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Preparation of a glucan from the roots of Rubus crataegifolius Bge. and its immunological activity
Carbohydrate Research 344 (2009) 2512–2518
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Preparation of a glucan from the roots of Rubus crataegifolius Bge. and its immunological activity Weihua Ni a, , Xu Zhang a, , Hongtao Bi a, Jeff Iteku a, Li Ji a, Chengxin Sun a, Jinbo Fang a, Guihua Tai a, Yifa Zhou a,*, Jimin Zhao b,* a b
School of Life Sciences, Northeast Normal University, Changchun 130024, PR China School of Life Sciences, Changchun Normal University, Changchun 130022, PR China
a r t i c l e
i n f o
Article history: Received 1 August 2009 Received in revised form 27 August 2009 Accepted 30 August 2009 Available online 3 September 2009 Keywords: Rubus crataegifolius Bge. Polysaccharide Glucan Immunological activity
a b s t r a c t A water-soluble glucan (RCP-1) was prepared from the roots of Rubus crataegifolius Bge. by extraction with hot-water, deproteination by Sevag reagent, a-amylase treatment and ultrafiltration. RCP-1 consisted of only glucose, and its molecular weight was determined to be 7 KD by high performance gel permeation chromatography (HPGPC). Fourier transform infra-red spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy (NMR), methylation and periodate oxidation analyses indicated that RCP1 was an a-D-glucan. Its main chains were composed of (1?4)- and (1?6)-linked a-glucopyranosyls, and side chains were single a-glucopyranosyl residues attached to the O-6 of glucosyls in the main chains. RCP-1 could increase both cytotoxic activity against B16 melanoma cells and the production of nitric oxide (NO) of macrophages in vitro. Furthermore, in vivo bioassay tests indicated that RCP-1 could remarkably enhance T and B lymphocyte proliferations, augment the phagocytosis of macrophages and increase the tumour necrosis factor-alpha (TNF-a) levels in serum. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Many polysaccharides isolated from medicinal plants exhibit immunomodulatory function and antitumour activities.1–3 These polysaccharides are often identified as biological response modifiers (BRMs), and their enhancement of the host defence has been recognised as a possible mean of inhibiting tumour growth without harming the host.4,5 The polysaccharides’ immunomodulatory effects and anticancer activity are influenced by the glycosidic linkage form, chain length, branch-point number, molecular size and tertiary structure.6 Previous studies of polysaccharide structure–activity relationships revealed that the 1,3-b-glucans have increased the potential to contribute to immunological and antitumour activities because of their interaction with the immunological cell surface receptors.7–10 Recently, some branched or linear a-1,4-/1,6-glucans isolated from medicinal plants Tinospora cordifolia,6,11 Cistanche deserticola,12 Strongylocentrotus nudus,13,14 Hedysarum polybotrys,15 Angelica sinensis16 and Aconitum carmichaeli17 have also been found to exhibit strong immunological and antitumour activities. Rubus crataegifolius Bge. is a species of raspberry that grows widely in north-eastern China, Japan, Korea and the Ussuri region of the Russian Far East.18 For many years, R. crataegifolius has been
* Corresponding authors. Tel./fax: +86 431 85098212 E-mail addresses: [email protected] (Y. Zhou), [email protected] (J. Zhao). These authors contributed equally to this paper. 0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2009.08.042
used in the treatment of various diseases such as rheumatic arthritis, hepatitis and lung cancer in China.19 Some small molecules in R. crataegifolius have been identified to be active components.20–24 Until now, there has been no report about polysaccharides extracted from R. crataegifolius. In this study, we describe the preparation of a low molecular weight a-glucan from R. crataegifolius and investigate its immunomodulatory activities. 2. Results and discussion 2.1. Isolation and purification A polysaccharide (RCP, yield 11.3% of dried material) was obtained from the roots of R. crataegifolius via hot-water extraction, ethanol precipitation and Sevag reagent deproteination. RCP exclusively contained glucose and exhibited a wide molecular weight distribution (from 20 KD to 210 KD) on Sepharose CL-6B chromatography. In an iodine test, RCP tested positive, indicating that it possesses starch structural features indicative of a starch-like glucan. Its molecular distribution was so wide that its structure and activity were difficult to study. Therefore, we treated RCP with a-amylase to break down some a-(1?4)-linked glucosidic bonds in an effort to obtain low molecular weight glucan. The enzymolysis products were fractionated by ultrafiltration (30–5 KD Mw cut-off membrane), yielding a main fraction RCP-1 (32% yield) that tested negative in an iodine test. RCP-1 was a water-soluble white powder. Its
2513
b 100
0.04
80
0.00
3417.3
1024.0
70 0.02
0
5
10
15 20 25 30 Retention time (min)
35
40
60 4000
840.8 1080.01151.3
0.06
90
1648.9
0.08
2931.3
% Transmittance
RI response
0.10
1454.1
0.12
2360.5
a
931.5
W. Ni et al. / Carbohydrate Research 344 (2009) 2512–2518
3500
3000
2500 2000
1500
1000
500
-1
Wavenumbers (cm )
Figure 1. (a) The HPGPC profile and (b) the FT-IR spectrum of RCP-1.
carbohydrate and protein contents were determined to be 97.3% and 1.2%. RCP-1 was homogeneous with Mw of 7 KD as demonstrated by HPGPC (Fig. 1a) and exclusively contained glucose. 2.2. Structural analysis The specific rotation of RCP-1 was ½a20 D +278.8 (c 1.0, H2O). The high positive rotation suggested the dominating presence of a-glycosidic linkages.15,25–27 The FT-IR spectrum of RCP-1 ( Fig. 1b) showed a-type glycosidic linkages at 932 and 841 cm1. The other absorption bands at 3417 cm1 (hydroxyl stretching vibration); 2931 cm1 (C–H stretching vibration); 1648 cm1 (bound water); 1151, 1079 and 1024 cm1 (pyranose ring) were from the corresponding sugar residues. The methylation of RCP-1 mainly produced four partially methylated alditol acetates, 1,5-di-acetyl-2,3,4,6-tetra-O-methyl glucitol (13.6%), 1,4,5-tri-acetyl-2,3,6-tri-O-methyl glucitol (62.8%), 1,5,6-tri-acetyl-2,3,4-tri-O-methyl glucitol (6.8%) and 1,4,5,6-tetra-acetyl-2,3-di-O-methyl glucitol (11.4%). Based on these results, RCP-1 correspondingly contained four glucosidic linkage forms, that is, (1?4)-, (1?6)-, (1?4, 6)-linked glucosyls and non-reducing terminals (Table 1). Periodate oxidation indicated that each mole of the residual glucosyl of RCP-1 consumed, on average, 1.32 mol of periodate and produced 0.29 mol of formic acid. GC analysis indicated that the hydrolysed products contained erythritol (70.2%) and glycerol (29.8%) but did not contain monosaccharides. These results supported the conjecture that RCP-1 is an a-(1?4), (1?6)-linked glucan. The NMR analyses, including 13C, 1H and 13C–1H HSQC (Fig. 2), were carried out to further characterise the structure of RCP-1
Table 1 Results of the methylation analysis of RCP-1 Partially methylated glycitol
Molar ratio
MS (m/z)
Linkage type
1,5-Di-acetyl-2,3,4,6-tetraO-methyl glucitol 1,4,5-Tri-acetyl-2,3,6-triO-methyl glucitol 1,5,6-Tri-acetyl-2,3,4-triO-methyl glucitol 1,4,5,6-Tetra-acetyl-2,3di-O-methyl glucitol
2
43, 45, 71, 87, 101, 117, 129, 145, 161, 205 43, 45, 87, 101, 117, 131, 161, 233 43, 45, 71, 87, 101, 117, 129, 161, 189, 233 43, 85, 101, 117, 127, 261
Glcp-(1?
9 1 2
?4)Glcp-(1? ?6)Glcp-(1? ?4,6)Glcp-(1?
