Article pubs.acs.org/JAFC
Immunomodulatory Activity in Vitro and in Vivo of Verbascose from Mung Beans (Phaseolus aureus) Zhuqing Dai, Di Su, Yun Zhang, Yi Sun, Bing Hu, Hong Ye, Saqib Jabbar, and Xiaoxiong Zeng* College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China ABSTRACT: In the present study, the immunostimulatory activity of verbascose from mung beans (Phaseolus aureus) was evaluated by using in vitro cell models and in vivo animal models. The results of in vitro experiments showed that verbascose could enhance the ability of devouring neutral red of peritoneal macrophages and promote the release of nitric oxide and immune reactive molecules such as interleukin (IL)-6, IL-1β, interferon (IFN)-α, and IFN-γ. Treatment with verbascose at a dose of 200 μg/mL exhibited the best effects. For assay in vivo, administration of verbascose at a medium dose of 90 mg/kg body weight could significantly increase the index of spleen, activity of lysozyme in spleen and serum, hemolysin level in serum, and swelling rate of earlap in the delayed type of hypersensitivity (DTH) of immunosuppressed mice. All of the results suggested that verbascose had potent immunostimulatory activity and could be explored as a potential natural immunomodulatory agent in functional foods. KEYWORDS: mung beans (Phaseolus aureus), verbascose, immunomodulating activity, in vitro, in vivo
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bacteria.18,25−28 Furthermore, it has been reported that oligosaccharide prebiotics may modulate the immune response through the modification of the intestinal microbiota and/or in a microbiota-independent manner by direct interaction on immune cells.3,18,29−31 Macrophage, one of the major immunocytes, exhibits many immune functions such as phagocytizing extraneous materials, secreting cytokines, and processing antigens. To execute these functions, macrophages classically produce and release pro-inflammatory cytokines and chemical mediators, such as nitric oxide (NO), interferon (INF), and prostaglandin E2 (PGE2).32−34 Pro-inflammatory cytokines, such as interleukin (IL)-1β and IL-6, are potentially capable of causing injury to host tissues in playing these key homeostatic functional roles.35 Verbascose, a member of the α-GOS and distributed in legume seeds, has a structure of three α-D-galactosyl residues linked (1→6) to the D-glucosyl residue of sucrose. It has been reported that mung bean (Phaseolus aureus), also known as green gram or golden gram that is commonly used in various cuisines across Asia, contains a high concentration of α-GOS, especially verbascose.20 As a kind of α-galacto-oligosaccharide, there are several papers available about verbascose composition, separation, synthesis, and structure analysis,36−38 whereas information about the immunomodulatory and prebiotic effects of verbascose is not available. In this study, therefore, highpurity verbascose was prepared from mung bean by extraction and purification through column chromatography. Afterward, the immunomodulatory activity in vitro of verbascose was evaluated by investigating the effects of cell proliferation, phagocytosis, and promotion of cykotines with RAW264.7 murine macrophages. Finally, the immunomodulatory activity
INTRODUCTION Prebiotics are defined as selectively fermented ingredients that allow specific changes, in both the composition and/or activity of the gut microflora, that confer benefits upon host well-being and health.1 It has been reported that the majority of prebiotics are low-digestible carbohydrates, including galacto-oligosaccharides (GOS), lactulose, inulin, and its fructo-oligosaccharide (FOS) derivatives.2,3 Currently, low-digestible carbohydrates have been widely studied and proved to be effective probiotic factors. They are highly selective for the growth of Bifidobacterium and Lactobacillus, which stimulates the production of short-chain fatty acids and plays a positive role in metabolic and inflammatory disorders such as obesity, diabetes, and inflammatory bowel diseases.4−6 GOS, generally considered to be one of the most common prebiotics, contain a mixture of oligosaccharides formed by one or more galactosyl moieties linked to a terminal glucose or by exclusively galactose units (galactobiose, galactotriose, etc.).7,8 In addition to being prebiotics, GOS exhibit various functions such as immunomodulatory and antitumor activities, preventing constipation, reducing the level of blood serum cholesterol, improving mineral absorption, and controlling some acute or chronic diseases.9−12 These properties make GOS applied widely as food ingredients. At present, there are numerous studies available on enzymatic synthesis and health benefits of β-GOS such as the prevention of allergic disease, reduction of cancer risk, and enhancement of calcium absorption.7,13−17 However, information on α-GOS is much scarcer than that of β-GOS. In fact, αGOS are widely distributed in legume seeds, for instance, soybean, mung bean, lupin, lentil, and chickpea.18−21 The wellknown α-GOS are raffinose, stachyose, verbascose, and ajugose, which are excellent dietary fibers and prebiotics and also known as commonly applied oligosaccharides in the food industry.22−24 α-GOS can significantly promote the growth of Bif idobacterium and inhibit the growth of pathogenic © 2014 American Chemical Society
