Preliminary Characterization of a Homogeneous Polysaccharide with Anticomplement Activity from Sijunzi Decoction

Sijunzi decoction (SJZD) is a classical herbal prescription in traditional Chinese medicine (TCM) used for enhancing the function of immune system. In previous studies, a polysaccharide fraction S-3 was screened from SJZD by assessment of immune system regulation, intestinal microbiota, and SCFA in order to explore the immune active ingredients in SJZD. In the present study, S-3 was further purified, and a homogeneous polysaccharide S-3-1 with a molecular mass of 13.5 × 10 Da was obtained after further fractionation by Sephadex G-150 size-exclusion chromatography.The immunological activities of S-3-1 were assayed in vitro for the first time. The determination of the anticomplement activity showed that S-3-1 displayed inhibitory effects on classical pathway of the complement system, with CH 50 values of 530 μg/mL. The FT-IR analysis showed that S-3-1 had absorptive peaks characteristic of polysaccharides. The methylation and GC-MS analysis showed that it is comprised of Rha, Ara, Xyl, Man, Gal, and Glc in a relative molar ratio of 0.35 : 0.37 : 1.4 : 0.31 : 3 : 0.8 and that it mainly contained 1,4-linked-Glc and 1,6-linked-Gal glycosidic bonds. The morphology of S-3-1 was observed by atomic force microscope (AFM). These results provided evidences for tracking the material basis of SJZD immune activity.


Introduction
Sijunzi decoction (SJZD) is a classic prescription used in traditional Chinese medicine (TCM) and is prepared using four kinds of Chinese herbs, Ginseng Radix, Atractylodes Macrocephalae Rhizoma, Poria, and Glycyrrhizae Radix.It is well known for curing spleen deficiency [1,2], characterized by the comprehensive manifestation of diseases in multiple systems, such as the digestive (digestion, absorption energy conversion) and the immune system.Modern pharmacological studies have shown that SJZD prescription affects the above physiological functions by strengthening the immune system [3].Although biologically active, low molecular mass compounds from SJZD such as flavone and saponin have previously been studied, they do not account for all of the components in SJZD.Polysaccharide is the most abundant and main effective component in SJZD [4].Polysaccharide extracted from SJZD is an effective immunomodulator and immune adjuvant [5].There are also reports of other TCM that contain polysaccharides and include Ginseng Radix [6], Atractylodes Macrocephalae Rhizoma [7], Poria [8], and Glycyrrhizae Radix [9], all of which are components of SJZD.Moreover, it has now been proven that the physiological/biological effects of polysaccharides realized through TCM prescriptions are considerably better than the effects that come from single herb prescriptions [10].
There is limited data currently available on the characterization of polysaccharides from SJZD prescriptions.Recent research has detected the presence of an active polysaccharide fraction (S-3) in SJZD that can improve the immune function of intestinal endothelial lymphocytes [11].However, information on the conformation of this polysaccharide fraction S-3 is limited.In this study therefore, we isolated and purified a new water-soluble polysaccharide from the S-3 fraction of SJZD prescription, characterized its structure, and investigated the potential activity of this polysaccharide against classical complement pathways.The results of this study will offer a theoretical basis for further investigation on the structure-activity relationship and development of SJZD polysaccharides.

Herbal Medicine and Chemicals
. SJZD was prepared by combining four herbs, Ginseng Radix, Atractylodes Macrocephalae Rhizoma, Poria, and Glycyrrhizae Radix, in the ratio of 3 : 3 : 3 : 2 by weight.The herbs were bought from Shanghai Huayu Pharmaceutical Co., Ltd.(Shanghai, China), their botanical origins were identified by the corresponding author, and voucher specimens were deposited at the School of Pharmacy, Shanghai Jiao Tong University.
Trifluoroacetic acid (TFA), dextrans, and monosaccharide standards were purchased from Aladdin Reagent Int.(Shanghai, China).All other chemicals used were of analytical grade as available.