(Table 2). According to the NMR data in the literature,15,26–30 four anomeric signals for typical a-glycosidic configurations appeared at d 99.38, 99.22, 99.01 and 98.05 ppm in the 13C NMR spectrum and at d 5.39, 5.33 and 4.96 ppm in the 1H NMR spectrum. Both of these results are consistent with the value of specific rotation and the absorptions in the FT-IR spectrum. The signals of O-substituted C-6 appeared at d 68.76;66.54/3.91 ppm, and those of unsubstituted C-6 appeared at d 59.92/3.86 and 60.12/3.70 ppm. The signals at d 77.23/3.57 and 77.12/3.67 ppm were assigned to O-substituted C-4, and those of unsubstituted C-4 appeared at d 70.71;70.42/3.42 ppm. The ratios of the signals from NMR were consistent with those of the sugar residues characterised by the chemical analysis described above and indicated that linear a-(1?4)-linked glucosyls were more than a-(1?6)-linked glucosyls. Summarising the comprehensive results, it was deduced that RCP-1 was composed of 43 a-glucopyranosyl residues. The main chains consisted of (1?4)- and (1?6)-linked a-glucopyranosyl residues, and the side chains contained one glucosyl attached to the O-6 of glucosyls on the main chains. The deduced structure of RCP-1 is shown in Figure 3, based on one hypothesis explaining the negative iodine test result of RCP-1 that (1?4)-linked a-glucopyranosyl residues were spaced by the (1?6)-linked side chain residues. Alternatively, it is possible that the (1?6)-linkages were in the main chain and could not form a helix structure. 2.3. Immunological activity The effects of RCP-1 on ConA- and LPS-induced lymphocyte proliferations were tested in vivo by the MTT method. Lymphocytes induced by ConA may be used as a method to evaluate T lymphocyte activity, while those lymphocytes induced by LPS may be used to evaluate B lymphocyte activity.12,17 As shown in Figure 4, in the presence of ConA or LPS, RCP-1 significantly increased lymphocyte proliferation in all the three testing doses (P <0.05). The increase in T cell proliferation was higher in the presence of 10 mg/kg of RCP-1 than 25 and 50 mg/kg. However, the increase in B cell proliferation peaked at 25 mg/kg. These results indicated that RCP-1 was able to activate both T and B cells. Like some other bioactive a-(1?4), (1?6)-glucans isolated from medicinal plants,6,14 RCP-1 might be a potent T/B-cell stimulator. Lymphocytes are considered to be one of the important components of the host defence against tumour growth and invading pathogens. These cells are able to produce
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60
59.92/3.70 60.12/3.86
65
68.76;66.54/3.91
70
70.64/3.84
98.05/4.96
100
99.01/5.33
95
90
85
80
77.12/3.67 77.23/3.57
ppm
75
70.71;70.42/3.42
105
99.38;99.22/5.39
5.8
5.6
5.4
5.2
Figure 2. HSQC spectrum of RCP-1: the measured
5.0
4.8
4.6
4.4
ppm
4.2
4.0
3.8
3.6 3.4
3.2
3.0
13
C spectrum is shown on the vertical axis and the 1H spectrum is shown on the top.
Table 2 1 H and 13C NMR chemical shift of RCP-1 in D2O Sugar residue
?4)-a-Glcp-(1?
a-Glcp-(1? ?4,6)-a-Glcp-(1? ?6)-a-Glcp-(1?
d
13
C/1H (ppm)
1
2
3
4
5
6
99.22 5.39 99.38 5.39 99.01 5.33 98.05 4.96
71.00 3.64 72.19 3.70 71.11 3.64 71.32 3.68
72.78 3.95 72.34 3.97 72.41 3.95 72.32 3.97
77.12 3.67 70.71 3.42 77.23 3.57 70.42 3.42
70.64 3.84 72.43 4.02 68.88 3.80 69.81 3.81
59.92 3.86 60.12 3.70 68.76 3.91 66.54 3.91
Figure 4. The effect of different doses of RCP-1 on ConA- or LPS-induced lymphocyte proliferation activity in mice. Each value represents the mean ± S.D. based on eight mice per group. Significant differences from the negative controls were evaluated using student’s t-test: *P <0.05, **P <0.01. Figure 3. The structure of RCP-1.
many kinds of cytokines after differentiation and activation. Stimulating proliferation of lymphocytes results in an increase in cytokine release, potentially accounting for the antitumour activity of the polysaccharides isolated from medicinal plants.31 The effects of RCP-1 on immunised murine macrophage phagocytosis were determined by using chicken red blood cells (CRBC).
As shown in Table 3, RCP-1 could significantly enhance phagocytosis at doses of 25 and 50 mg/kg, compared to the control. Both the rates of phagocytosis and the phagocytic indices were increased by RCP-1 treatment in a dose-dependent manner. TNF-a is an important mediator in the destruction of tumour cells. It has tumour-selective cytotoxicity by combining with the receptor on the tumour cell surface, and kills tumour cells specifically but does not injure normal cells.32 RCP-1 increased TNF-a
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Table 3 Effects of RCP-1 polysaccharide on macrophage-mediated phagocytosis in mice Animal groups Control RCP-1
Dose (mg/kg)
Phagocytosis rate (%)
Phagocytic index
10 25 50
29.34 ± 2.32 31.94 ± 1.97 41.18 ± 3.43* 51.88 ± 4.66**
0.43 ± 0.11 0.49 ± 0.19 0.73 ± 0.23* 0.77 ± 0.22**
The phagocytic rate was measured by counting the number of macrophages containing CRBCs found in the total macrophage cell population; the phagocytosis index was measured by counting the number of phagocytosed CRBCs present in the total macrophage cell population. Each value represents the mean ± S.D. based on eight mice per group. Significant differences from the negative controls were evaluated using student’s t-test: *P <0.05, **P <0.01.
Figure 6. Effect of RCP-1 on macrophage NO (as nitrite) production. RCP-1 (5– 500 lg/mL) and LPS (10 lg/mL) were added to peritoneal macrophages (0.5 106 cells/well) for 24 h, respectively. The isolated supernatants were mixed with an equal volume of Griess reagent, and nitrite production was measured by an ELISA reader at 540 nm. The reported values are the mean ± SD, (n = 6). *P <0.05, **P <0.01 versus the medium-treated group.
Figure 5. TNF-a levels in serum of RCP-1-immunised mice. Each value represents the mean ± S.D. based on eight mice per group. Significant differences from the negative controls were evaluated using student’s t-test: *P <0.05, **P <0.01.
levels of serum in RCP-1-immunised mice. As shown in Figure 5, the levels of TNF-a in all RCP-1 treatment groups were much higher than in the control group (P <0.01). The TNF-a levels (77.3 pg/ mL) peaked at a dose of 25 mg/kg. Nitric oxide (NO) has been identified as another major effector molecule involved in the destruction of tumour cells by activated macrophages. The production of NO in vitro is assessed by the Griess reaction. As shown in Figure 6, RCP-1 significantly increased NO production at concentrations of 50–500 lg/mL in a dosedependent manner. However, NO production was weaker after stimulation with RCP-1 than LPS, a potent activator of murine macrophages. To examine whether RCP-1 enhances the ability of macrophages to kill B16 melanoma cells, which are sensitive to either TNF-a or NO, the macrophages were treated with various concentrations of RCP-1 for 24 h and then co-cultured with B16 melanoma cells for 16 h. The cell viability was then evaluated by the MTT method. As shown in Figure 7, RCP-1 markedly increased the cytotoxicity of macrophages in a concentration-dependent manner. These data indicated that RCP-1 could stimulate the tumouricidal activities of macrophages. The antitumour function of macrophages may comprise two mechanisms. One mechanism is cell-to-cell contact between the macrophage and the target cell; and the other is secretion of factors such as cytokines and nitrogen intermediates.33 RCP-1 induced TNF-a and NO productions in B16 melanoma cells, which are either TNF-a or NO sensitive. Therefore, it was deduced that the enhancement of macrophage cytotoxicity by RCP-1 may be at least partially due to the secretion of cytokines and nitrogen intermediates.