Received: Revised: Accepted: Published: 10727
July 24, 2014 October 8, 2014 October 15, 2014 October 15, 2014 dx.doi.org/10.1021/jf503510h | J. Agric. Food Chem. 2014, 62, 10727−10735
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chromogenic TAL/TAL end point assay kit according to the manufacturer’s instruction. Cell Culture. The RAW264.7 cells cryopreserved in liquid nitrogen were rapidly thawed in a water bath at 37 °C, transferred to sterile tissue culture dishes, suspended in DMEM supplemented with 10% newborn calf serum, 100 IU/mL penicillin, and 100 IU/mL streptomycin, and incubated in a humidified 5% CO2 incubator at 37 °C. When the cells spread to >80% of the bottom of the culture dishes, the adherent cells were kept for cell passage and used for further culture. Assay of RAW264.7 Cell Viability. Measurement of RAW264.7 cell viability was done according to the MTT-based colorimetric method.39 Briefly, the RAW264.7 cells, suspended at a density of 1 × 105 cells/mL in DMEM supplemented with 10% newborn calf serum, 100 IU/mL penicillin, and 100 IU/mL streptomycin, were seeded into a 96-well flat-bottom plate (100 μL/well) and inoculated in a humidified 5% CO2 incubator at 37 °C for 12 h. The nonadherent cells in the wells were removed by washing three times with DMEM. After that, a series of concentrations of verbascose solution (final concentrations of 25.0, 50.0, 100.0, 200.0, and 400.0 μg/mL) were added (100 μL/well). LPS (final concentration of 10.0 μg/mL) and complete DMEM alone were used as positive control and blank control, respectively. The cells were incubated as mentioned above for 12, 24, 36, and 48 h, respectively. After each incubation, 200 μL of MTT solution (0.5 mg/mL) was added to each well, and the plate was further incubated at 37 °C for 4 h. Finally, the reaction was stopped by adding 150 μL of DMSO to each well for the dissolution of formazan crystals. The absorbance (Abs) at 570 nm was determined by a Synergy 2 Multimode Microplate Reader (BioTeK Instruments, Inc., Winooski, VT, USA), and the cell viability rate was calculated according to the formula below:
in vivo of verbascose was evaluated by using animal models through the measurement of immune organ index, lysozyme activity, hemolysin level, and swelling rate of earlap in delayedtype hypersensitivity (DTH) reaction.
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MATERIALS AND METHODS
Materials and Chemicals. Mung beans were obtained from a local market (Nanjing, China). The RAW264.7 murine macrophage cell line was purchased from Nanjing University of Chinese Medicine (Nanjing, China). The Kunming mice (8 weeks old), grade of specific pathogen free (SPF) with body weights (BWs) of 20 ± 2 g, were purchased from the Experiment Animal Center of Academy of Military Medical Sciences (Beijing, China). Mulbecco’s modified Eagle medium (DMEM), newborn calf serum, 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES), and trypan blue dye were purchased from Gibco/Invitrogen (Carlsbad, CA, USA). Sheep blood and guinea pig serum were purchased from Chuanxiang Biotechnology Co. (Shanghai, China). Lipopolysaccharide (LPS), neutral red, penicillin− streptomycin stock solution, and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were obtained from SigmaAldrich Chemical Co., Ltd. (St. Louis, MO, USA). Cyclophosphamide (CPA) was purchased from Jiangsu Hengrui Medcine Co. (Nanjing, China). Dinitrofluorobenzene (DNFB) was purchased from Xiya Reagent Co., Ltd. (Chengdu, China). Levamisole hydrochloride was purchased from Jinan Aodekai Pharmaceutical Co., Ltd. (Jinan, China). Lysozyme kit and NO kit, as well as IL-6, IL-1β, INF-α, and INF-γ ELISA kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Chromogenic TAL/TAL end point assay kit for determination of endotoxin was purchased from Chinese Horseshoe Crab Reagent Manufactory Co., Ltd. (Xiamen, China). All other reagents used were of analytical grade. Preparation of Verbascose from Mung Beans. The preparation of