INTRODUCTION
Pulses have been part of human diet for thousands of years. However, the interest in and potential dietary impact of pulses on human health have only been revived during the past two to three decades. Chickpea (Cicer arietinum L.) is pivotal pulse crop produced and utilized globally, especially in African and Asian countries. It has been reported to be produced in more than 45 countries, ranking the third important pulse crop in the world (1, 2). From economic point of view, kabuli chickpea is considered more important than desi chickpea because it gets higher market price (3, 4).
Chickpea contains many bioactive compounds such as oligosaccharides, enzyme inhibitors, phenolic compounds and phytates that could assist in reducing the chronic disease risk. It serves as a worthwhile source of nutrients in vegetarian diets, especially in developing countries. Moreover, chickpea is rich in fibre and low in fat (5, 6), being important source for a balanced diet. Some important vitamins are found in chickpea such as folate, thiamin, niacin, riboflavin, β-carotene and vitamin A precursor (4). Chickpea consumption has numerous physiological health advantages and in combination with other cereals and pulses, it has favourable effects on alleviating the symptoms of some major human diseases, such as digestive disorder, cardiovascular disease, type 2 diabetes and cancer (2, 6).
Polysaccharides are high-molecular-mass polymers, which can be found as storage carbohydrates (starch) or as structural carbohydrates (cellulose). Natural polysaccharides are widely distributed in microorganisms and plants. Some of them have been confirmed to have various bioactive properties, such as antioxidative, antifatigue, immunomodulatory, antiatherosclerotic and hepatoprotective activities (7). Furthermore, polysaccharides extracted from yeasts, fungi, plants and algae have been increasingly utilized in food, biochemistry and pharmacology because of their wide range of biological functions (8).
There has been interest in recent decades in using polysaccharides as ideal immunomodulators (potential therapeutic agents) that can act by inducing the immune response of macrophages (7, 9). Research shows excellent biological functions and nutritional value of natural polysaccharides from legumes and cereals (9-11). However, the information on the immunomodulatory activity of polysaccharides from chickpea hull is unavailable so far. In our prior study, we investigated the chickpea hull polysaccharide (CHPS) extraction optimization, characterization and in vitro antioxidant activities (12). In present study, therefore, first we determined the in vitro immunomodulation of CHPS using RAW264.7 murine macrophages, and then performed in vivo study using specific pathogen-free (SPF) BALB/c mouse animal model.
MATERIALS AND METHODS
Materials
The kabuli chickpea seeds were acquired from Urumchi (local market), Xinjiang Uygur Autonomous Region of China. Lipopolysaccharide (LPS), penicilin, streptomycin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) were purchased from Shanghai Bioengineering Co., Ltd (Shanghai, PR China). Trichloroacetic acid, bovine serum albumin and dimethyl sulphoxide (DMSO) were procured from Solarbio Co. (Beijing, PR China). Guinea pig serum and 2% sheep red blood cell (SRBC) were obtained from Shanghai Chuanxiang Biological Technology Co., Ltd. (Shanghai, PR China). Cyclophosphamide (Cy) and levamisole hydrochloride were purchased from Shanghai Ruiyong Biological Technology Co. Ltd. (Shanghai, PR China). Assay kits for lysozyme, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC) and malondialdehyde (MDA) were bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, PR China). Dulbecco’s modified Eagle complete medium (DMEM) 1640 and newborn calf serum and Triton X-100 were obtained from Gibco/Invitrogen (Carlsbad, CA, USA). RAW264.7 cell line was arranged from Nanjing Keygen Company (Nanjing, PR China). Specific pathogen-free (SPF) grade BALB/c mice were purchased from Beijing Weitong Lihua Experimental Technology Co., Ltd. (Beijing, PR China). Nitrogen monoxide (NO) kit was obtained from Beyotime Institute of Biotechnology (Shanghai, PR China), while enzyme-linked immunosorbent assay (ELISA) kits for cytokines (interleukins IL-6, IL-1β and tumor necrosis factor-alpha (TNF-α)) were bought from Neobioscience Technology Company (Shenzhen, PR China). Sephadex G-100 column and neutral red were procured from Sigma-Aldrich Chemical Co., Merck (St. Louis, MO, USA). All other used chemicals were of analytical grade.