The macrophages are considered the pivotal immunocytes of the host defence. Many polysaccharides from plants have been shown to activate macrophages by binding to the receptors on the surface of immune cells. It has been known that macrophages can interact with botanical polysaccharides and/or glycoproteins via Toll-like receptor 4 (TLR4), complement receptor 3, the scavenger receptor, dectin-1 and the mannose receptor.5 Activation of these receptors leads to intracellular signalling cascades, resulting in transcriptional activation and production of pro-inflammatory cytokines. In our experiment, RCP-1 augments cytotoxic activity against tumour cells and increases phagocytosis and cytokine (TNF-a) and NO production in macrophages. These data are consistent with the findings that (1,4)-a-glucans isolated from T. cordifolia augmented the phagocytic ability of macrophages and induced TNF-a production in a concentration-dependent manner in vitro,11 and the (1,4)-a-glucans isolated from S. nudus-induced NO production from macrophages in vitro.14 The differences in these data
Figure 7. Tumouricidal activities of RCP-1-treated murine peritoneal macrophages against B16 melanoma cells. The macrophages were treated with various doses of RCP-1 for 24 h. Antitumour activity was determined as described in materials and methods at an initial effector: target ratio of 10:1. The reported values are the mean ± SD, (n = 6). *P <0.05, **P <0.01 versus the medium-treated group.
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may result from the glucan structures, specifically molecular weight, chain length and tertiary structure.6 In conclusion, a low molecular weight glucan RCP-1 prepared by a-amylase treatment of the polysaccharides isolated from the roots of R. crataegifolius was characterised to be a branched a-D-glucan with a backbone of (1?4)- and (1?6)-linked a-D-glucopyranosyls. The side chains consisted of one a-D-glucopyranosyl residue attached to O-6 of (1?4)-linked a-D-glucopyranosyls. RCP-1 could significantly enhance lymphocyte proliferation and augment the phagocytic ability of macrophages. RCP-1 increased macrophage NO production and TNF-a levels in serum, resulting in the inhibitory effects of murine peritoneal macrophages against B16 melanoma cells in vitro. Therefore, RCP-1 may be useful in tumour therapy.
3. Experimental 3.1. General methods The total carbohydrate content was determined by the phenol– sulfuric acid method, using glucose as the standard.34 Uronic acid content was determined by the m-hydroxydiphenyl method, using galacturonic acid as the standard.35 Protein content was determined by the Bradford assay, using bovine serum albumin as the standard.36 The presence of starch was analysed with the iodine test.37 Dialysis was carried out using a tube with Mw cut-off of 3500 Da (for globular protein). Gas chromatography was performed using a Shimadzu GC-14C instrument equipped with a hydrogen flame ionisation detector on an Rtx-2330 column (0.32 mm 15 m i.d., 0.2 lm) at a temperature programme of 175 °C (hold 2 min) followed by 8 °C/min to 240 °C (hold 1 min) and 8 °C/min to 265 °C (hold 17 min). The hydrogen flow rate was 10 mL/min, and the ion-source temperature was 275 °C. High performance liquid chromatography (HPLC) was performed using a Shimadzu 10Avp HPLC system equipped with 10Avp HPLC Pump, SPD-10Avp UV–vis Detector and RID-10A Refractive Index Detector. The specific rotation was determined at 20 ± 1 °C with an automatic polarimeter (Model WZZ-2B, China). UV–vis absorbance spectra were recorded with a UV–vis spectrophotometer (Model SP-752, China). Sephadex G-75, standard monosaccharides and gel filtration standard dextrans were purchased from Sigma Co. All other chemicals were of analytical grade made in China. 3.2. Preparation of glucan RCP-1 The roots of R. crataegifolius were collected from Tonghua, Jilin province, China and identified by Professor Hongxing Xiao, School of Life Sciences, Northeast Normal University in Changchun, China. The roots of R. crataegifolius were extracted with distilled water (100 °C, 1:10 w/v) three times (4 h each). The aqueous filtrates were combined and concentrated to a small volume (one-tenth of the original volume). To precipitate the polysaccharides, 95% ethanol was added to the aqueous filtrates, totalling 80% of the final volume. The polysaccharides were collected by centrifugation and dried in vacuum. The collected polysaccharides were dissolved in water (15% w/v) to remove the insoluble substances originating from centrifugation. The supernatant was treated with Sevag reagent (1:4 n-butanol/chloroform, v/v) to remove the proteins.38 After removing the proteins and Sevag reagent by centrifugation, the water phase was dialysed against distilled water and lyophilised to yield polysaccharide (RCP). RCP was dissolved in PBS (0.1 M, pH 6.9) and treated with a-amylase from Bacillus subtilis (E.C. 3.2.1.1, Sigma, St. Louis, USA) at 37 °C. The remaining starch was assessed using the iodine test every 15 min. When the poly-
saccharide was tested negative by the iodine test, the solution was boiled to inactivate the a-amylase, centrifuged to remove the insoluble substances and subsequently purified by ultrafiltration through 30 KD and 5 KD Mw cut-off membranes to yield the purified, degraded polysaccharide RCP-1. Lipopolysaccharide (LPS) contamination was tested by Limulus amoebocytes lysate (LAL) assay using an E-TOXATE kit (Sigma, St. Louis, USA) according to the manufacturer’s instruction. The quantity of endotoxin in RCP-1 was less than 0.015 EU/mg (negative). 3.3. Homogeneity and molecular weight Determination of homogeneity and molecular weight was carried out by an HPLC-linked gel filtration column of TSK-G3000 PWXL, eluting with 0.2 M NaCl at 0.6 mL/min, 35.0 ± 0.1 °C. The gel filtration column was calibrated by standard dextrans (50 KD, 25 KD, 12 KD, 5 KD, 1 KD) using linear regression. The sample concentration was 5 mg/mL, and the injection amount was 20 lL. 3.4. Monosaccharide composition The monosaccharide analysis was performed as described by Honda39 and Yang.40 The polysaccharide RCP-1 (2 mg) was hydrolysed with anhydrous methanol (0.5 mL) containing 2 M HCl at 80 °C for 16 h and then with 2 M CF3COOH (0.5 mL) at 120 °C for 1 h.41 The hydrolysis product was derivatised with 0.5 M 1-phenyl-3-methyl-5-pyrazolone (PMP) and 0.3 M NaOH. After neutralisation with 0.3 M HCl, the derivatives were analysed by HPLC on a DIKMA Inertsil ODS-3 column (150 4.6 mm i.d.) with a guard column on a Shimadzu HPLC system (LC-10ATvp pump and UV–vis detector) and monitored by UV absorbance at 245 nm. 3.5. Periodate oxidation The periodate oxidation was performed according to the procedure described by Chaplin and Kennedy.42 RCP-1 (25 mg) was dissolved in 0.015 M NaIO4 (25 mL), and the solution was kept at 4 °C in the dark. The A223 nm of reaction solution was determined every 6 h by spectrophotometer. After the oxidation was completed (64 h), the excessive NaIO4 was decomposed with ethylene glycol (0.1 mL). The NaIO4 consumption was calculated according to the decrease of absorbance (A223 nm). The formic acid production was determined by titration with 0.1 M NaOH. The reaction mixture was dialysed against tap water and distilled water in turn, and then the retentate was reduced with NaBH4 overnight. After neutralisation and dialysis, the retentate was freeze-dried, hydrolysed with 2 M CF3COOH (1 mL) at 120 °C for 2 h, reduced by NaBH4, acetylated with pyridine (0.5 mL) and acetic anhydride (0.5 mL) at 90 °C for 1 h and then analysed for sugar composition by GC. 3.6. Methylation analysis The methylation analysis was carried out according to the method of Needs and Selvendran.43 In brief, RCP-1 (10 mg) was dissolved in DMSO (1.5 mL) and methylated by the treatment with NaOH/DMSO suspension (1 mL) and iodomethane (1.0 mL). The reaction mixture was extracted with CHCl3, and then the solvent was removed by vacuum evaporation. The completion of methylation was confirmed by the disappearance of the –OH band (3200– 3400 cm1) in the FT-IR spectrum. The per-O-methylated polysaccharide was hydrolysed subsequently by HCOOH (85%, 0.5 mL) for 4 h at 100 °C and then CF3COOH acid (2 M, 1 mL) for 6 h at 100 °C. The partially methylated sugars in the hydrolysate were reduced by NaBH4 and acetylated.44 The resulting alditol acetates were analysed by GC–MS.
