2 Institute of Chinese Medicine, Chinese University of Hong Kong, Shatin, N.T., Hong Kong, People's Republic of China; 3 Department of Biochemistry, Chinese University of Hong Kong, Shatin, N.T., Hong Kong, People's Republic of China; 4 South China Institute of Botany, Chinese Academy of Sciences, Guangzhou, People's Republic of China; and 5 Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, People's Republic of China
Received on July 31, 2003; revised on December 2, 2003; accepted on December 19, 2003
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Abstract |
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Introduction |
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Polysaccharide BRMs can be prepared from bacteria, fungi, and plants. Bacterial polysaccharide BRMs can be membrane proteoglycan from Klebsiella pneumoniae (Sironi et al., 1990), cell wall lipopolysaccharide (LPS) from Gram-negative bacteria (Blackstock et al., 2000
; Heinzelmann et al., 2000
), and exopolysaccharide from Paenibacillus jamilae CP-7 (Ruiz-Bravo et al., 2001
). Fungal polysaccharide BRMs are mainly derived from cell wall or cytoplasmic reserve (Domer et al., 1988
; Liu et al., 1993
) and botanical polysaccharide BRMs are mainly pectic substances in origin (Ebringerova et al., 2003
; Guo et al., 2000
; Nose et al., 1998
; Yaneva et al., 2002
).
The polysaccharides may have phosphate, acetyl group, carboxylic group, peptide, and lipid attached. The sugar composition of the polysaccharides might contain variable amounts of glucose, mannose, galactose, arabinose, and xylose. The linkages of the polysaccharides can be -, ß-, (1
2), (1
3), (1
4), or (1
6)-linkages. The polysaccharides are thus very diverse in their components and linkages except those derived from fungi and aloe. In fungi, the majority polysaccharide BRMs are ß-D-glucans and those from yeast are
-D-mannan and glucomannan. Polysaccharide BRMs derived from aloe are mainly mannose polymer with ß-(1
4)-D linkage.
Although a lot of polysaccharide BRMs have been reported, there is little information regarding to the structure and potency relationship of the polysaccharides. Such relationship study is hindered by the very complex sugar compositions and linkages of the polysaccharides. However, aloe polysaccharides having simple sugar composition and linkage are ideal models for the studies of the structure and potency relationship. The simplicity of the polysaccharides makes the structure determination feasible, and then the studies between structure and potency can be performed. In this study, three purified polysaccharide fractions were prepared from Aloe vera L. var. chinensis (Haw.) Berg. and were characterized chemically and biologically to delineate the effect of sugar content and molecular weight on the potency of the polysaccharide fractions.
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Results |
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Discussion |
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CE results of PAC-I, PAC-II, and PAC-III suggested the purified polysaccharide fractions might still contain some impurities. However, the purity was good enough for chemical and biological study. Protein or peptide, which can be immunostimulatory, was undetectable in PAC-I, PAC-II, and PAC-III. The result accorded with elemental analysis. Elemental analysis demonstrated that there existed no nitrogen in PAC-I, PAC-II, and PAC-III (unpublished data). Methylation analysis of the polysaccharides had been attempted. However the sizes of the polysaccharide fractions were too large for all the free hydroxyl groups to be methylated completely. The 13C NMR spectrum of PAC-I was found to be very similar to that of legume-seed galactomannan (Grasdalen and Painter, 1980). Legume-seed galactomannan has manopyranosides in the main skeleton linked together by ß-(1-4)-D-linkage and galactopyranoside attached at C-6 of the skeletal manopyranosides.
PAC-II, which had nearly the same composition and NMR profiles as PAC-I, had the same main skeletal structure as PAC-I but smaller in size. PAC-I had molecular weight 7.8 times that of PAC-II. The deduced structure of the main linkage of PAC-I and PAC-II is illustrated in Figure 7. The main skeleton of PAC-I and PAC-II was ß-(14)-D-linked mannopyranosyl units. The manopyranosyl units in the polymer were O-acetylated at carbon six. The composition and linkages of PAC-III were more complicated than PAC-I and PAC-II. The possibility of impurities in PAC-III that contributed to the complex NMR spectra of PAC-III was rather low. CE results indicated that the purity of PAC-III was comparable to that of PAC-I and PAC-II.