Preparation of chickpea hull polysaccharides
The CHPS were prepared as per the reported procedure of Ye et al. (12). Briefly, chickpea hull (206 g) was separated manually from chickpea seeds (6 kg) with a yield of 3.43%. The hulls were soaked in water (at room temperature) for 4-5 min and then removed with tweezers. The hull was ground to powder, sieved through 60 mesh and extracted with hot water. The extract was then centrifuged at 3260×g (centrifuge model TDZW-S; Changsha Xiangzhi Centrifuge Instruments Co., Changsha, PR China) for 15 min. The resulting supernatant was concentrated, mixed with four volumes of absolute ethanol and kept overnight at 4 °C. The precipitates were centrifuged at 3260×g for 15 min, washed twice with dehydrated ethanol and lyophilized, obtaining crude CHPS. For purification, the crude CHPS was dissolved in distilled water, then loaded to DEAE Fast Flow column (Sigma-Aldrich, Merck, Chicago, IL, USA) and stepwise eluted with 0, 0.1, 0.3 and 0.5 M NaCl solution according to Ye et al. (12). The fractions were collected and the absorbance (A) was determined at 490 nm using the phenol-sulfuric acid method (13). Three fractions having carbohydrates were collected, concentrated, dialyzed, lyophilized and then further purification was done with Sephadex G-100 column (Sigma Chemical Co., Merck, St. Louis, MO, USA) chromatography by eluting with deionized water, respectively. The resulting fractions detected as mentioned above were accumulated, concentrated, then dialyzed and lyophilized, respectively, to obtain CHPS-1, CHPS-2 and CHPS-3.
Immunomodulatory activity assay in vitro of chickpea hull polysaccharide
RAW264.7 cell culture
RAW264.7 cells were taken out of the liquid nitrogen tank and immediately thawed at 37 °C in the water bath. The cells were then transferred to a tube containing 5 mL of DMEM and centrifuged at 405×g (TDZW-S; Changsha Xiangzhi Centrifuge Instruments Co, Ltd.) for 5 min. The supernatant was removed, and the cell sediment was suspended with 1 mL medium containing 10% (V/V) newborn calf serum and 1% (V/V) penicillin-streptomycin mixture, transferred to a flat bottom culture box containing 3 mL of medium and incubated at 37°C in a humidified atmosphere of 5% CO2. When the cell growth reached 70-80% of the bottom of culture box, subculture was carried out.
Cell viability assay
RAW264.7 cell viability was measured in accordance with MTT-based colourimetric method (14) with minor changes. The cells were cultured on a 96-well culture plate (200 µL per well) at a density of 105 cells/mL for 12 h. The non-adherent cells from the wells were aspirated by washing three times with phosphate-buffered saline (PBS) without cell rupture, and 100 µL of the medium were added to each well containing different concentrations (20.0, 40.0, 80.0, 160.0 and 320.0 µg/mL) of CHPS. LPS (10.0 µg/mL) and medium alone were used as positive and blank control, respectively. The cells were incubated for 24, 36 and 48 h. After incubation, 200 µL of MTT solution (0.5 mg/mL) were added to each well of the 96-well plate and further incubated for 4 h at 37 °C. Finally, the MTT solution was taken out, and 150 µL of DMSO were added to each well to terminate the reaction and dissolution of formazan crystals. After shaking for 5 min, the absorbance (A) at 570 nm was measured using a microplate reader (model 800 TS; BioTek Instruments Inc., Agilent, Winooski, VT, USA). Cell viability rate was measured as a ratio of Asample and Ablank control.
Phagocytosis assay
Phagocytosis index of RAW264.7 was measured based on the findings of Zhang et al. (15), with some modifications. RAW264.7 cells were cultured as previously mentioned. CHPS solutions of different concentrations (20.0, 40.0, 80.0, 160.0 and 320.0 µg/mL) as well as a blank control (medium) and positive control (γ(LPS)=10.0 µg/mL) were added to a 96-well plate, and the plate was incubated at 37 °C in an incubator set at 5% CO2 for 24 h. After removing the medium, a neutral red solution (100 µL) was added to each well. After 1 h of incubation, the cells were washed twice with PBS to remove the excess neutral red. After that, a mixture of 100 µL of cell lysate (0.1 M acetic acid/ethanol=1:1) was added to each well and the plate was incubated again overnight at 37 °C. The absorbance was measured at 540 nm with microplate reader (model 800 TS; BioTek Instruments Inc.). The phagocytosis index was calculated as a ratio of Asample and Ablank control.