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3.7. Fourier transformed infra-red (FT-IR) analysis FT-IR spectra were obtained on a Nicolet 560 FT-IR spectrometer with DTGS detector in a range of 400–4000 cm1. The sample was ground with KBr powder and then pressed into 1mm pellet for FT-IR measurement. 3.8. NMR analyses 1 H, 13C NMR and HSQC spectra were recorded on a Bruker Avance 600 MHz spectrometer (Germany), operating at 600 MHz for 1H NMR and 150 MHz for 13C NMR. The sample (20 mg) was dissolved in D2O (99.8%, 0.5 mL), freeze-dried, re-dissolved in D2O (0.5 mL) and centrifuged to remove the excess sample. All the data were analysed using standard Bruker software.
3.9. Immunological assays 3.9.1. Animals and treatment Male C57BL/6 mice (6–8 weeks old) were purchased from the Pharmacology Experimental Center of Jilin University (Changchun, China). The mice were housed on a 12/12-h light–dark cycle at room temperature and allowed free access to standard rodent food and water during the experiments. Animal handling procedures were conducted under National Institutes of Health animal care and use guidelines. All efforts were made to minimise animals’ suffering and to reduce the number of animals used. The mice were classified into four groups. RCP-1 was dissolved in physiological saline and administered intraperitoneally (ip) into mice at different dosages (10, 25, 50 mg/kg) daily for five days. The control group was given physiological saline instead of polysaccharide solution. The dose volume was 0.2 mL. 3.9.2. Macrophage phagocytosis assay CRBC (1%, 1 mL) was injected ip into RCP-1-immunised mice on day 6. After 30 min, the mice were sacrificed, and D-Hank’s solution (2.5 mL) was injected.45 The activated macrophages were obtained by peritoneal lavage onto a microscope slide. After centrifugation at 150g for 10 min, the supernatant was removed, and the free CRBCs were lysed by sterile 0.16 mM NH4Cl buffer. The macrophages were fixed with methanol and stained with Giemsa-Wright for 7–10 min. The microscope slides were washed with PBS and counted using a microscope (Olympus CX21, Japan, 100 objective lens). The rate of phagocytosis was calculated as follows:
Rate of phagocytosis ¼ ðNo: of macrophages containing CRBC= Total macrophages countedÞ 100: The phagocytosis index was measured as follows:
Phagocytosis index ¼ ðNo: of CRBC in macrophages= Total macrophages countedÞ 100: 3.9.3. Lymphocyte proliferation assay Spleens were aseptically extirpated from the immunised mice. Spleen single cell suspensions were pooled in ice-cold Hank’s solution by filtering through sieve mesh. The spleen cells were depleted of erythrocytes by Tris2NH4Cl (0.16 M, Tris2NH4Cl, pH 7.2) owing to low-osmosis, followed by washing twice in Hank’s solution and resuspending in complete RPMI 1640 medium. The viability of the splenocytes was >95%, as assessed by the trypan blue dye exclusion method described by Lee et al.46 The spleen cells (5 106 cells/mL) were seeded in a 96-well plate and cultured with mitogen ConA (5.0 lg/mL; Sigma, St. Louis, USA) or LPS (10.0 lg/mL; Sigma, St. Louis, USA). The cells were cul-
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tured at 37 °C, 5% CO2 for 44 h. MTT (5 mg/mL, 20 lL; Sigma, St. Louis, USA) was added to each well and incubated for an additional 4 h for the MTT cellular viability assay. The absorbance at 570 nm was measured using a Bio-Rad microplate reader (Model 550, USA). All determinations were conducted in triplicate. 3.9.4. Measurement of TNF-a activity The serum collected from the immunised mice was measured using an enzyme-linked immunosorbent assay kit (Pharmingen, San Diego, CA, USA) for tumour necrosis factor-alpha according to the manufacturer’s instructions. 3.9.5. Activation of peritoneal macrophages in vitro The cells from the peritoneal exudate were collected from normal C57BL/6 mice (male, 6–8 weeks) by peritoneal lavage with serum-free RPMI 1640 medium (5 mL; Sigma, St. Louis, USA). The collected cells were washed with D-Hank’s and cultured in complete RPMI 1640 medium (Sigma, St. Louis, USA) and supplemented with 25 mM HEPES, 10% heat-inactivated foetal calf serum (FCS), 1 105 IU/L penicillin G and 100 mM streptomycin. The cells were placed in a flat-bottomed culture plate and then incubated for 2 h at 37 °C and 5% CO2. After removal of the nonadherent cells, the mono-layer of macrophages was collected. 3.9.6. Assay for nitrite Peritoneal macrophages were prepared as described above and were plated at a density of 0.5 106 cells/well in 48-well plates and then treated with filter-sterilised samples or LPS (10.0 lg/mL; Sigma, St. Louis, USA) as a positive control. After 24 h of incubation, the culture supernatant was collected. The isolated supernatants were mixed with an equal volume of Griess reagent (1% sulfanilamide in 2.5% phosphoric acid, 0.1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid) and incubated at room temperature for 10 min. Using NaNO2 to generate a standard curve, the concentration of nitrite was measured by OD at 540 nm.47 3.9.7. Macrophage-mediated cytotoxicity The macrophage-mediated cytotoxicity was determined by the modification of the technique described previously.48 Peritoneal macrophages (1.0 105 cells/well) prepared as described above were first incubated in either medium alone or in medium supplemented with RCP-1 for 24 h in 96-well plates, washed with RPMI 1640 medium to remove the RCP-1 and then co-incubated with B16 melanoma cells (ATCC, Rochville, MD, USA) (1.0 104 cells/ wells; an initial effector: target cell ratio of 10:1) at 37 °C in a 5% CO2 incubator for 16 h. The cell density was then assessed by incubating the cells with MTT (25 lg/mL) for another 4 h.49 After aspirating the supernatant from the wells, DMSO (100 lL) was added for dissolution of formazan crystal. The absorbance at 570 nm was measured using a microplate reader. Cytolytic activity is expressed as the percentage of tumour cytotoxicity, where % Cytotoxicity = {1 O.D. of [(target + macrophages) macrophages]/O.D. of target (nontreated)} 100. Acknowledgements This work was supported by the National Natural Science Foundation of China(Nos. 30670478 and 30770489) and the Natural Science Foundation of Jilin Province (No. 20070710). References 1. Wasser, S. Appl. Microbiol. Biotechnol. 2003, 60, 258–274. 2. Tzianabos, A. O. Clin. Microbiol. Rev. 2000, 13, 523–533. 3. Moradali, M. F.; Mostafavi, H.; Ghods, S.; Hedjaroude, G. A. Int. Immunopharmacol. 2007, 7, 701–724. 4. Leung, M. Y. K.; Liu, C.; Koon, J. C. M.; Fung, K. P. Immunol. Lett. 2006, 105, 101– 114.