Extraction and Purification of Polysaccharide from SJZD.
SJZPS (crude SJZD polysaccharide) and polysaccharide fraction S-3 were prepared as described previously [11].S-3 was further purified by gel filtration chromatography on a Sephadex G-150 column (16 mm × 70 cm) and eluted with deionized water at flow rate 30 mL/h.Two polysaccharides were obtained through elution, named S-3-1 and S-3-2, and the yield was 24.5% and 1.2%, respectively.

Determination of Homogeneity and Relative Molecular
Mass.The molecular mass of S-3-1 and S-3-2 were determined by high performance gel permeation chromatography (HPGPC) on a TSK-GEL G4000PWxl column (7.5 × 300 mm, Tosoh Co., Japan) as described previously [12].Deionized water was used as the elution and flow rate was maintained at 1 mL/min.A 20 L aliquot was injected for each run.The calibration curve of log(Mw) versus elution time () is given by log(Mw) = −0.1264+ 5.6361.

General Analysis Methods.
Carbohydrate content of the polysaccharide was determined by phenol-sulfuric acid colorimetric methods [13] using D-Glc as the standard.
Protein content was measured by the Lowry method [14] using BSA as the standard.Uronic acid content was measured by photometry with m-hydroxydiphenyl at 525 nm using dgalacturonic acid as the standard [15].

Monosaccharide Composition Analysis.
Monosaccharide composition analyses of S-3-1 and S-3-2 were performed by GC-MS (model 7890/5975C-GC/MSD, Agilent Technologies; Santa Clara, CA, USA) as previously described [16].10 mg of each sample was hydrolyzed with 2 mol/L trifluoroacetic acid at 95 ∘ C for 2 h.The excess acid was removed by four successive washes with methyl alcohol.For GC-MS analysis, the complete hydrolysate was mixed with 5 mg hydroxylamine hydrochloride, 0.5 mL pyridine, and 4 mg inositol as the internal standard and incubated at 90 ∘ C for 30 min.The mixture was cooled to room temperature and 0.5 mL acetic anhydride was added, mixed thoroughly, and incubated at 90 ∘ C for 30 min.After cooling, the resulting aldononitrile acetate derivatives were analyzed by GC-MS.Aldononitrile acetate derivatives of seven authentic monosaccharide standards with inositol as internal standard were prepared and subjected to GC-MS analysis the same way.By comparison based on retention times, monosaccharide compositions of S-3-1 and S-3-2 were determined.[18].The amount of NaIO 4 consumed was calculated according to the NaIO 4 standard curve.Production of formic acid was determined by titration.The remaining periodate product was treated with ethylene glycol and dialyzed for 24 h.The content inside the dialysis sack was concentrated and reduced with NaBH 4 overnight, and pH was adjusted to 5.5-7.0 with 0.1 mol/L acetic acid and further dialyzed against deionized water for 48 h.The dialysate was collected and concentrated to dryness by rotary evaporation at 50 ∘ C. Then 4.0 mL methanol was added and evaporated to dryness for four times.The resulting product was hydrolyzed with trifluoroacetic acid and aldononitrile acetate derivatives were prepared by procedures described in previous section and subjected to GC-MS analysis.

Methylation Analysis.
Methylation of S-3-1 sample was performed according to the method of Ciucanu and Kerek [19], with minor modifications.An S-3-1 sample was methylated with CH 3 I in dimethyl sulfoxide in the presence of dry sodium hydroxide.2 mL distilled water was added to stop the reaction and the methylated sample was then extracted with 2 mL methyl dichloride.The methylene chloride extract was separated by centrifugation and evaporation.The disappearance of the OH band (3200 cm −1 to 3700 cm −1 ) in FT-IR spectrum was used to confirm complete methylation [18].Methyl derivatives were prepared subsequently as follows: hydrolyzing the premethylated polysaccharide sample by treatment with 88% formic acid solution (3 ml) at 100 ∘ C for (model 7890/5975C-GC/MSD, Agilent Technologies; Santa Clara, CA, USA).

NMR Analysis.
Dried polysaccharide sample (20 mg) was dissolved in 1 mL of D 2 O and kept at room temperature for 3 h. 13C and 1 H NMR spectra were recorded with a Bruker AMX-600 NMR spectrometer using sodium trimethylsilyl propionate (TSP) as internal standard, D 2 O as the solvent at 25 ∘ C, and the HOD signal fixed at 4.79 ppm.