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Both PAC-I and PAC50 mediated a significant regression of Sc-180 in vivo (Figure 6). The in vivo tumor regression properties of the aloe BRMs might be due to direct toxicity of the polysaccharide fractions, tumor-sensitive toxic metabolic derivatives of the polysaccharide fractions, or immunostimulatory properties of the polysaccharide fractions. The direct toxicity of the polysaccharide fractions was excluded because antitumor effect was only observed in vivo but not in vitro (unpublished data). The polysaccharide fractions mediated no tumor regression in nude mice, which suggested that aloe polysaccharides were not metabolized to tumor-sensitive toxic substances. Therefore, aloe BRMs must mediate tumor regression in vivo by the activation of host immune effector mechanisms.
Similar to PAC-I and PAC-II, ß-(13)-D-glucans have simple monosaccharide composition and linkage. The in vivo antitumor properties of fungal ß-(1
3)-D-glucans are initiated by the binding of the glucans to ß-glucan receptor, such as dectin-1 (Brown and Gordon, 2001
; Taylor et al., 2002
), on immune cells. The target receptor for aloe BRMs might be mannose receptor (MR) on macrophages and dendritic cells. Aloe polysaccharides might bind to MR of dendritic cells and macrophages and lead to activation of immunity. The stimulation of B cells by PAC polysaccharides might through another mechanism, because the presence of MR on B cells was not reported. The potent B cell stimulatory effect of PAC polysaccharides in vitro might be related to its highly repeating structures, which cross-link surface IgM of polysaccharide-responding B cells in a multivalent fashion. The cross-linking activates the B cells to proliferate (Anderson and Blomgren, 1971
; Dorries et al., 1974
).
The mechanism of activation of immunity by ß-glucan is well established. Lesson from ß-glucan study may provide clues to the immune activation by mannose-rich polysaccharide at a molecular level. Dectin-1 is a pattern recognition receptor. Dectin-1 cooperates with Toll-like receptors (TLRs), and many other surface receptors for the recognition of different microbial products such as fungal cell wall, lipopolysaccaharide, lipoprotein, flagellin, and bacterial DNA (Gantner et al., 2003; Underhill, 2003
). The repertoire of receptors that recognize microbial products determines the immune effector mechanism to be activated. Zymosan particle, which is composed mainly of ß-glucan, mannan, mannoproteins, and chitin, is recognized by dectin-1, TLRs, and CD14. The receptors work together to enhance immune response to the particles. Dectin-1 can trigger phagocytosis and reactive oxygen production directly. TLRs induce signaling through NF-
B results in the secretion of inflammatory cytokines. The TLR induction is enhanced by dectin-1. Mannose-rich polysaccharide may also collaborate with TLRs for the activation of immunity.
Altogether, mannan from aloe has a simple composition, and the linkage is an excellent model for the study of structure and activity relationship. PAC-I and PAC-II prepared from A. vera L. var. chinensis (Haw.) Berg. has potent BRMs. The polysaccharide fractions mediated indirect tumor regression in vivo through the activation of antitumor effector cells and the secretion of antitumor cytokines. The skeletons of the polysaccharide fractions were ß-(14)-D-linked mannose with O-acetylation at C-6. High molecular weight and high mannose content may be essential for high antitumor potency of aloe polysaccharide.
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Materials and methods |
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Protein content determination
Bradford micro-protein assay (Bradford, 1976) was used to determine protein content. Bovine serum albumin (Sigma, St. Louis, MO) was used as protein standard. Filtered Bradford reagent (BioRad, Hercules, CA) (0.2 ml) was reacted with 0.8 ml sample at 1 mg/ml at room temperature for 15 min. Absorbance of the reaction mixture was measured at optical density 595.