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28. Rodrigo, A. R.; Cesar, A. T.; Philip, A. J.; Iacomini, M. FEMS Microbiol. Lett. 2002, 210, 1–5. 29. Wu, Y.; Sun, C.; Pan, Y. Carbohydr. Polym. 2006, 63, 251–256. 30. Ruthes, A. C.; Komura, D. L.; Carbonero, E. R.; Cordeiro, L. M. C.; Reis, R. A.; Sassaki, G. L.; Gorin, P. A. J.; Iacomini, M. Plant Physiol. Biochem. 2008, 46, 500– 505. 31. Leung, M. Y. K.; Liu, C.; Zhu, L. F.; Hui, Y. Z.; Yu, B.; Fung, K. P. Glycobiology 2004, 14, 501–510. 32. Kim, G. Y.; Oh, Y. H.; Park, Y. M. Biochem. Biophys. Res. Commun. 2003, 309, 399– 407. 33. Liu, C.; Leung, M. Y. K.; Koon, J. C. M.; Zhu, L. F.; Hui, Y. Z.; Yu, B.; Fung, K. P. Int. Immunopharmacol. 2006, 6, 1634–1641. 34. Dubois, M.; Gilles, K. A.; Hamilton, J. K.; Rebers, P. A.; Smith, F. Anal. Chem. 1956, 28, 350–356. 35. Blumenkrantz, N.; Asboe-Hansen, G. Anal. Chem. 1973, 54, 484–489. 36. Sedmak, J. J.; Grossberg, S. E. Anal. Biochem. 1977, 79, 544–552. 37. Swanson, M. A. J. Biol. Chem. 1948, 172, 825–837. 38. Sevag, M. G.; Lackman, D. B.; Smolens, J. J. J. Biol. Chem. 1938, 124, 425–436. 39. Honda, S.; Akao, E.; Suzuki, S.; Okuda, M.; Kakehi, K.; Nakamura, J. Anal. Biochem. 1989, 180, 351–357. 40. Yang, X. B.; Zhao, Y.; Wang, Q. W.; Wang, H. F.; Mei, Q. B. Anal. Sci. 2005, 21, 1177–1180. 41. De Ruiter, G. A.; Schols, H. A.; Voragen, A. G. J.; Rombouts, F. M. Anal. Biochem. 1992, 207, 176–185. 42. Chaplin, F. M.; Kennedy, J. F. Carbohydrate Analysis: A Practical Approach, 2nd ed.; IRL Press: London, 1994. pp 81–95. 43. Needs, P. W.; Selvendran, R. R. Carbohydr. Res. 1993, 245, 1–10. 44. Sweet, D. P.; Shapiro, R. H.; Albersheim, P. Carbohydr. Res. 1975, 40, 217– 225. 45. Li, X.; Jiao, L.; Zhang, X.; Tian, W.; Chen, S.; Zhang, L. Int. Immunopharmacol. 2008, 8, 909–915. 46. Lee, S. J.; Saiki, I.; Hayakawa, Y.; Nunome, S.; Yamada, H.; Kim, S. H. Int. Immunopharmacol. 2003, 3, 147–157. 47. Zheng, R.; Jie, S.; Hanchuan, D.; Moucheng, W. Int. Immunopharmacol. 2005, 5, 811–820. 48. Flick, D. A.; Gifford, G. E. J. Immunol. Methods 1984, 68, 167–175. 49. Choi, H. S.; Kim, K. H.; Sohn, E.; Park, J. D.; Kim, B. O.; Moon, E. Y.; Rhee, D. K.; Pyo, S. Biosci. Biotechnol. Biochem. 2008, 72, 1817–1825.
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Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Preparation of a glucan from the roots of Rubus crataegifolius Bge. and its immunological activity Weihua Ni a, , Xu Zhang a, , Hongtao Bi a, Jeff Iteku a, Li Ji a, Chengxin Sun a, Jinbo Fang a, Guihua Tai a, Yifa Zhou a,*, Jimin Zhao b,* a b
School of Life Sciences, Northeast Normal University, Changchun 130024, PR China School of Life Sciences, Changchun Normal University, Changchun 130022, PR China
a r t i c l e
i n f o
Article history: Received 1 August 2009 Received in revised form 27 August 2009 Accepted 30 August 2009 Available online 3 September 2009 Keywords: Rubus crataegifolius Bge. Polysaccharide Glucan Immunological activity
a b s t r a c t A water-soluble glucan (RCP-1) was prepared from the roots of Rubus crataegifolius Bge. by extraction with hot-water, deproteination by Sevag reagent, a-amylase treatment and ultrafiltration. RCP-1 consisted of only glucose, and its molecular weight was determined to be 7 KD by high performance gel permeation chromatography (HPGPC). Fourier transform infra-red spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy (NMR), methylation and periodate oxidation analyses indicated that RCP1 was an a-D-glucan. Its main chains were composed of (1?4)- and (1?6)-linked a-glucopyranosyls, and side chains were single a-glucopyranosyl residues attached to the O-6 of glucosyls in the main chains. RCP-1 could increase both cytotoxic activity against B16 melanoma cells and the production of nitric oxide (NO) of macrophages in vitro. Furthermore, in vivo bioassay tests indicated that RCP-1 could remarkably enhance T and B lymphocyte proliferations, augment the phagocytosis of macrophages and increase the tumour necrosis factor-alpha (TNF-a) levels in serum. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Many polysaccharides isolated from medicinal plants exhibit immunomodulatory function and antitumour activities.1–3 These polysaccharides are often identified as biological response modifiers (BRMs), and their enhancement of the host defence has been recognised as a possible mean of inhibiting tumour growth without harming the host.4,5 The polysaccharides’ immunomodulatory effects and anticancer activity are influenced by the glycosidic linkage form, chain length, branch-point number, molecular size and tertiary structure.6 Previous studies of polysaccharide structure–activity relationships revealed that the 1,3-b-glucans have increased the potential to contribute to immunological and antitumour activities because of their interaction with the immunological cell surface receptors.7–10 Recently, some branched or linear a-1,4-/1,6-glucans isolated from medicinal plants Tinospora cordifolia,6,11 Cistanche deserticola,12 Strongylocentrotus nudus,13,14 Hedysarum polybotrys,15 Angelica sinensis16 and Aconitum carmichaeli17 have also been found to exhibit strong immunological and antitumour activities. Rubus crataegifolius Bge. is a species of raspberry that grows widely in north-eastern China, Japan, Korea and the Ussuri region of the Russian Far East.18 For many years, R. crataegifolius has been
* Corresponding authors. Tel./fax: +86 431 85098212 E-mail addresses: [email protected] (Y. Zhou), [email protected] (J. Zhao). These authors contributed equally to this paper. 0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2009.08.042
used in the treatment of various diseases such as rheumatic arthritis, hepatitis and lung cancer in China.19 Some small molecules in R. crataegifolius have been identified to be active components.20–24 Until now, there has been no report about polysaccharides extracted from R. crataegifolius. In this study, we describe the preparation of a low molecular weight a-glucan from R. crataegifolius and investigate its immunomodulatory activities. 2. Results and discussion 2.1. Isolation and purification A polysaccharide (RCP, yield 11.3% of dried material) was obtained from the roots of R. crataegifolius via hot-water extraction, ethanol precipitation and Sevag reagent deproteination. RCP exclusively contained glucose and exhibited a wide molecular weight distribution (from 20 KD to 210 KD) on Sepharose CL-6B chromatography. In an iodine test, RCP tested positive, indicating that it possesses starch structural features indicative of a starch-like glucan. Its molecular distribution was so wide that its structure and activity were difficult to study. Therefore, we treated RCP with a-amylase to break down some a-(1?4)-linked glucosidic bonds in an effort to obtain low molecular weight glucan. The enzymolysis products were fractionated by ultrafiltration (30–5 KD Mw cut-off membrane), yielding a main fraction RCP-1 (32% yield) that tested negative in an iodine test. RCP-1 was a water-soluble white powder. Its
2513
b 100
0.04
80
0.00
3417.3
1024.0
70 0.02
0
5
10
15 20 25 30 Retention time (min)
35
40
60 4000
840.8 1080.01151.3
0.06
90
1648.9
0.08
2931.3
% Transmittance
RI response
0.10
1454.1
0.12
2360.5
a
931.5
W. Ni et al. / Carbohydrate Research 344 (2009) 2512–2518
3500
3000
2500 2000
1500
1000
500
-1
Wavenumbers (cm )
Figure 1. (a) The HPGPC profile and (b) the FT-IR spectrum of RCP-1.
carbohydrate and protein contents were determined to be 97.3% and 1.2%. RCP-1 was homogeneous with Mw of 7 KD as demonstrated by HPGPC (Fig. 1a) and exclusively contained glucose. 2.2. Structural analysis The specific rotation of RCP-1 was ½a20 D +278.8 (c 1.0, H2O). The high positive rotation suggested the dominating presence of a-glycosidic linkages.15,25–27 The FT-IR spectrum of RCP-1 ( Fig. 1b) showed a-type glycosidic linkages at 932 and 841 cm1. The other absorption bands at 3417 cm1 (hydroxyl stretching vibration); 2931 cm1 (C–H stretching vibration); 1648 cm1 (bound water); 1151, 1079 and 1024 cm1 (pyranose ring) were from the corresponding sugar residues. The methylation of RCP-1 mainly produced four partially methylated alditol acetates, 1,5-di-acetyl-2,3,4,6-tetra-O-methyl glucitol (13.6%), 1,4,5-tri-acetyl-2,3,6-tri-O-methyl glucitol (62.8%), 1,5,6-tri-acetyl-2,3,4-tri-O-methyl glucitol (6.8%) and 1,4,5,6-tetra-acetyl-2,3-di-O-methyl glucitol (11.4%). Based on these results, RCP-1 correspondingly contained four glucosidic linkage forms, that is, (1?4)-, (1?6)-, (1?4, 6)-linked glucosyls and non-reducing terminals (Table 1). Periodate oxidation indicated that each mole of the residual glucosyl of RCP-1 consumed, on average, 1.32 mol of periodate and produced 0.29 mol of formic acid. GC analysis indicated that the hydrolysed products contained erythritol (70.2%) and glycerol (29.8%) but did not contain monosaccharides. These results supported the conjecture that RCP-1 is an a-(1?4), (1?6)-linked glucan. The NMR analyses, including 13C, 1H and 13C–1H HSQC (Fig. 2), were carried out to further characterise the structure of RCP-1
Table 1 Results of the methylation analysis of RCP-1 Partially methylated glycitol
Molar ratio
MS (m/z)
Linkage type
1,5-Di-acetyl-2,3,4,6-tetraO-methyl glucitol 1,4,5-Tri-acetyl-2,3,6-triO-methyl glucitol 1,5,6-Tri-acetyl-2,3,4-triO-methyl glucitol 1,4,5,6-Tetra-acetyl-2,3di-O-methyl glucitol
2
43, 45, 71, 87, 101, 117, 129, 145, 161, 205 43, 45, 87, 101, 117, 131, 161, 233 43, 45, 71, 87, 101, 117, 129, 161, 189, 233 43, 85, 101, 117, 127, 261
Glcp-(1?