Atomic Force Microscopy.
The molecular morphology of S-3-1 in solution was observed using a Nanonavi E-Sweep Environment Control Scanning Probe Microscope [20] (Seiko, Japanese).The polysaccharide (50 g/mL) was dissolved in deionized water with gentle stirring and serially diluted to 10 ng/mL.Next, a drop of 5 L polysaccharide solution was placed on the mica and dried at 25 ∘ C for at least 1 h.Imaging of samples was performed by the microscope in tapping mode.Nanoscope software was used to obtain and process AFM images.

Complement-Fixing Assay.
The complement-fixing assay was performed as previously described [21,22].Antibodysensitized sheep erythrocytes were washed three times with BBS containing 2.8 mM barbitone sodium and 0.8 mM barbital before use.The erythrocytes were prepared to a concentration of 2% cell suspension in BBS and used the same day.Guinea pig serum was diluted with BBS to a concentration giving about 50% hemolysis.Triplicate samples containing 50 L of each serially diluted polysaccharide fraction were mixed with 50 L diluted serum, added to microplate wells, and incubated at 37 ∘ C.After 10 min, sensitized sheep erythrocytes (100 L) and BBS (100 L) were added to each well, and the samples were incubated for an additional 30 min at 37 ∘ C.After centrifugation (2152 g for 10 min), 200 L of each supernatant was added in flat-bottom microplates, and absorbance was measured at 405 nm.100% lysis was obtained by adding distilled H 2 O to sensitized sheep erythrocytes.Samples containing BBS, serum, and sensitized sheep erythrocytes were used as background controls ( control ), while heparin served as a positive control.Inhibition of hemolysis induced by the test samples was calculated by the formula: [( control −  sample )/ control ] × 100%.A doseresponse curve (6-7 points) was constructed to calculate the concentration of test sample able to give 50% inhibition of hemolysis (CH 50 ).A low CH 50 meant high complementfixing activity.

Molecular Mass and Chemical Composition Analysis of
Homogeneous Polysaccharides Purified from S-3. Fractionation of polysaccharides S-3-1 and S-3-2 by Sephadex G-150 size-exclusion chromatography resulted in a HPGPC profile as shown in Figure 1.The single, symmetrically sharp peak showed by each polysaccharide in the HPGPC profile revealed that they were homogeneous polysaccharides.Based on the retention times of each polysaccharide, average molecular mass of S-3-1 and S-3-2 was estimated to be 13.5 × 10 4 Da and 7.1 × 10 4 Da, respectively.
The total sugar content of S-3-1 was higher (99.67%) than that of S-3-2 (97.42%).No protein content was detected in S-3-1 and S-3-2.The uronic acid content in the two polysaccharides was found to be 1.03% and 6.25%, respectively, suggesting that S-3-2 belonged to the class of acidic polysaccharides.