Carbohydrate content determination
Dubois method (Dubois et al., 1956) was used to measure carbohydrate content. D-mannose was used as standard. A sample of 400 µl at 500 µg/ml was mixed with 200 µl 6% phenol solution and 1 ml concentrated sulfuric acid. The mixture was reacted for 20 min at room temperature. Absorbance of the reaction mixture was measured at optical density 490.
HPLC of PAC50
PAC50 was dissolved in 0.1 M NaCl at 5 mg/ml. Five milliliters of PAC50 were loaded onto a Gel filtration column (Jordi Aqueous GPC Glucose Bound 10,000 Å 5 U SS column; Alltech, Nicholasville, KY) with dimensions of 22 mm x 500 mm. NaCl (0.1 M) was the mobile phase, and the flow rate was maintained at 3 ml/min. Dextrans of different molecular weights were used as standard. The eluate was collected as 3-ml fractions. The carbohydrate content in each fraction was determined by Dubois's method.
CE
Honda's method (Honda et al., 1989) was used to determine the purity of column separated polysaccharides. Sample of 20 µl at 200 mg/ml eluant (H3BO3-KOH, pH 10) was applied to Quanta 400E (Waters, Milford, CT) CE system and electrophoresed at 20 kV. Polysaccharide was detected by measuring at optical density 254.
Monosaccharide composition analysis
Monosaccharide composition analysis followed the method of Wozniewski et al. (1990). A polysaccharide fraction of 3 mg was hydrolyzed with 10 ml 2 M aqueous trifluoroacetic acid in a sealed tube at 120°C for 1 h. The hydrolysate was concentrated with rotary evaporator under reduced pressure at 60°C, and remaining traces of trifluoracetic acid was removed by coevaporating with methanol (3 x 10 ml). The concentrated hydrolysate was dried over P2O5 in vacuum overnight. The dried hydrolysate was dissolved in 5 ml dH2O, and 10 mg sodium borohydride (NaBH4) was then added. The mixture was stirred at room temperature for 18 h. Excess NaBH4 was removed by acetic acid. The mixture was then concentrated and dried under reduced pressure. Dried residue was dissolved in 6 ml anhydrous pyridine. Acetic anhydride (6 ml) was added, and the mixture was stirred overnight at room temperature. After incubation, the mixture was coevaporated with toluene (3 x 10 ml) at 55°C under reduced pressure and finally dried in vacuum. Dried residue was dissolved in minimal amount of chloroform for gas chromatographymass spectrometry (GC-MS) analysis with a GC analyzer (Varian VISTA 6000, Palo Alto, CA). The GC column was OV-17 with dimension of 0.2 mm x 300 mm (Agilent Technologies, Wilmington, DE). The injector and column temperature was 250°C.
13C NMR and 1H NMR spectroscopy
Samples were dissolved in deuteriated water at 50 mg/ml for both 13C NMR and 1H NMR (Agrawal, 1992). 1H NMR spectra were recorded with an INOVA-600 spectroscopy at 30°C in D2O, and chemical shifts were referred to HOD. 13C NMR spectra were recorded with an INOVA-600 spectroscopy at 30°C in D2O, and chemical shifts were referred to Me4Si.
LAL coagulation test
The LAL test is commonly employed to determine the presence of LPS or endotoxin (lipopolysaccharide) in herbal preparation. The Pyrotell (Associates of Cape Cod) testing reagent was used according to instructions. Pyrogen-free water and LPS (0.05 ng/ml) from Escherichia coli of serotype 0127:B8 (Sigma) were used as controls.