verbascose from mung beans was carried out according to the reported method.21 Full grains of pest-free mung beans were selected and crushed with a grinder, and the powder was extracted two times with 80% aqueous ethanol in a ratio of 10:1 (ethanol solution to material, mL/g) at 70 °C for 60 min. The extract was centrifuged at 2800g for 10 min. The supernatants of two extractions were combined and concentrated by using a rotary vacuum evaporator (Heidolph, Germany). The resulting residue was loaded onto a column (2.0 × 30.0 cm) of charcoal/Celite (1:1, w/w), and the column was successively eluted with a linear gradient of 0−50% aqueous ethanol. The fractions were collected (40 mL/tube) and screened by reaction with naphthoresorcinol, and those with sugars were further analyzed by using an Agilent 1100 series HPLC system (Agilent, USA) with a refraction index detector (RID). The separation of sugars was completed on a Sugar-D column (4.6 × 250 mm, Nacalai Tesque Inc., Kyoto, Japan) by using acetonitrile/water (75:25, v/v) as the mobile phase at a flow rate of 1.0 mL/min. Sugars were identified by comparing the retention times with those of standard sugars. The fractions containing verbascose only were combined, concentrated in vacuo, and further purified by gel filtration chromatography of Biogel P-2 (1.5 × 100.0 cm). The fractions (2.5 mL/tube) containing verbascose checked by HPLC were combined, concentrated/ and freeze-dried, affording high-purity verbascose. The structure of verbascose prepared was characterized by HPLC, mass spectrometry (Mariner system 5304 mass spectrometer, Applied Biosystems, Foster City, CA, USA), and nuclear magnetic resonance (NMR) spectroscopy. 1H and 13C NMR spectra of the sample, dissolved in deuterated water, were recorded on a Burker Avance DRX-500 spectrometer (Bruker, Karlsruhe, Germany) at a temperature of 298 K. Data for verbascose: 1H NMR (500 MHz, D2O) δ 5.42 (d, 1H, J = 3.7 Hz), 4.98 (t, 2H); 13C NMR (500 MHz, D2O) δ 106.44, 100.98, 100.57, 100.41, 94.74, 83.98, 79.00, 76.64, 75.39, 73.90, 73.59, 72.14, 72.03, 71.88, 71.48, 71.30, 71.08, 70.91, 69.10, 68.95, 68.49, 65.12, 64.08, 63.80; MS m/z 851.27 for [M + Na]+ (calcd for C30H52O26 [M + Na]+, 851.2639). Determination of Endotoxin Contamination. The concentration of endotoxin in the verbascose sample was measured by using a
cell viability rate (%) = Abssample /Abscontrol × 100 Assay of Phagocytosis of RAW264.7 Cells. Phagocytosis of RAW264.7 cells was measured according to the reported method with modification.40 The prior stage culture of RAW264.7 cells was done as previously described. The verbascose solutions with different concentration (25.0, 50.0, 100.0, 200.0, and 400.0 μg/mL) as well as a blank control (complete DMEM) and positive control (LPS, 10.0 μg/mL) were added to a 96-well plate (100 μL/well), and the plate was incubated at 37 °C in a 5% CO2 incubator for 24 h. Subsequently, the neutral red solution at a concentration of 0.075% was added to each well (100 μL/well). After further incubation for 1 h, the cells were washed with phosphate-buffered saline (PBS) two times to remove excess neutral red. After that, a mixture of glacial acetic acid (0.1 M) and ethanol (1:1, v/v) was added to each well (100 μL/well), and the plate was incubated again overnight at 37 °C. Finally, Abs at 540 nm of each well was measured by using an ELISA plate reader. The phagocytosis index of devouring neutral red of peritoneal macrophages was calculated by using the following equation: phagocytosis rate (%) = Abssample /Abscontrol × 100 Assay of NO and Cytokines. The NO amount released by peritoneal macrophages was determined by measuring the nitrite level in the cell supernatant with Greiss reaction.41 Briefly, the collected cell supernatant from the assay of phagocytosis of peritoneal macrophages was mixed in 96-well cell plates with Griess reagent (1% sulfanilamide and 0.1% N-1-naphthylethylenediamine dihydrochloride in 5% phosphoric acid) in equal volume and incubated at room temperature for 10 min. Abs at 492 nm was then measured by an ELISA plate reader by using sodium nitrite (NaNO2) as a standard. The levels of cytokines (IL-6, IL-1β, IFN-α, and IFN-γ) were measured by using an ELISA kit according to the manufacturer’s protocol. Animal Grouping and Experimental Design. All animals were treated according to the previously reported method,42 and the procedures involving animals were conducted in strict accordance with the Chinese legislation on the use and care of latoratory animals. Briefly, female Kunming mice were housed in an air-conditioned animal room with constant temperature (21 ± 1 °C), 50−60% relative 10728