9 1 2
?4)Glcp-(1? ?6)Glcp-(1? ?4,6)Glcp-(1?
(Table 2). According to the NMR data in the literature,15,26–30 four anomeric signals for typical a-glycosidic configurations appeared at d 99.38, 99.22, 99.01 and 98.05 ppm in the 13C NMR spectrum and at d 5.39, 5.33 and 4.96 ppm in the 1H NMR spectrum. Both of these results are consistent with the value of specific rotation and the absorptions in the FT-IR spectrum. The signals of O-substituted C-6 appeared at d 68.76;66.54/3.91 ppm, and those of unsubstituted C-6 appeared at d 59.92/3.86 and 60.12/3.70 ppm. The signals at d 77.23/3.57 and 77.12/3.67 ppm were assigned to O-substituted C-4, and those of unsubstituted C-4 appeared at d 70.71;70.42/3.42 ppm. The ratios of the signals from NMR were consistent with those of the sugar residues characterised by the chemical analysis described above and indicated that linear a-(1?4)-linked glucosyls were more than a-(1?6)-linked glucosyls. Summarising the comprehensive results, it was deduced that RCP-1 was composed of 43 a-glucopyranosyl residues. The main chains consisted of (1?4)- and (1?6)-linked a-glucopyranosyl residues, and the side chains contained one glucosyl attached to the O-6 of glucosyls on the main chains. The deduced structure of RCP-1 is shown in Figure 3, based on one hypothesis explaining the negative iodine test result of RCP-1 that (1?4)-linked a-glucopyranosyl residues were spaced by the (1?6)-linked side chain residues. Alternatively, it is possible that the (1?6)-linkages were in the main chain and could not form a helix structure. 2.3. Immunological activity The effects of RCP-1 on ConA- and LPS-induced lymphocyte proliferations were tested in vivo by the MTT method. Lymphocytes induced by ConA may be used as a method to evaluate T lymphocyte activity, while those lymphocytes induced by LPS may be used to evaluate B lymphocyte activity.12,17 As shown in Figure 4, in the presence of ConA or LPS, RCP-1 significantly increased lymphocyte proliferation in all the three testing doses (P <0.05). The increase in T cell proliferation was higher in the presence of 10 mg/kg of RCP-1 than 25 and 50 mg/kg. However, the increase in B cell proliferation peaked at 25 mg/kg. These results indicated that RCP-1 was able to activate both T and B cells. Like some other bioactive a-(1?4), (1?6)-glucans isolated from medicinal plants,6,14 RCP-1 might be a potent T/B-cell stimulator. Lymphocytes are considered to be one of the important components of the host defence against tumour growth and invading pathogens. These cells are able to produce
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60
59.92/3.70 60.12/3.86
65
68.76;66.54/3.91
70
70.64/3.84
98.05/4.96
100
99.01/5.33
95
90
85
80
77.12/3.67 77.23/3.57
ppm
75
70.71;70.42/3.42
105
99.38;99.22/5.39
5.8
5.6
5.4
5.2
Figure 2. HSQC spectrum of RCP-1: the measured
5.0
4.8
4.6
4.4
ppm
4.2
4.0
3.8
3.6 3.4
3.2
3.0
13
C spectrum is shown on the vertical axis and the 1H spectrum is shown on the top.
Table 2 1 H and 13C NMR chemical shift of RCP-1 in D2O Sugar residue
?4)-a-Glcp-(1?
a-Glcp-(1? ?4,6)-a-Glcp-(1? ?6)-a-Glcp-(1?
d
13
C/1H (ppm)
1
2
3
4
5
6
99.22 5.39 99.38 5.39 99.01 5.33 98.05 4.96
71.00 3.64 72.19 3.70 71.11 3.64 71.32 3.68
72.78 3.95 72.34 3.97 72.41 3.95 72.32 3.97
77.12 3.67 70.71 3.42 77.23 3.57 70.42 3.42
70.64 3.84 72.43 4.02 68.88 3.80 69.81 3.81
59.92 3.86 60.12 3.70 68.76 3.91 66.54 3.91
Figure 4. The effect of different doses of RCP-1 on ConA- or LPS-induced lymphocyte proliferation activity in mice. Each value represents the mean ± S.D. based on eight mice per group. Significant differences from the negative controls were evaluated using student’s t-test: *P <0.05, **P <0.01. Figure 3. The structure of RCP-1.
many kinds of cytokines after differentiation and activation. Stimulating proliferation of lymphocytes results in an increase in cytokine release, potentially accounting for the antitumour activity of the polysaccharides isolated from medicinal plants.31 The effects of RCP-1 on immunised murine macrophage phagocytosis were determined by using chicken red blood cells (CRBC).
As shown in Table 3, RCP-1 could significantly enhance phagocytosis at doses of 25 and 50 mg/kg, compared to the control. Both the rates of phagocytosis and the phagocytic indices were increased by RCP-1 treatment in a dose-dependent manner. TNF-a is an important mediator in the destruction of tumour cells. It has tumour-selective cytotoxicity by combining with the receptor on the tumour cell surface, and kills tumour cells specifically but does not injure normal cells.32 RCP-1 increased TNF-a
W. Ni et al. / Carbohydrate Research 344 (2009) 2512–2518
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Table 3 Effects of RCP-1 polysaccharide on macrophage-mediated phagocytosis in mice Animal groups Control RCP-1
Dose (mg/kg)
Phagocytosis rate (%)
Phagocytic index
10 25 50
29.34 ± 2.32 31.94 ± 1.97 41.18 ± 3.43* 51.88 ± 4.66**
0.43 ± 0.11 0.49 ± 0.19 0.73 ± 0.23* 0.77 ± 0.22**
The phagocytic rate was measured by counting the number of macrophages containing CRBCs found in the total macrophage cell population; the phagocytosis index was measured by counting the number of phagocytosed CRBCs present in the total macrophage cell population. Each value represents the mean ± S.D. based on eight mice per group. Significant differences from the negative controls were evaluated using student’s t-test: *P <0.05, **P <0.01.
Figure 6. Effect of RCP-1 on macrophage NO (as nitrite) production. RCP-1 (5– 500 lg/mL) and LPS (10 lg/mL) were added to peritoneal macrophages (0.5 106 cells/well) for 24 h, respectively. The isolated supernatants were mixed with an equal volume of Griess reagent, and nitrite production was measured by an ELISA reader at 540 nm. The reported values are the mean ± SD, (n = 6). *P <0.05, **P <0.01 versus the medium-treated group.