Acid phosphatase activity
CHPS effect on acid phosphatase activity of RAW264.7 cells was determined as per previously reported method (16). The cultured cells (RAW264.7) were treated with complete medium alone or CHPS solutions (20.0, 40.0, 80.0, 160.0 and 320.0 µg/mL) or LPS (10.0 µg/mL). The supernatant was removed from the wells after incubation for 48 h at 37 °C. After that, 25 µL of 1% Triton X-100 and 150 µL of 1.0 mg/mL p-nitrophenyl phosphate were added into each well, and then the plate was incubated for 1 h at 37 °C. Then, 50 µL of 3.0 M NaOH solution were added to terminate the reaction. The absorbance at 405 nm was measured by a microplate reader (model 800 TS; BioTek Instruments Inc.). Acid phosphatase activity was measured as a ratio of Asample and Ablank control.
Nitrogen monoxide and cytokine assays
The colourimetric assay with Griess reagent was used to measure the released level of nitrogen monoxide in the medium (14). In a word, RAW264.7 cells (106 cells/mL) were cultured for 24 h in 96-well plates with different concentrations of CHPS-3 (20.0, 40.0, 80.0, 160.0, 320.0 µg/mL), blank control (medium) and positive control (γ(LPS)=10.0 µg/mL). After that, 100 µL of culture supernatant were mixed with an equal volume of Griess reagent (1% sulfanilamide and 0.1% N-[1-naphthyl]ethylenendiamine dihydrochloride in 5% phosphoric acid). The samples were incubated for 15 min at room temperature. The absorbance was measured at 540 nm using a microplate reader (model 800 TS; BioTek Instruments Inc.). Standard curve was generated by using NaNO2. The cytokines (IL-6, IL-1β and TNF-α) were calculated by ELISA kit (Neobioscience Technology Company, Beijing, PR China) as per the manufacturer’s instructions.
Immunomodulatory activity of CHPS-3 assay in vivo
In our previous study, we found that CHPS-3 had the strongest reducing power, highest scavenging activity against 2,2'-azinobis-(3-ethyl-benzothiazolin-6-sulfonic acid) and 1,1-diphenyl-2-picrylhydrazyl radicals, and prevention of H2O2-induced oxidative injury in PC12 cells (12). In the present study, CHPS-3 exhibited superior immunomodulatory activity in vitro. Hence, it was used for further study of immunomodulatory activity in vivo.
Animal grouping and experimental design
The experimental design followed the method by Liu et al. (17) with slight modifications. SPF BALB/c mice, 6-week-old with body mass (bm) of (20±2) g, were used. After a 7-day acclimatizing period, all mice were randomly divided into 6 groups (10 mice in each). The animal housing conditions were approved by the Ethical Committee of the College of Food Science and Technology, Nanjing Agricultural University, Nanjing, PR China, and strictly followed in accordance with the Chinese experimental animal management regulations. The temperature and relative humidity were maintained at (21±1) °C and 50-60% respectively. All mice were kept under standard maintenance conditions of 12 h light/dark cycle and were given free access to feed and water. Group I mice (normal control) were treated with 0.9% NaCl once a day for 17 days. Group II mice (model control) were treated with intraperitoneal injection of cyclophosphamide (Cy) administered at a dose of 80 mg/kg daily for the first 3 days of the experiment, followed by intraperitoneal injection of 0.9% NaCl for 14 days. Mice in group III (positive control) were treated with levamisole hydrochloride (dose 10 mg/kg b) instead of 0.9% NaCl administered in group II. Mice in groups IV, V and VI (low, medium and high-dose of CHPS-3 respectively) were treated with Cy for the first three days of experiment at the dose of 80 mg/kg followed by 50, 100 and 150 mg/kg bm of CHPS-3 per day for 14 days, respectively. After 12 h of the last feeding, all mice were sacrificed by cervical dislocation. During the experiment, it was essential to reduce the number of the used animals and their suffering.