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Figure 1. HPLC chromatograms of soluble sugars (A) and purified verbascose (B) from mung beans. HPLC conditions: column, Sugar-D (4.6 × 250 mm); mobile phase, acetonitrile/water (75:25, v/v); flow rate, 1.0 mL/min; detector, RID. Peaks: 1, fructose; 2, glucose; 3, sucrose; 4, melibiose; 5, raffinose; 6, unknown compound; 7, stachyose; 8, verbascose. Assay for Serum Hemolysis to Sheep Red Blood Cell (SRBC). On the fourth day of treatment, the mice were sensitized by intraperitoneal injection of 0.2 mL of 2% SRBC suspension. At 24 h after the last inspection, the serum was collected through the eye orbit of the mouse and diluted with normal saline. The diluted serum (200 μL) was taken into a centrifuge tube, and 200 μL of SRBC (2%) and 200 μL of guinea pig serum were added in sequence. The diluted serum was substituted with normal saline as a blank control. The mixtures were incubated at 37 °C for 30 min and then centrifuged at 10000g for 5 min. The Abs of the supernatant was measured at 540 nm (Abssample). In addition, the Abs at which SRBC reached the half hemolytic situation was measured (AbsSRBC). Briefly, 2% SRBC (1.0 mL) was centrifuged and the sediment was diluted with distilled water to a volume of 950 μL. After 10 min, 17% NaCl solution (50 μL) was added to afford a 0.2% hemoglobin solution. To the resulting hemoglobin solution (50 μL), 50 μL of SRBC suspension and 400 μL of normal saline were added. Finally, the Abs at 540 nm of the supernatant from centrifugation was measured as AbsSRBC. The content of serum hemolysin antibody was expressed by HC50 as follows:
humidity, and a 12 h light/dark cycle with free acess to laboratory chow and water. After acclimatization for 7 days, the mice were randomly assigned to six groups (eight for each group). The mice in group I (normal control) were given a hypodermic injection of 0.9% NaCl once daily for 8 days. The mice in group II (model control group) were given a hypodermic injection of 0.9% NaCl once daily as well as a hypodermic injection of CPA (100 mg/kg BW) on days 2, 3, and 4. Group III was positive control, in which 0.9% NaCl was replaced with levamisole hydrochloride on the basis of group II. Groups IV−VI were verbascose treatment groups at doses of 30, 90, and 270 mg/kg BW, respectively. Different doses of verbascose were given for 8 consecutive days, and a hypodermic injection of CPA (100 mg/kg BW) was performed on days 2, 3, and 4. At 12 h after the last administration, all of the mice were sacrificed by using a cervical dislocation method and the spleen index, thymus index, swelling rate of earlap, lysozyme activity, and serum 50% hemolytic value (HC50) were measured. Lymphoid Organ and Body Weights. The BWs of mice in each group were measured during the experimental period. Furthermore, the weights of vital organs (spleen and thymus) were recorded after cervical dislocation of the mice. The viscera index was calculated as
HC50 = Abssample /AbsSRBC
viscera index (mg/g) = W1/W0
Determination of Lysozyme Activity in Spleen and Serum. The spleen and serum were obtained as previously described. The lysozyme activity was measured by an ELISA kit according to the manufacturer’s protocol. Statistical Analysis. Data are expressed as means ± SD and subjected to one-way analysis of variance (ANOVA). Duncan’s new multiple-range test was performed to determine the significant difference using SPSS 18.0 software (SPSS Inc., Chicago, IL, USA).
where W1 is the weight of the spleen or thymus and W0 is the BW of mouse. DTH Response. DNFB solution was prepared with acetone and vegetable oil in a ratio of 1:1 (v/v) in advance. On the third day of inspection, abdomenal hair on the mice was cut off (3.0 × 3.0 cm), and 30 μL of 1% DNFB was smeared slowly on the shaved abdomen. After 7 days of treatment, both sides of the right ears of the mice were embrocated with 20 μL of 1% DNFB, whereas the left ears were not treated (as control). After cervical dislocation of the mice, a 6 mm diameter round ear piece was taken from each earlap by using a puncher. The swelling rate of the earlap was calculated as follows:
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RESULTS AND DISCUSSION
Preparation of Verbascose. As shown in Figure 1A, the soluble sugars extracted from mung bean were effectively separated by HPLC. According to the retention times of standard sugars, fructose, glucose, sucrose, melibiose, raffinose,
swelling rate of earlap (%) = (Wright ear − Wleft ear)/ Wleft ear × 100 10729
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Figure 2. Mass spectrum (A) and 1H (B) and 13C NMR (C) spectra of verbascose prepared from mung bean.
