Preliminary Characterization of a Homogeneous Polysaccharide with Anticomplement Activity from Sijunzi Decoction.
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 . 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 . Polysaccharide extracted from SJZD is an effective immunomodulator and immune adjuvant . There are also reports of other TCM that contain polysaccharides and include Ginseng Radix , Atractylodes Macrocephalae Rhizoma , Poria , and Glycyrrhizae Radix , 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 .
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 . 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 S3 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.
2. Materials and Methods
2.1. 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.
2.2. Extraction and Purification of Polysaccharide from SJZD. SJZPS (crude SJZD polysaccharide) and polysaccharide fraction S-3 were prepared as described previously . S-3 was further purified by gel filtration chromatography on a Sephadex G-150 column (16 mm x 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.
2.3. Determination of Molecular Mass and Analysis of Monosaccharide Composition of Polysaccharides
2.3.1. 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 x 300 mm, Tosoh Co., Japan) as described previously . Deionized water was used as the elution and flow rate was maintained at 1mL/min. A 20 [micro]L aliquot was injected for each run. The calibration curve of log(Mw) versus elution time (T) is given by log(Mw) = -0.1264T+ 5.6361.
2.3.2. General Analysis Methods. Carbohydrate content of the polysaccharide was determined by phenol-sulfuric acid colorimetric methods  using D-Glc as the standard. Protein content was measured by the Lowry method  using BSA as the standard. Uronic acid content was measured by photometry with m-hydroxydiphenyl at 525 nm using dgalacturonic acid as the standard .
2.3.3. 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 . 10 mg of each sample was hydrolyzed with 2mol/L trifluoroacetic acid at 95[degrees]C for 2h. 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[degrees]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[degrees]C for 30 min. After cooling, the resulting aldononitrile acetate derivatives were analyzed by GCMS. 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.
2.4. Structural Characterization of S-3-1
2.4.1. UV and IR Spectra Analysis. The impurity content in polysaccharide was determined by ultraviolet spectra analysis. Absorbance of wavelengths in the 200-400 nm regions was recorded with a UV spectrophotometer. The organic functional groups of polysaccharide were identified by Fourier transform infrared (FT-IR) method. Polysaccharide (2 mg) was dried at 35-45[degrees]C in vacuum over [P.sub.2][O.sub.5] for 48 h prior to grinding with spectroscopic grade KBr powder and then pressed into a 1mm pellet for FT-IR measurement by a Thermo Fisher Nicolet 6700 FT-IR spectrometer .
2.4.2. Periodate Oxidation and Smith Degradation. S-3-1 sample (25 mg) was oxidized with 15 mM NaI[O.sub.4] (25 mL) and kept in the dark at 4[degrees]C. At 12 h intervals, 30 [micro]L aliquots were taken, diluted 250 times with distilled water, and read by spectrophotometry at 223 nm . The amount of NaI[O.sub.4] consumed was calculated according to the NaI[O.sub.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 NaB[H.sub.4] overnight, and pH was adjusted to 5.5-70 with 0.1mol/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[degrees]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.
2.4.3. Methylation Analysis. Methylation of S-3-1 sample was performed according to the method of Ciucanu and Kerek , with minor modifications. An S-3-1 sample was methylated with CH3I in dimethyl sulfoxide in the presence of dry sodium hydroxide. 2mL 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.sup.-1] to 3700 [cm.sup.-1]) in FTIR spectrum was used to confirm complete methylation . Methyl derivatives were prepared subsequently as follows: hydrolyzing the premethylated polysaccharide sample by treatment with 88% formic acid solution (3 ml) at 100[degrees]C for 3h and 2M TFA (3mL) at 100[degrees]C for 6h. The hydrolysate was reduced with sodium borodeuteride (NaB[H.sub.4]) solution (5% in water) at room temperature (25[degrees]C) for 4 h and acetylation with acetic anhydride [(Ac.sub.2]O) at 100[degrees]C for 1 h. The resulting methylated derivatives were detected by GC-MS (model 7890/5975C-GC/MSD, Agilent Technologies; Santa Clara, CA, USA).
2.4.4. NMR Analysis. Dried polysaccharide sample (20 mg) was dissolved in 1mL of [D.sub.2]O and kept at room temperature for 3 h. [sup.13]C and [sup.1]H NMR spectra were recorded with a Bruker AMX-600 NMR spectrometer using sodium trimethylsilyl propionate (TSP) as internal standard, [D.sub.2]O as the solvent at 25[degrees] C, and the HOD signal fixed at 4.79 ppm.
