 5.0 mg/mL) was mixed with 100 μL of 4.0 M trifluoroacetic acid (TFA) and hydrolyzed at 120 °C for 2 h. The resulting solution was cooled to room temperature, and the excess TFA was removed by repeated codistillations with methanol. The residue was dissolved with deionized water (100 μL) and mixed with 100 μL of 0.6 M NaOH, and half of the mixture was derivatized with 0.5 M methanol solution of PMP (100 μL) for 100 min at 70 °C. After neutralization with 0.3 M HCl, the resultant solution was concentrated to dryness under reduced pressure. Deionized water and chloroform were added to the residue, followed by vortexing. The water phase was filtered through a 0.45 μm membrane and then analyzed by an Agilent HPLC using Eclipse Plus C18 column (4.6 × 250 mm, 5 μm, Agilent) and ultraviolet (UV) detector at a flow rate of 1.0 mL/min at 30 °C. The mobile phase contained 83% phosphate-buffered saline (PBS, 0.1 M, pH 6.7) and 17% acetonitrile (v/v). In a similar manner, the monosaccharide standards were PMP-labeled and analyzed by HPLC. FT-IR Spectral Analysis. The polysaccharide sample was incorporated into potassium bromide (KBr, spectroscopic grade) and pressed into a 1 mm pellet. The IR spectrum of SCP-1 in a range of 400−
cell viability = Abssample /Abs blank control Assay of Phagocytosis. The influence of SCP-1 on phagocytic activity was investigated by using the neutral red phagocytosis assay.31 As described above, confluent cultures of RAW264.7 cells in 96-well plates were exposed to complete medium alone or different concentrations of sample or LPS solution and incubated for 48 h at 37 °C. After removal of supernatant, the nonadherent cells were removed by washing twice with PBS, and 100 μL of neutral red solution was added to each well. The plates were incubated for 1 h, followed by the removal of the supernatant. To remove the excess neutral red solution, the cells were washed with PBS twice. After that, 100 μL of cell lysate (1.0 M acetic acid/ethanol = 1:1, v/v) was then added to each well. After incubation overnight at room temperature, the Abs at 540 nm of each well was measured by a microplate reader. The phagocytosis index of macrophages was calculated by using the following equation:
phagocytosis index = Abssample /Abs blank control Assay of Acid Phosphatase. The effect of SCP-1 on acid phosphatase activity of RAW264.7 cells was investigated according to the reported method.32 The cultured RAW264.7 cells were treated with complete B
DOI: 10.1021/acs.jafc.5b03306 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry medium alone or SCP-1 solution (12.5, 25.0, 50.0, 100.0, and 200.0 μg/ mL) or LPS (10.0 μg/mL). After incubation for 48 h at 37 °C, the supernatant was aspirated from the wells, 25 μL of 1% Triton X-100 and 150 μL of 1.0 mg/mL p-nitrophenyl phosphate (substrate of acid phosphatase) were added to each well, and the plate was incubated at 37 °C for 1 h. The reaction was terminated by the addition of 50 μL of 3.0 M NaOH solution, and the Abs of the culture well was measured at 405 nm by spectrophotometer. The index of acid phosphatase activity was calculated by using the following equation:
exclusion chromatography. As shown in Figure 1B, it yielded a single elution peak, named SCP-1. Characterization of SCP-1. Contents of Carbohydrate, Protein, Uronic Acid, and Sulfuric Radical in SCP-1. The contents of carbohydrate, uronic acid, and sulfuric radical in SCP1 were 99.96, 0.13, and 0.20%, respectively. However, SCP-1 had a negative response to the Bradford test and showed no absorption at 280 or 260 nm in the UV spectrum, indicating the absence of protein or nucleic acid in SCP-1. Molecular Weight and Monosaccharide Composition of SCP-1. As shown in Figure 2, SCP-1 was eluted as a single and
Downloaded by TEXAS A&M INTL UNIV on September 6, 2015 | http://pubs.acs.org Publication Date (Web): September 4, 2015 | doi: 10.1021/acs.jafc.5b03306
index of acid phosphatase activity = Abssample /Abs blank control Assay of Cytokine and NO. The levels of NO, TNF-α, IFN-γ, and IL1β in the supernatants of the RAW264.7 cells from each group were determined by using ELISA kits according to the manufacturers’ protocols. Statistical Analysis. Data were expressed as means ± standard deviations (SD), and a one-way ANOVA was performed, followed by Duncan’s multiple-range tests. P values of