stachyose, and verbascose were identified in mung beans. Notably, the three most abundant sugars were verbascose, sucrose, and raffinose, accounting for 41.8, 26.7, and 12.5% of the total soluble sugars in mung beans, respectively. The contents of monosaccharides and disaccharides including
fructose, glucose, sucrose, and melibiose accounted for only 42.4% of the total soluble sugars, only slightly greater than the content of verbascose. Despite the low level of stachyose, αGOS still accounted for >50% of total soluble sugars. The results clearly demonstrate that nodigestible α-GOS are the 10730
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Table 1. Effects of Verbascose on RAW264.7 Cell Viabilitya cell viability verbascose incubation time (h) 12 24 36 48 a
control 1 1 1 1
± ± ± ±
0.08a 0.09a 0.12a 0.05a
LPS 1.29 1.76 1.67 1.50
± ± ± ±
0.14c 0.19cd 0.17c 0.14d
25 μg/mL 1.04 1.21 1.29 1.23
± ± ± ±
0.02a 0.10b 0.17b 0.28b
50 μg/mL 1.13 1.25 1.34 1.26
± ± ± ±
0.02ab 0.30b 0.29b 0.13b
100 μg/mL 1.23 1.56 1.43 1.31
± ± ± ±
0.07bc 0.21c 0.20b 0.22bc
200 μg/mL 1.34 1.82 1.60 1.44
± ± ± ±
0.12c 0.25d 0.15c 0.11cd
400 μg/mL 1.34 1.78 1.62 1.41
± ± ± ±
0.16c 0.11cd 0.24c 0.12cd
Different lower case letters represent a significant difference (p < 0.05) between different samples of the same time.
Effects of Verbascose on Phagocytic Activity of RAW264.7 Cells. Activated macrophage not only participates in both specific and nonspecific immune reactions but also is the “bridge cell” of these two kinds of immune reactions.43 The increase in phagocytic activity is one of the most distinguished features of macrophage activation. Therefore, the neutral red phagocytosis assay was applied to evaluate the effects of verbascose on phagocytic activity of RAW264.7 cells, and the results are presented in Figure 3. The phagocytosis indices of
major oligosaccharides present in mung bean. Therefore, mung bean is one of the most suitable materials for verbascose preparation, which is consistent with the previous study.20 To prepare verbascose, the soluble sugars extracted from mung bean were separated by charcoal/Celite column chromatography combined with Biogel P-2 gel filtration chromatography. Charcoal/Celite column chromatography can isolate different carbohydrates mainly through their hydrophobic properties. However, stachyose and verbascose showed similar hydrophobicities, and both were eluted out by 15% aqueous ethanol in the present study. The mixture of stachyose and verbascose was further separated by Biogel P-2 gel filtration chromatography, due to the distinct difference in their molecular sizes. The data of MS and NMR (Figure 2) for the resulting fractions of verbascose clearly demonstrated that it was verbascose,36 and the purity of verbascose obtained was 98.5% (Figure 1B). Endotoxin Contents. Endotoxin, as a contaminant in biological preparations, has a capacity of immune stimulation. In the present work, we measured the endotoxin content in verbascose sample by TAL chromogenic method for the sake of endotoxin influence. As a result, the endotoxin contamination of verbascose sample was quite low (0.0056%), suggesting that the interference from endotoxin to the immunity could be ignored. Effects of Verbascose on RAW264.7 Cell Viability. The viability of RAW264.7 cells is an indicator for further experiments of immune activation. The assay is dependent on the ability of viable cells to metabolize a water-soluble tetrazolium salt into a water-insoluble formazan product. Therefore, we investigated the effects of verbascose on the viability of RAW264.7 cells. As shown in Table 1, the viabilities of treatments by verbascose (100.0, 200.0, and 400.0 μg/mL) were significantly increased (p < 0.05) as compared with that of blank control after 12 h of incubation. However, after incubation for 24, 36, and 48 h, all of the doses of verbascose showed significant differences (p < 0.05) compared with the blank control, indicating that they all had positive influences on the viability of RAW264.7 cells. The cell viability increased with the increase of sample concentration and reached the maximum value at a concentration of 200.0 μg/mL. Furthermore, both treatments at concentrations of 200.0 and 400.0 μg/mL showed striking differences compared with the other treatments, but there was no significant difference between the two concentrations. The results suggested that the influence of verbascose on RAW264.7 cell viability was in a dose-dependent manner at lower concentrations. The treatments at 100.0, 200.0, and 400.0 μg/mL of verbascose with incubation time of 24 h exhibited the best effects on cell viability. Therefore, 24 h was selected for the cell culture time in the following experiments.