Figure 5. TNF-a levels in serum of RCP-1-immunised mice. Each value represents the mean ± S.D. based on eight mice per group. Significant differences from the negative controls were evaluated using student’s t-test: *P <0.05, **P <0.01.
levels of serum in RCP-1-immunised mice. As shown in Figure 5, the levels of TNF-a in all RCP-1 treatment groups were much higher than in the control group (P <0.01). The TNF-a levels (77.3 pg/ mL) peaked at a dose of 25 mg/kg. Nitric oxide (NO) has been identified as another major effector molecule involved in the destruction of tumour cells by activated macrophages. The production of NO in vitro is assessed by the Griess reaction. As shown in Figure 6, RCP-1 significantly increased NO production at concentrations of 50–500 lg/mL in a dosedependent manner. However, NO production was weaker after stimulation with RCP-1 than LPS, a potent activator of murine macrophages. To examine whether RCP-1 enhances the ability of macrophages to kill B16 melanoma cells, which are sensitive to either TNF-a or NO, the macrophages were treated with various concentrations of RCP-1 for 24 h and then co-cultured with B16 melanoma cells for 16 h. The cell viability was then evaluated by the MTT method. As shown in Figure 7, RCP-1 markedly increased the cytotoxicity of macrophages in a concentration-dependent manner. These data indicated that RCP-1 could stimulate the tumouricidal activities of macrophages. The antitumour function of macrophages may comprise two mechanisms. One mechanism is cell-to-cell contact between the macrophage and the target cell; and the other is secretion of factors such as cytokines and nitrogen intermediates.33 RCP-1 induced TNF-a and NO productions in B16 melanoma cells, which are either TNF-a or NO sensitive. Therefore, it was deduced that the enhancement of macrophage cytotoxicity by RCP-1 may be at least partially due to the secretion of cytokines and nitrogen intermediates.
The macrophages are considered the pivotal immunocytes of the host defence. Many polysaccharides from plants have been shown to activate macrophages by binding to the receptors on the surface of immune cells. It has been known that macrophages can interact with botanical polysaccharides and/or glycoproteins via Toll-like receptor 4 (TLR4), complement receptor 3, the scavenger receptor, dectin-1 and the mannose receptor.5 Activation of these receptors leads to intracellular signalling cascades, resulting in transcriptional activation and production of pro-inflammatory cytokines. In our experiment, RCP-1 augments cytotoxic activity against tumour cells and increases phagocytosis and cytokine (TNF-a) and NO production in macrophages. These data are consistent with the findings that (1,4)-a-glucans isolated from T. cordifolia augmented the phagocytic ability of macrophages and induced TNF-a production in a concentration-dependent manner in vitro,11 and the (1,4)-a-glucans isolated from S. nudus-induced NO production from macrophages in vitro.14 The differences in these data
Figure 7. Tumouricidal activities of RCP-1-treated murine peritoneal macrophages against B16 melanoma cells. The macrophages were treated with various doses of RCP-1 for 24 h. Antitumour activity was determined as described in materials and methods at an initial effector: target ratio of 10:1. The reported values are the mean ± SD, (n = 6). *P <0.05, **P <0.01 versus the medium-treated group.
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may result from the glucan structures, specifically molecular weight, chain length and tertiary structure.6 In conclusion, a low molecular weight glucan RCP-1 prepared by a-amylase treatment of the polysaccharides isolated from the roots of R. crataegifolius was characterised to be a branched a-D-glucan with a backbone of (1?4)- and (1?6)-linked a-D-glucopyranosyls. The side chains consisted of one a-D-glucopyranosyl residue attached to O-6 of (1?4)-linked a-D-glucopyranosyls. RCP-1 could significantly enhance lymphocyte proliferation and augment the phagocytic ability of macrophages. RCP-1 increased macrophage NO production and TNF-a levels in serum, resulting in the inhibitory effects of murine peritoneal macrophages against B16 melanoma cells in vitro. Therefore, RCP-1 may be useful in tumour therapy.
3. Experimental 3.1. General methods The total carbohydrate content was determined by the phenol– sulfuric acid method, using glucose as the standard.34 Uronic acid content was determined by the m-hydroxydiphenyl method, using galacturonic acid as the standard.35 Protein content was determined by the Bradford assay, using bovine serum albumin as the standard.36 The presence of starch was analysed with the iodine test.37 Dialysis was carried out using a tube with Mw cut-off of 3500 Da (for globular protein). Gas chromatography was performed using a Shimadzu GC-14C instrument equipped with a hydrogen flame ionisation detector on an Rtx-2330 column (0.32 mm 15 m i.d., 0.2 lm) at a temperature programme of 175 °C (hold 2 min) followed by 8 °C/min to 240 °C (hold 1 min) and 8 °C/min to 265 °C (hold 17 min). The hydrogen flow rate was 10 mL/min, and the ion-source temperature was 275 °C. High performance liquid chromatography (HPLC) was performed using a Shimadzu 10Avp HPLC system equipped with 10Avp HPLC Pump, SPD-10Avp UV–vis Detector and RID-10A Refractive Index Detector. The specific rotation was determined at 20 ± 1 °C with an automatic polarimeter (Model WZZ-2B, China). UV–vis absorbance spectra were recorded with a UV–vis spectrophotometer (Model SP-752, China). Sephadex G-75, standard monosaccharides and gel filtration standard dextrans were purchased from Sigma Co. All other chemicals were of analytical grade made in China. 3.2. Preparation of glucan RCP-1 The roots of R. crataegifolius were collected from Tonghua, Jilin province, China and identified by Professor Hongxing Xiao, School of Life Sciences, Northeast Normal University in Changchun, China. The roots of R. crataegifolius were extracted with distilled water (100 °C, 1:10 w/v) three times (4 h each). The aqueous filtrates were combined and concentrated to a small volume (one-tenth of the original volume). To precipitate the polysaccharides, 95% ethanol was added to the aqueous filtrates, totalling 80% of the final volume. The polysaccharides were collected by centrifugation and dried in vacuum. The collected polysaccharides were dissolved in water (15% w/v) to remove the insoluble substances originating from centrifugation. The supernatant was treated with Sevag reagent (1:4 n-butanol/chloroform, v/v) to remove the proteins.38 After removing the proteins and Sevag reagent by centrifugation, the water phase was dialysed against distilled water and lyophilised to yield polysaccharide (RCP). RCP was dissolved in PBS (0.1 M, pH 6.9) and treated with a-amylase from Bacillus subtilis (E.C. 3.2.1.1, Sigma, St. Louis, USA) at 37 °C. The remaining starch was assessed using the iodine test every 15 min. When the poly-
saccharide was tested negative by the iodine test, the solution was boiled to inactivate the a-amylase, centrifuged to remove the insoluble substances and subsequently purified by ultrafiltration through 30 KD and 5 KD Mw cut-off membranes to yield the purified, degraded polysaccharide RCP-1. Lipopolysaccharide (LPS) contamination was tested by Limulus amoebocytes lysate (LAL) assay using an E-TOXATE kit (Sigma, St. Louis, USA) according to the manufacturer’s instruction. The quantity of endotoxin in RCP-1 was less than 0.015 EU/mg (negative). 3.3. Homogeneity and molecular weight Determination of homogeneity and molecular weight was carried out by an HPLC-linked gel filtration column of TSK-G3000 PWXL, eluting with 0.2 M NaCl at 0.6 mL/min, 35.0 ± 0.1 °C. The gel filtration column was calibrated by standard dextrans (50 KD, 25 KD, 12 KD, 5 KD, 1 KD) using linear regression. The sample concentration was 5 mg/mL, and the injection amount was 20 lL. 3.4. Monosaccharide composition The monosaccharide analysis was performed as described by Honda39 and Yang.40 The polysaccharide RCP-1 (2 mg) was hydrolysed with anhydrous methanol (0.5 mL) containing 2 M HCl at 80 °C for 16 h and then with 2 M CF3COOH (0.5 mL) at 120 °C for 1 h.41 The hydrolysis product was derivatised with 0.5 M 1-phenyl-3-methyl-5-pyrazolone (PMP) and 0.3 M NaOH. After neutralisation with 0.3 M HCl, the derivatives were analysed by HPLC on a DIKMA Inertsil ODS-3 column (150 4.6 mm i.d.) with a guard column on a Shimadzu HPLC system (LC-10ATvp pump and UV–vis detector) and monitored by UV absorbance at 245 nm. 3.5. Periodate oxidation The periodate oxidation was performed according to the procedure described by Chaplin and Kennedy.42 RCP-1 (25 mg) was dissolved in 0.015 M NaIO4 (25 mL), and the solution was kept at 4 °C in the dark. The A223 nm of reaction solution was determined every 6 h by spectrophotometer. After the oxidation was completed (64 h), the excessive NaIO4 was decomposed with ethylene glycol (0.1 mL). The NaIO4 consumption was calculated according to the decrease of absorbance (A223 nm). The formic acid production was determined by titration with 0.1 M NaOH. The reaction mixture was dialysed against tap water and distilled water in turn, and then the retentate was reduced with NaBH4 overnight. After neutralisation and dialysis, the retentate was freeze-dried, hydrolysed with 2 M CF3COOH (1 mL) at 120 °C for 2 h, reduced by NaBH4, acetylated with pyridine (0.5 mL) and acetic anhydride (0.5 mL) at 90 °C for 1 h and then analysed for sugar composition by GC. 3.6. Methylation analysis The methylation analysis was carried out according to the method of Needs and Selvendran.43 In brief, RCP-1 (10 mg) was dissolved in DMSO (1.5 mL) and methylated by the treatment with NaOH/DMSO suspension (1 mL) and iodomethane (1.0 mL). The reaction mixture was extracted with CHCl3, and then the solvent was removed by vacuum evaporation. The completion of methylation was confirmed by the disappearance of the –OH band (3200– 3400 cm1) in the FT-IR spectrum. The per-O-methylated polysaccharide was hydrolysed subsequently by HCOOH (85%, 0.5 mL) for 4 h at 100 °C and then CF3COOH acid (2 M, 1 mL) for 6 h at 100 °C. The partially methylated sugars in the hydrolysate were reduced by NaBH4 and acetylated.44 The resulting alditol acetates were analysed by GC–MS.