Structural Characterization of S-3-1.
In the ultraviolet spectra analysis S-3-1 did not display significant absorption  peak between wavelengths 280 nm and 260 nm (Figure 2), indicating that protein and nucleic acid impurity have been reduced to a minimum.The FT-IR spectra of S-3-1 revealed polysaccharide specific absorption bands at 3600-3200 cm −1 , 3000-2800 cm −1 , and 1400-1200 cm −1 (Figure 3).The strong broad absorption peak at 3429-3430 cm −1 was due to the hydroxyl stretching vibration of the polysaccharide, and the peak at 2923-2920 cm −1 was due to the C-H stretching vibration band.The peak at 1630 cm −1 was attributed to the bending vibration of O-H, and the signal at 1383-1384 cm −1 band was attributed to the stretching vibration of C-H bending in the CH 2 group [12,[23][24][25].The bands at 1148 cm −1 and 1116 cm −1 were attributed to the vibration of C-O.The weak 1028-1093 cm −1 band corresponded to C-O-C stretching in the pyranose ring.The absorption peaks at 870-860 cm −1 , indicated that mannose was present [16].The characteristic absorption band at 834 cm −1 suggested the presence of D-Glc pyranoside configurations in S-3-1 [26].
The locations of glycosidic linkages in the polysaccharides are preliminarily determined by periodate consumption and production of formic acid in periodate oxidation [27].As shown in Table 1, results from periodate oxidation showed that, per sugar residue, 1.04 mol of periodate was consumed and 0.45 mol formic acid was produced.The production of formic acid indicated that some of the residue may exist in the pyranohexose 1-linked or 1,6-linked form.Since the amount of periodate consumed was more than twice the amount of formic acid produced, it is possible that many linkages exist in the pyranose 1,2-linked, 1,4-linked, 1,2,6-linked, 1,4,6-linked, and 1,2,4-linked forms or furanose 1-linked forms.Moreover, the ratio of the amount of monosaccharide to amount of periodate was nearly 1 : 1 for polysaccharide S-3-1.This proved that the 1,3-linked forms that do not consume periodate were either not present or few in number [28].The periodateoxidized products of S-3-1 were further reduced, hydrolyzed, and analyzed by GC-MS.Results showed that the degradation process completely removed glucose, Rha, and Gal residues and produced a large amount of glycol, methylglycol, glycerol, and erythritol, indicating that these 4 glycoside residues may exist in the 1,4-linked, 1,4,6-linked, 1,2-linked, 1,2,6linked, or 1,6-linked forms, which could be oxidized.Ratios of Smith degradation products detected by GC-MS were Glycerol : Erythritol : Man : Gal = 5.57 : 0.82 : 0.60 : 0.28 in S-3-1.A large proportion of galactose and mannose were probably from 1,3-linkages, erythritol from 1 → 4 linkages, and glycerol from 1,6-and/or 1,2-linkages.
From Table 2, based on the results of the methylation analysis on S-3-1, the following conclusions can be drawn: (1) the Gal residues were present as 1,4-linked and 1,4,6linked Galp residues; (2) the Glc residues were present as terminal, 1,4-linked, and 1,6-linked Glcp residues; (3) the Rha residues were present as terminal; (4) the Man residues were present as 1,2-linked, 1,3-linked terminal, and 1,3,6linked Manp residue; (5) the Xyl residues were present as 1,5linked Xylf residue.These residues mentioned above except terminal Rhap, Glcp, and Xylf account for 93% of total   methylated sugars, suggesting that the backbone of S-3-1 may be composed of 1,6-linked Galp, 1,4-linked Glcp, and 1,2linked Manp residues, with branches attached to the O-6 of some of the 1,4,6-linked Galp and 1,3,6-linked Manp.This inference also agrees with the results obtained from periodate oxidation and Smith degradation analysis.600-MHz NMR spectra of S-3-1 are shown in Figure 4.The 1 H and 13 C-NMR spectra of polysaccharide S-3-1 were crowded in narrow regions ranging from 3.0 to 5.5 ppm ( 1 H NMR) and 60 to 110 ppm ( 13 C NMR) which were typical of polysaccharides [18].The 1 H NMR signals at 5.0-5.3ppm and 4.4-5.0ppm were assigned as the anomeric protons of the alpha-glycosidic configuration and beta-glycosidic configuration [18].The weak resonances at 1.3 and 1.26 ppm were attributed to the methyl group of Rha [12].In the anomeric region, the signals at 110.24 ppm were assigned to alpha-Araf, the signals at 105.1 and 98.35 ppm were assigned to beta-Glcp and alpha-Glcp, the signals at 101.27 and 101.24 ppm were assigned to beta-Galp [29], and the signals at 100.51 and 100.31 ppm were assigned to alpha-Manp.The signals for carbons of alpha-Araf and beta-Glc (1→6) were obviously stronger than those of beta-Galp and Glc [30]; this suggested that Ara and beta-Glc (1→6) residues were present in flexible side chains.The chemical shift at 19.4 ppm was assigned to the methyl group of Rha, which was consistent with the detected sugar compositions.
AFM is a powerful tool for directly observing the surface topography or morphology of macromolecule [31,32] and was used to provide direct evidence of the chain conformation of S-3-1 polysaccharide extracted from SJZD.The S-3-1 image taken from AFM is shown in Figure 5.The S-3-1 molecular chain was found to be a spherical distribution polysaccharide with winding coil and multiple nodules held together by intermolecular interactions.
The AFM figure of S-3-1 at different concentrations showed that the morphology of the polysaccharide changed with concentration.At high concentrations of the polysaccharide solution, the molecules wrapped around each other, held together by molecular interatomic forces and van der Waals forces.At low concentrations of the polysaccharide solution, the molecules curled into a globular structure.Figure 5(c) shows the images of the polysaccharide in water, captured at a concentration of 10 ng/mL.As expected, the polysaccharides that existed as flexible chains turned into the branched and knotted state as observed.This indicated that the morphology of the polysaccharide changed with change in the surrounding environment.Hence it was speculated that molecular interatomic forces and van der Waals forces play a major role in the morphology of polysaccharide chains.