Figure 3. Effects of verbascose on phagocytosis of RAW264.7 cells. Letters a−f represent a significant difference (p < 0.05) between different concentrations of samples.
different doses of verbascose on devouring neutral red of peritoneal macrophages all exceeded 1.0, and all of the stimulating indices at different concentrations were comparable to that stimulated by LPS (positive control). The results demonstrated that verbascose had notable activity of promoting peritoneal macrophages to devour neutral red at a low dose. Furthermore, the promoting effects increased with the increases of sample concentration ranging from 25 to 200 μg/mL. Nevertheless, the phagocytosis index for treatment at 400 μg/ mL was significantly lower (p < 0.05) than that of treatment at 200 μg/mL, which confirmed that verbascose exhibited its maximum activity of cell phagocytosis at a concentration of 200 μg/mL. Effects of Verbascose on NO and Cytokine Production. Once activated, macrophages release a large number of cell factors, such as NO, INF, and PGE2, to exert joint efficacy against microbial infections and cancer.44 NO is a modulator of neurotransmission that can act as a vasorelaxant and defend against pathogens. It has been reported that the antitumor and antiviral activities, inflammatory reaction, and immune response 10731
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have a strong relationship with the release of NO.45 Thus, we considered NO as an index to evaluate the immunomodulatory effects of verbascose. As shown in Figure 4, a minimum amount
every cell type and often in concert with other cytokines or small mediator molecules. IFN-α, produced by leukocytes, is mainly involved in innate immune response against viral infection.34 Accordingly, the effects of verbascose on cytokine production (IL-1β, IL-6, IFN-α, and IFN-γ) were investigated in the present study. Compared with those of the normal control group (Table 2), the concentrations of cytokines (IL1β, IL-6, IFN-α, and IFN-γ) secreted by RAW264.7 cells in all treatments of verbascose were significantly higher (p < 0.05). In the range of 25−200 μg/mL verbascose, the production of cytokines was enhanced in a dose-dependent manner, whereas there was no significant difference in the secretion of IL-6, IFNα, and IFN-γ between the treatments of verbascose at concentrations of 400 and 200 μg/mL. The results indicated that verbascose could stimulate immune responses through the release of immune cell factors (IL-1β, IL-6, IFN-α, and IFN-γ) as well as inflammatory mediators (NO). Effects of Verbascose on the Immune Organ Index. Thymus decline is an important marker of immune system aging because the immune system degeneration is expressed as an aging process from center to periphery piloted by thymus involution. Thus, the immunomodulatory effect is closely related to the change of immune organ index.48 CPA, a DNA alkylating agent with striking immunomodulatory properties being used as a chemotherapeutic and immunosuppression drug, may cause the decline of lymphocyte percent, peripheral blood leucocyte, macrophage phagocytosis, spleen index, thymus index, DTH earlap swelling, carbon clearance, and serum hemolysin. Therefore, it is often used to establish the immunosuppressed model.49 In this study, the effects of verbascose on the thymus and spleen indices were investigated. Compared with the normal control group (Figure 5), significant decreases in indices of spleen and thymus were observed after treatment by CPA in model control group (p < 0.05). However, treatment of verbascose could improve the spleen index as compared with the model control group. Furthermore, the medium-dose treatment (90 mg/kg BW) exhibited the best effect (significant difference with the other treatments at p < 0.05), even though no significant difference was observed for medium-dose treatment and the normal group. For the thymus index, there were no significant difference between the treatment and model control group (p > 0.05), but the medium-dose treatment showed some improvements on the thymus index. The results suggested that verbascose was able to act against immunosuppression induced by CPA in vivo to some extent. Effects of Verbascose on DTH. DTH is a protective localized cell-mediated immune response mediated by CD4+ and CD8+ T-cells, primarily against intracellular pathogens. In
Figure 4. Effects of verbascose on nitric oxide level of RAW264.7 cells. Letters a−f represent a significant difference (p < 0.05) between different concentrations of samples.