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3.7. Fourier transformed infra-red (FT-IR) analysis FT-IR spectra were obtained on a Nicolet 560 FT-IR spectrometer with DTGS detector in a range of 400–4000 cm1. The sample was ground with KBr powder and then pressed into 1mm pellet for FT-IR measurement. 3.8. NMR analyses 1 H, 13C NMR and HSQC spectra were recorded on a Bruker Avance 600 MHz spectrometer (Germany), operating at 600 MHz for 1H NMR and 150 MHz for 13C NMR. The sample (20 mg) was dissolved in D2O (99.8%, 0.5 mL), freeze-dried, re-dissolved in D2O (0.5 mL) and centrifuged to remove the excess sample. All the data were analysed using standard Bruker software.
3.9. Immunological assays 3.9.1. Animals and treatment Male C57BL/6 mice (6–8 weeks old) were purchased from the Pharmacology Experimental Center of Jilin University (Changchun, China). The mice were housed on a 12/12-h light–dark cycle at room temperature and allowed free access to standard rodent food and water during the experiments. Animal handling procedures were conducted under National Institutes of Health animal care and use guidelines. All efforts were made to minimise animals’ suffering and to reduce the number of animals used. The mice were classified into four groups. RCP-1 was dissolved in physiological saline and administered intraperitoneally (ip) into mice at different dosages (10, 25, 50 mg/kg) daily for five days. The control group was given physiological saline instead of polysaccharide solution. The dose volume was 0.2 mL. 3.9.2. Macrophage phagocytosis assay CRBC (1%, 1 mL) was injected ip into RCP-1-immunised mice on day 6. After 30 min, the mice were sacrificed, and D-Hank’s solution (2.5 mL) was injected.45 The activated macrophages were obtained by peritoneal lavage onto a microscope slide. After centrifugation at 150g for 10 min, the supernatant was removed, and the free CRBCs were lysed by sterile 0.16 mM NH4Cl buffer. The macrophages were fixed with methanol and stained with Giemsa-Wright for 7–10 min. The microscope slides were washed with PBS and counted using a microscope (Olympus CX21, Japan, 100 objective lens). The rate of phagocytosis was calculated as follows:
Rate of phagocytosis ¼ ðNo: of macrophages containing CRBC= Total macrophages countedÞ 100: The phagocytosis index was measured as follows:
Phagocytosis index ¼ ðNo: of CRBC in macrophages= Total macrophages countedÞ 100: 3.9.3. Lymphocyte proliferation assay Spleens were aseptically extirpated from the immunised mice. Spleen single cell suspensions were pooled in ice-cold Hank’s solution by filtering through sieve mesh. The spleen cells were depleted of erythrocytes by Tris2NH4Cl (0.16 M, Tris2NH4Cl, pH 7.2) owing to low-osmosis, followed by washing twice in Hank’s solution and resuspending in complete RPMI 1640 medium. The viability of the splenocytes was >95%, as assessed by the trypan blue dye exclusion method described by Lee et al.46 The spleen cells (5 106 cells/mL) were seeded in a 96-well plate and cultured with mitogen ConA (5.0 lg/mL; Sigma, St. Louis, USA) or LPS (10.0 lg/mL; Sigma, St. Louis, USA). The cells were cul-
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tured at 37 °C, 5% CO2 for 44 h. MTT (5 mg/mL, 20 lL; Sigma, St. Louis, USA) was added to each well and incubated for an additional 4 h for the MTT cellular viability assay. The absorbance at 570 nm was measured using a Bio-Rad microplate reader (Model 550, USA). All determinations were conducted in triplicate. 3.9.4. Measurement of TNF-a activity The serum collected from the immunised mice was measured using an enzyme-linked immunosorbent assay kit (Pharmingen, San Diego, CA, USA) for tumour necrosis factor-alpha according to the manufacturer’s instructions. 3.9.5. Activation of peritoneal macrophages in vitro The cells from the peritoneal exudate were collected from normal C57BL/6 mice (male, 6–8 weeks) by peritoneal lavage with serum-free RPMI 1640 medium (5 mL; Sigma, St. Louis, USA). The collected cells were washed with D-Hank’s and cultured in complete RPMI 1640 medium (Sigma, St. Louis, USA) and supplemented with 25 mM HEPES, 10% heat-inactivated foetal calf serum (FCS), 1 105 IU/L penicillin G and 100 mM streptomycin. The cells were placed in a flat-bottomed culture plate and then incubated for 2 h at 37 °C and 5% CO2. After removal of the nonadherent cells, the mono-layer of macrophages was collected. 3.9.6. Assay for nitrite Peritoneal macrophages were prepared as described above and were plated at a density of 0.5 106 cells/well in 48-well plates and then treated with filter-sterilised samples or LPS (10.0 lg/mL; Sigma, St. Louis, USA) as a positive control. After 24 h of incubation, the culture supernatant was collected. The isolated supernatants were mixed with an equal volume of Griess reagent (1% sulfanilamide in 2.5% phosphoric acid, 0.1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid) and incubated at room temperature for 10 min. Using NaNO2 to generate a standard curve, the concentration of nitrite was measured by OD at 540 nm.47 3.9.7. Macrophage-mediated cytotoxicity The macrophage-mediated cytotoxicity was determined by the modification of the technique described previously.48 Peritoneal macrophages (1.0 105 cells/well) prepared as described above were first incubated in either medium alone or in medium supplemented with RCP-1 for 24 h in 96-well plates, washed with RPMI 1640 medium to remove the RCP-1 and then co-incubated with B16 melanoma cells (ATCC, Rochville, MD, USA) (1.0 104 cells/ wells; an initial effector: target cell ratio of 10:1) at 37 °C in a 5% CO2 incubator for 16 h. The cell density was then assessed by incubating the cells with MTT (25 lg/mL) for another 4 h.49 After aspirating the supernatant from the wells, DMSO (100 lL) was added for dissolution of formazan crystal. The absorbance at 570 nm was measured using a microplate reader. Cytolytic activity is expressed as the percentage of tumour cytotoxicity, where % Cytotoxicity = {1 O.D. of [(target + macrophages) macrophages]/O.D. of target (nontreated)} 100. Acknowledgements This work was supported by the National Natural Science Foundation of China(Nos. 30670478 and 30770489) and the Natural Science Foundation of Jilin Province (No. 20070710). References 1. Wasser, S. Appl. Microbiol. Biotechnol. 2003, 60, 258–274. 2. Tzianabos, A. O. Clin. Microbiol. Rev. 2000, 13, 523–533. 3. Moradali, M. F.; Mostafavi, H.; Ghods, S.; Hedjaroude, G. A. Int. Immunopharmacol. 2007, 7, 701–724. 4. Leung, M. Y. K.; Liu, C.; Koon, J. C. M.; Fung, K. P. Immunol. Lett. 2006, 105, 101– 114.
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