Assay of thymus and spleen indices
The body mass of all the mice was measured in each group during the experiment. Moreover, thymus and spleen were removed and washed with normal saline to get rid of blood stains. The mass (m) of vital organs (thymus and spleen) was recorded. The mouse thymus or spleen indices (in mg/g) were calculated using the following equation:
Thymus or spleen index=(m(thymus or spleen)/m(body))∙100 /1/
Serum haemolysin assay
On the tenth day of administration, the mice were injected with 0.2 mL of 2% sheep red blood cell (SRBC) suspension intraperitoneally. Twelve hours after the last administration, the serum was collected from the eye orbit of mice and normal saline was added for dilution. A volume of 200 µL of diluted serum, 200 µL of SRBC (2%) and 200 µL of guinea pig serum were added in sequence into a centrifugal tube. Furthermore, as a blank control the diluted serum was replaced with normal saline. The mixtures were placed in an incubator for 30 min at 37 °C and then centrifuged for 5 min at 6520×g (TDZW-S; Changsha Xiangzhi Centrifuge Instruments Co., Ltd.). The supernatant was collected and absorbance was measured at 540 nm (Asample) using a microplate reader (model 800 TS; BioTek Instruments Inc.). Moreover, the absorbance at which SRBC reached half the haemolytic condition (ASRBC) was determined. In short, 2% SRBC (1.0 mL) was centrifuged (600×g) with distilled water and then sediment was diluted to 950 µL. After resting for 10 min, NaCl solution (17%, 50 µL) was added to obtain haemoglobin solution 0.2%. The resulting (50 µL) haemoglobin solution, 50 µL of SRBC suspension and 400 µL of normal saline were added. Finally, after the centrifugation the absorbance of the supernatant at 540 nm was calculated as ASRBC. Serum haemolysin antibody content was expressed by HC50 as a ratio of Asample and ASRBC.
Serum lysozyme activity assay
The lysozyme activity of mice serum was determined by commercial kit (Sigma-Aldrich, Merck, Chicago) and protocol provided with the kit was followed. Lysozyme activity is defined as its dual activity as a lytic enzyme and a small cationic protein that damages or kills bacteria by lysing their cell wall peptidoglycan, by disrupting bacterial membranes, and by activating autolytic enzymes in the bacterial cell wall.
Assay of antioxidant index in liver
After the mice were sacrificed, the liver was placed on ice and the tissue homogenate was prepared with physiological saline at a m/V ratio of 1:9. Centrifugation was done at 6520×g (TDZW-S; Changsha Xiangzhi Centrifuge Instruments Co., Ltd.) for 5 min, and the supernatant was collected for use. The levels of CAT, GSH-Px, T-AOC and SOD (U/mg), and MDA (nmol/mg) in mice liver protein were measured in accordance with the manufacturer’s instructions for the commercial kit (Sigma-Aldrich, Merck, Chicago).
Statistical analysis
Data are expressed as mean values±standard deviation (S.D.). One-way ANOVA was carried out using Duncan’s multiple range test. Difference was considered to be statistically significant at p<0.05. SPSS software v. 19.0 (18) was used to perform the statistical analysis.
CONCLUSIONS
In the present study, we demonstrated that the three purified fractions of chickpea hull polysaccharides (CHPS) could promote the cell proliferation rate, phagocytosis index and enhance the activity of acid phosphatase in murine macrophages of RAW264.7 in vitro. Among them, CHPS-3 exhibited superior immunomodulatory activity in vitro. Additionally, it was observable that CHPS-3 enhanced NO secretion and cytokine (IL-6, IL-1β and TNF-α) production. In the study in vivo, CHPS-3 improved the thymus and spleen indices in cyclophosphamide-induced immunodeficient mice. It increased the lysozyme activity, serum haemolysin content, CAT, GSH-Px, T-AOC and T-SOD activities, and reduced the content of MDA in the liver. In a word, CHPS exhibited strong antioxidant ability and immunomodulatory activity. Further studies to elucidate detailed mechanism of CHPS and its structure are in process.