In vitro lymphocyte blast transformation assay
Splenic lymphocytes were prepared from female albino BALB/c mice by Ficoll-Plaque centrifugation. Three spleens were ground with a sterilized sieve and a 2.5-ml syringe plunger in 10 ml RPMI-1640 medium (Invitrogen, Carlsbad, CA). The ground cell suspension was transferred to a 50-ml centrifuge tube, and volume was adjusted to 20 ml with medium. Equal volume of Ficoll was then added to the bottom of the cell suspension. The tube was centrifuged at 2200 x g for 20 min. Cells at interface were splenic lymphocytes and were collected. T cells were prepared by B cells depleting column (Cedarlane Labs, Hornby, Ontario, Canada), and B cells were prepared by T cells depleting column (Cedarlane Labs, Carlsbad, CA). Prepared T and B cells were resuspended in medium supplemented with 10% fetal calf serum (Invitrogen, Carlsbad, CA) at 5 x 106 cells/ml. Cell suspension of 100 µl was distributed per well of a 96-well plate. Cells were incubated with filter-sterilized polysaccharide at 37°C for 48 h. Cell proliferation was monitored by thymidine incorporation.
In vivo lymphocyte blast transformation assay
Polysaccharide fraction of 0.2 ml at 5 mg/ml phosphate buffered saline (PBS) was injected IP into female albino BALB/c mice. Spleens were obtained from mice on 3 days after sample administration. Splenic lymphocytes were prepared as described. Cell suspension of 100 µl at 5 x 106 cells/ml was distributed per well of a 96-well plate. Cell proliferation was monitored by thymidine incorporation at 48 h after incubation.
In vitro IL-2, IL-6, and INF- secretion
Splenic lymphocytes were prepared from female albino BALB/c mice. Cell suspension of 100 µl at 5 x 106 cells/ml concentration was plated per well of a 96-well plate. Cells were incubated with filter-sterilized polysaccharide at 37°C for 48 h. After the incubation, 100 µl medium was removed from each well, and the level of IL-2, IL-6, and INF- in removed medium was assayed by enzyme-linked immunoasorbent assay (ELISA) kit (Pharmingen, San Diego, CA).
In vitro IL-1ß and TNF- secretion
Thioglycollate broth (Difco Laboratories, Detroit, MI) of 1.5 ml at 0.3% concentration was injected IP per male BALB/c mouse. Three days after injection, the peritoneal cavity was washed with 20 ml PBS. Cells in the peritoneal wash were washed twice with RPMI-1640 medium. Cell concentration was adjusted to 108 cells/ml medium. Cell suspension of 100 µl was added per well of a 96-well plate. The cells were incubated at 37°C for 3 h. After incubation, nonadherent cells were removed by washing. The adherent cells, which were peritoneal macrophages, were incubated with filter-sterilized polysaccharide at 37°C for 48 h. After the incubation, 100 µl medium was removed from each well. The level of IL-1ß and the level of TNF- in removed media were assayed by murine IL-1ß and TNF-
ELISA kit (Pharmingen), respectively.
In vivo TNF- secretion
Polysaccharide of 0.2 ml at 500 mg/ml was injected IV per male BALB/c mouse. LPS at 10 µg/ml was used as positive control. Blood was collected 1.5 h after polysaccharide administration by heart puncture. Collected blood was clotted at room temperature and then centrifuged at 4000xg for 30 min. Serum was collected for TNF- measurement by murine TNF-
ELISA kit (Pharmingen).
In vivo antitumor assay
Sarcoma 180 (Sc-180) cells (ATCC) of 50 µl at a cell concentration of 107 cells per ml PBS were injected SC into the right groin of male ICR albino mice (10 mice/group) on day 1. Mice were then injected IP with 0.2 ml polysaccharide fraction at 1 mg/ml PBS or 0.2 ml PBS (PBS as negative control) for 10 consecutive days from day 1. On day 21, mice were euthanized, and solid tumors were excised for weight determination.
Statistical analysis
Student's t-test was used for the statistical analysis of results at a level of significance of p < 0.05.
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Footnotes |
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Abbreviations |
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References |
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