of NO was produced in the blank control group, which might be due to the low expression of inducible NO synthase (iNOS) in static status RAW264.7 cells, whereas treatments with verbascose induced significant increases of NO production (p < 0.05), indicating that verbascose had the ability to activate macrophages to release NO. At concentrations of verbascose from 25.0 to 200.0 μg/mL, the levels of NO production showed a dose-dependent effect. In addition, between every two concentrations there was a significant difference (p < 0.05). It might be related to the expression of iNOS induced by verbascose. Cytokines are small molecule proteins produced by immune cells with immunomodulatory and anti-inflammatory abilities. It is well-known that mature T cells, following antigen recognition, are induced to differentiate into three distinct functional subsets of Th1, Th2, and Th0 cells, which can be discriminated on the basis of the array of cytokines produced. Th1 cells secrete IL-2, IFN-γ, and TNF-β, and Th2 cells secrete IL-4, IL-5, and IL-6, whereas Th0 cells, having the capacity to produce both Th1 and Th2 cytokines, are believed to represent a stage of differentiation before commitment to the Th1 or Th2 lineage. Thus, the section of IFN-γ and IL-6 may represent the function of mature T cells.46,47 IL-1 (IL-1α and IL-1β) is the prototypic “multifunctional” cytokine, which affects nearly
Table 2. Effects of Verbascose on Cytokine (IL-6, IL-1β, IFN-α, and IFN-γ) Levels of RAW264.7 Cellsa treatment control LPS verbascose 25 μg/mL 50 μg/mL 100 μg/mL 200 μg/mL 400 μg/mL a
concn of IL-6 (pg/mL)
concn of IL-1β (pg/mL)
concn of INF-α (pg/mL)
concn of INF-γ (pg/mL)
113.33 ± 2.32a 231.26 ± 12.31d
53.51 ± 1.83a 137.02 ± 3.69cd
63.77 ± 4.54a 118.98 ± 7.73b
142.22 ± 11.98a 350.24 ± 13.20d
158.17 158.33 162.67 198.33 195.00
± ± ± ± ±
8.16b 2.85b 6.69b 4.00c 1.21c
108.15 118.19 142.55 143.76 150.31
± ± ± ± ±
0.26b 1.14bc 1.83d 9.41d 7.33d
118.30 128.53 136.12 134.82 132.31
± ± ± ± ±
3.38b 8.31bc 10.88c 3.46c 6.57c
237.78 251.22 255.56 280.67 274.00
± ± ± ± ±
0.45b 5.35bc 9.43bc 8.17c 6.25bc
Different lower case letters represent a significant difference (p < 0.05) between different concentrations of samples. 10732
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Figure 6. Effects of verbascose on the swelling rate of earlap in delayed type of hypersensitivity reaction. Letters a−f represent a significant difference (p < 0.05) between different concentrations of samples. Normal, normal control group, given a hypodermic injection of 0.9% NaCl once daily for 8 days; CPA-Model, model control group, given 0.9% NaCl once daily as well as a hypodermic injection of cyclophosphamide (CPA, 100 mg/kg body weight (BW)) on days 2, 3, and 4; Positive, positive control group, which replaced NaCl with levamisole hydrochloride on the basis of model control group; LVG30, low dose of verbascose (30 mg/kg BW); MVG-90, medium dose of verbascose (90 mg/kg BW); HVG-30, high dose of verbascose (270 mg/kg BW).
Figure 5. Effects of verbascose on the spleen and thymus indices. Letters a−c represent a significant difference (p < 0.05) between different concentrations of samples. Normal, normal control group, given a hypodermic injection of 0.9% NaCl once daily for 8 days; CPA-Model, model control group, given 0.9% NaCl once daily as well as a hypodermic injection of cyclophosphamide (CPA, 100 mg/kg body weight (BW)) on days 2, 3, and 4; Positive, positive control group, which replaced NaCl with levamisole hydrochloride on the basis of model control group; LVG-30, low dose of verbascose (30 mg/kg BW); MVG-90, medium dose of verbascose (90 mg/kg BW); HVG-30, high dose of verbascose (270 mg/kg BW).