Anticomplement Activities of S-3-1 and S-3-2.
The effects of polysaccharide S-3-1 in activation of human complement through the classical pathway were examined in 1 : 40-diluted NHS, using heparin as a reference and as shown in Table 3.The S-3-1 polysaccharide exhibited inhibitory effects on the classical pathway of the complement system, evidencing CH 50 values of 530 g/mL, which was close to the CH 50 value of heparin (500 g/mL).This showed that S-3-1 exhibited inhibitory effects comparable to heparin on the complement system.However, no anticomplement activity was detected for polysaccharide S-3-2.

Discussions and Conclusion
In this study, S-3, a polysaccharide fraction from SJZD, was purified, and the homogeneous polysaccharide S-3-1 separated from it.S-3-1 was assayed for immunological activities in vitro and structural characterization.The results showed that S-3-1 is comprised of Rha, Ara, Xyl, Man, Gal, and Glc and mainly contained 1,4-linked-Glc and 1,6-linked-Gal glycosidic bonds.It has been reported that macrophage phagocytosis activity of ginseng is due to a polysaccharide that is a type I rhamnogalacturonan (RG-I) pectin (WGPA-2-RG) with the arabinogalactan side chains [33].The Atractylodis macrocephalae Koidz polysaccharides are found to be composed of Rha, Ara, Xyl, Man, Glc, and Gal, with a backbone constructed by Glc and the branches constituted by Rha [34].It has also been reported that polysaccharides extracted from P. cocos and fractionated with size-exclusion column chromatography gave a water-soluble 1,6-branched 1,3-alpha-D-Gal structure [35].Polysaccharides from Liquorice roots have been found to contain about 81% carbohydrates and 19% protein, with the polysaccharide content to mainly contain Ara, Gal, Glc, and GlcA [9].This showed that the polysaccharides isolated from the four crude drugs comprising SJZD were different from S-3-1 which contains six kinds of monosaccharides.Thus, it was speculated that the active polysaccharide of SJZD originated from the decocting process of the four crude drugs.
The complement system is a vital part of the body's immune system and is important for maintaining health.The complement pathway plays an essential role in the host's defense against pathogens [36].Many active ingredients from TCM have been proved to possess widespread anticomplementary effect [37].In the present study of natural products, polysaccharide was found to be one of the best complement inhibitors.Our results have shown that the purified polysaccharide S-3-1 displayed a certain inhibitory effect on the complement system, with CH 50 values of 530 g/mL.It is evident that the homogeneous polysaccharide S-3-1 plays an important role in SJZD's immunologically activities.
In this study, for the first time, the immune active anti-inflammatory mechanism of polysaccharide was proved through anticomplement, and the structure of S-3-1 was characterized as well.Based on review of data from previous literature, it was found that the chemical composition of S-3-1 and sugar residue connection have not been reported yet, indicating that S-3-1 is produced from the decocted SJZD prescription process rather than from any of the four single herbs.Polysaccharide structure analysis of SJZD will provide a theoretical basis for research on the functions of TCM prescriptions in future.

Figure 5 :
Figure 5: AFM topographic image of S-3-1 deposited on mica.(a) At a concentration of 50 g/ml.(b) At a concentration of 1 g/ml.(c) At a concentration of 10 ng/ml.