2.4.5. Atomic Force Microscopy. The molecular morphology of S-3-1 in solution was observed using a Nanonavi ESweep Environment Control Scanning Probe Microscope  (Seiko, Japanese). The polysaccharide (50 [micro]g/mL) was dissolved in deionized water with gentle stirring and serially diluted to 10 ng/mL. Next, a drop of 5 [micro]L polysaccharide solution was placed on the mica and dried at 25[degrees]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.
2.5. Complement-Fixing Assay. The complement-fixing assay was performed as previously described [21, 22]. Antibody-sensitized 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 [micro]L of each serially diluted polysaccharide fraction were mixed with 50 [micro]L diluted serum, added to microplate wells, and incubated at 37[degrees]C. After 10 min, sensitized sheep erythrocytes (100 [micro]L) and BBS (100 [micro]L) were added to each well, and the samples were incubated for an additional 30 min at 37[degrees]C. After centrifugation (2152 g for 10 min), 200 [micro]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.sub.2]O to sensitized sheep erythrocytes. Samples containing BBS, serum, and sensitized sheep erythrocytes were used as background controls ([A.sub.control]), while heparin served as a positive control. Inhibition of hemolysis induced by the test samples was calculated by the formula: [([A.sub.control] - [A.sub.sample])/[A.sub.control] x 100%. A dose-response curve (6-7 points) was constructed to calculate the concentration of test sample able to give 50% inhibition of hemolysis (C[H.sub.50]). A low C[H.sub.50] meant high complement-fixing activity.
3.1. 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 x [10.sup.4] Da and 7.1 x [10.sup.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.
Alditol acetates of each polysaccharide hydrolysate were subjected to GC-MS analysis. The neutral monosaccharide composition of S-3-1, based on retention times and peak areas, was determined to be Rha, Ara, Xyl, Man, Gal, and Glc in molar ratio 0.35 : 0.37 : 1.4 : 0.31 : 3 : 0.8. The monosaccharide composition of S-3-2 was determined to be Rha, Ara, Xyl, Man, Gal, and Glc in molar ratio 0.75: 0.24 : 1.2: 0.17: 0.89 : 0.23. Thus, it was concluded that S3-1 and S-3-2 are heteropolysaccharides with the same kind of monosaccharide composition.
3.2. 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.sup.-1], 3000-2800 [cm.sup.-1], and 1400-1200 [cm.sup.-1] (Figure 3). The strong broad absorption peak at 3429-3430 [cm.sup.-1] was due to the hydroxyl stretching vibration of the polysaccharide, and the peak at 2923-2920 [cm.sup.-1] was due to the C-H stretching vibration band. The peak at 1630 [cm.sup.-1] was attributed to the bending vibration of O-H, and the signal at 13831384 [cm.sup.-1] band was attributed to the stretching vibration of C-H bending in the CH2 group [12, 23-25]. The bands at 1148 [cm.sup.-1] and 1116 [cm.sup.-1] were attributed to the vibration of C-O. The weak 1028-1093 [cm.sup.-1] band corresponded to CO-C stretching in the pyranose ring. The absorption peaks at 870-860 [cm.sup.-1], indicated that mannose was present . The characteristic absorption band at 834 [cm.sup.-1] suggested the presence of D-Glc pyranoside configurations in S-3-1 .
The locations of glycosidic linkages in the polysaccharides are preliminarily determined by periodate consumption and production of formic acid in periodate oxidation . 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 . 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 ofglycol, methylglycol, glycerol, and erythritol, indicating that these 4 glycoside residues may exist in the 1,4-linked, 1,4,6-linked, 1,2-linked, 1,2,6-linked, 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 S3-1. A large proportion of galactose and mannose were probably from 1,3-linkages, erythritol from 1[right arrow] 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 1,3,6-linked Manp residue; (5) the Xyl residues were present as 1,5-linked 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 1,2-linked 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 [sup.1]H and [sup.13]C-NMR spectra of polysaccharide S-3-1 were crowded in narrow regions ranging from 3.0 to 5.5 ppm ([sup.1]H NMR) and 60 to 110 ppm ([sup.13]C NMR) which were typical of polysaccharides . The [sup.1]H NMR signals at 5.0-5.3 ppm and 4.4-5.0ppm were assigned as the anomeric protons of the alpha-glycosidic configuration and beta-glycosidic configuration . The weak resonances at 1.3 and 1.26 ppm were attributed to the methyl group of Rha . 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 , 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 [right arrow] 6) were obviously stronger than those of beta-Galp and Glc ; this suggested that Ara and beta-Glc (1 [right arrow] 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-31 image taken from AFM is shown in Figure 5. The S-31 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 10ng/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.