the case of allergenic effect, T-lymphocyte may become sensitized lymphocyte and will generate a regional abnormal reactive inflammation when it encounters the allergen again. This allergic inflammation is delayed (starting after 12 h and peaking at 24−72 h) and characterized by cell degeneration and necrosis.50 It has been reported that DNFB (a hapten) may become a complete antigen when binding with skin protein. It will then stimulate T-lymphocyte to form sensitized lymphocyte. After it is painted on the earlap of mice, regional earlap swelling would be observed 4−7 days later and the swelling rate of earlap is associated with the degree of DTH reaction. As shown in Figure 6, a significant decrease of earlap swelling rate was observed between the model control group compared and the normal control group (p < 0.05), which indicated the feasibilty of the present immunosuppressed model. When mice received positive drug or verbascose, they showed a significant DTH response in vivo (p < 0.05) compared with the normal control group. Compared with the positive control group, all of the treatments of different verbascose doses exhibited significant DTH responses. However, a notably significant immunological effect was observed for the treatment with the middle dose (90 mg/kg) of verbascose compared to the other two dose treatments (p < 0.05). The results suggested that verbascose might strengthen immune function by increasing DTH earlap swelling. Effects of Verbascose on Serum Hemolysin (HC50). Serum hemolysin antibody, secreted by B-lymphocytes when stimulated by a variety of antigens, is a reliable indicator reflected by the level of humoral immunity.51 Therefore, we evaluated the levels of serum hemolysin antibody in response to SRBC in mice. As shown in Figure 7, the daily treatments of mice with 30 or 90 mg/kg doses of verbascose resulted in a significant enhancement of serum hemolysin level as compared with the model control group (p < 0.05). Particularly, there was
Figure 7. Effects of verbascose on hemolysin in serum (HC50). Letters a−c represent a significant difference (p < 0.05) between different concentrations of samples. Normal, normal control group, given a hypodermic injection of 0.9% NaCl once daily for 8 days; CPA-Model, model control group, given 0.9% NaCl once daily as well as a hypodermic injection of cyclophosphamide (CPA, 100 mg/kg body weight (BW)) on days 2, 3, and 4; Positive, positive control group, which replaced NaCl with levamisole hydrochloride on the basis of model control group; LVG-30, low dose of verbascose (30 mg/kg BW); MVG-90, medium dose of verbascose (90 mg/kg BW); HVG30, high dose of verbascose (270 mg/kg BW).
no significant difference between the treatments of the middle dose of verbascose and positive control, and both treatments exhibited the best effects. However, the high-dose treatment (270 mg/kg BW) showed a sharp decrease in hemolysin level, indicating that the high-dose verbascose treatment might suppress the production of serum hemolysin to SRBC. From the above results, it was concluded that low-dose verbascose 10733
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Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
treatment possessed the potential of immune-enhancing activity. Effects of Verbascose on Lysozyme in Serum and Spleen. Lysozyme, also called muramidase or peptidoglycan N-acetylmuramoyl-hydrolase, existing extensively in animals, plants, and microorganisms, is considered to be a potent innate immunity molecule.52 In vivo, lysozyme catalyzes the hydrolysis of the β-(1→4) glycosidic linkage between N-acetylmuramic acid and N-acetylglucosamine, alternates the sugar residues in the bacterial peptidoglycan, and causes bacterial cell lysis.53 As a consequence, lysozyme occupies an important position in normal and nonspecific immunity. In the present study, the effects of verbascose on lysozyme activity in serum and spleen were evaluated, and the results are presented in Table 3.
Notes
The authors declare no competing financial interest.
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Table 3. Effects of Verbascose on Activity of Lysozyme in Mouse Spleen and Seruma sample normal CPA-model levamisole low dose of verbascose (30 mg/kg BW) medium dose of verbascose (90 mg/kg BW) high dose of verbascose (270 mg/kg BW)
lysozyme in serum (U/mL) 347.2 182.66 486.15 272.69
± ± ± ±
19.14bcd 25.91a 39.82d 43.12abc
lysozyme in spleen (U/mg) 142.96 77.23 273.31 90.68
± ± ± ±
29.80d 7.93a 15.08f 3.26b
427.63 ± 26.76cd
146.91 ± 12.56e
236.89 ± 20.35ab
105.14 ± 15.19c
a
Different lower case letters represent a significant difference (p < 0.05) between different concentrations of samples.
Compared with the normal control group, the activities of lysozyme in the three doses of verbascose and positive drug were significantly enhanced (p < 0.05). In addition, it was found that treatment with a medium dose of verbascose (90 mg/kg BW) could significantly improve the activity of lysozyme compared with the other two treatment doses (p < 0.05) and restored the activity of lysozyme to normal level. The results suggested that verbascose could reinforce the immune function by raising the activity of lysozyme. In conclusion, we demonstrated that verbascose could promote the proliferation in vitro of RAW264.7 cells, strengthen peritoneal macrophages to devour neutral red, and enhance the production of NO and cytokines (IL-1β, IL-6, IFN-α, and IFN-γ). In in vitro experiments, treatment with 200 μg/mL verbascose exhibited the most significant immunomodulatory activity. As for in vivo experiments, the results demonstrated that verbascose could raise the spleen index, elevate the activity of lysozyme in serum, and increase the level of serum hemolysin and the swelling rate of earlap in DTH reaction. The results suggested that verbascose had potent immunostimulatory activity, and further works on its potential mechanisms are in progress.
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AUTHOR INFORMATION
Corresponding Author
*(X.Z.) Phone/fax: +86 25 84396791. E-mail: zengxx@njau. edu.cn. Funding
This work was partly supported by Grants-in-Aid for Scientific Fesearch from the National Natural Science Foundation of China (31171750) and a project funded by the Priority 10734
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