3.3. 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 C[H.sub.50] values of 530 [micro]g/mL, which was close to the C[H.sub.50] value of heparin (500 [micro]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.
4. Discussions and Conclusion
In this study, S-3, a polysaccharide fraction from SJZD, was purified, and the homogeneous polysaccharide S-31 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 . 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 . 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 . 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 . 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 . Many active ingredients from TCM have been proved to possess widespread anticomplementary effect . 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 C[H.sub.50] values of 530 [micro]g/mL. It is evident that the homogeneous polysaccharide S-3-1 plays an important role in SJZDs 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-31 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.
Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.
This work was supported by the National Natural Science Foundation of China (no. 81473318).
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Ruijun Wang (iD), Yunfei Ji (iD), Ying Peng (iD), and Xiaobo Li (iD)
School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China
Correspondence should be addressed to Xiaobo Li; firstname.lastname@example.org
Received 27 August 2017; Revised 14 November 2017; Accepted 5 December 2017; Published 16 January 2018
Academic Editor: Wieslaw Wiczkowski
Caption: Figure 1: HPGPC profile of polysaccharide S-3-1 and S-3-2.
Caption: Figure 2: UV spectra of S-3-1.
Caption: Figure 3: FT-IR spectra of S-3-1.
Caption: Figure 4: 600-MHz NMR spectra of S-3-1 in [D.sub.2]O. (a) [sup.1]H NMR spectrum of S-3-1. (b) [sup.13]C NMR spectrum of S-3-1.
Caption: Figure 5: AFM topographic image of S-3-1 deposited on mica. (a) At a concentration of 50 [micro]g/ml. (b) At a concentration of 1 [micro]g/ml. (c) At a concentration of 10 ng/ml.
Table 1: Results of periodate oxidation/Smith degradation of S-3-1. Linkage Consumption Yield offormic Products of ofperiodate acid (mol/mol Smith (mol/mol Glc) Glc) degradation 1 [right arrow] 2 1 nd Glycerol 1 [right arrow] 3 nd nd Gal/Man 1 [right arrow] 4 1 nd Erythritol 1 [right arrow] 6 2 1 Glycerol 1.047 0.4533 Glycerol: Erythritol: Man: Gal = 5.57: 0.82 : 0.60: 0.28 Table 2: Methylation analysis of S-3-1. Retention Linkage Methylated sugar Molar time (min) ratios 9.433 1 [right arrow] 6 2,3,4-[Me.sub.3]-Glc 1.45 11.036 1 [right arrow] 2 3,4,6-[Me.sub.3]-Man 4.04 12.909 1 [right arrow] 2,3,4,6-[Me.sub.4]-Glc 2.55 13.199 1 [right arrow] 2,3,4-[Me.sub.3]-Rha 1.6 13.385 1 [right arrow] 3 2,4,6-[Me.sub.3]-Man 2.28 14.287 1 [right arrow] 4 2,3,6-[Me.sub.3]-Glc 9.49 14.499 1 [right arrow] 4,6 2,3-[Me.sub.2]-Gal 2.16 14.614 1 [right arrow] 6 2,3,4-[Me.sub.3]-Gal 38.3 16.681 1 [right arrow] 3,6 2,4-[Me.sub.2]-Man 3.16 33.526 1 [right arrow] 2,3,4-[Me.sub.3]-Xyl 0.32 Table 3: Anticomplement activity of two polysaccharides (S-3-1 and S-3-2) isolated from SJZD through the classical pathway. Compounds C[H.sub.50] ([micro]g/mL) S-3-1 530 [+ or -] 20 S-3-2 NA Heparin 500 [+ or -] 17 Data are expressed as mean [+ or -] SD of triplicate measurements; NA indicated no inhibitory effect. Heparin was used as the positive control.
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|Title Annotation:||Research Article|
|Author:||Wang, Ruijun; Ji, Yunfei; Peng, Ying; Li, Xiaobo|
|Publication:||Journal of Chemistry|
|Date:||Jan 1, 2018|
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