A Pectic Polysaccharide from Sijunzi Decoction Promotes the Antioxidant Defenses of SW480 Cells

Sijunzi Decoction (SJZD) is a formula used for the treatment of spleen deficiency and gastrointestinal diseases in Traditional Chinese Medicine. Polysaccharides are reported to be the main components of SJZD responsible for its bio-functions. However, highly purified and clearly characterized polysaccharides from SJZD are not well described. Here we obtained a purified polysaccharide (SJZDP-II-I) from SJZD using ion exchange chromatography and gel filtration. Structure analysis by FT-IR and NMR identified SJZDP-II-I as a typical pectic polysaccharide with homogalacturonan and rhamnogalacturonan type I regions and arabinogalactan type I and II as side chains. In vitro studies indicated that SJZDP-II-I treatment could significantly enhance the total antioxidant capacity of SW480 cells, resulting from the promoted expressions of antioxidant enzymes and their master regulator PGC-1α, which would be valuable for further research and applications.


Introduction
As a conventional Traditional Chinese Medicine Formula (TCMF), Sijunzi Decoction (SJZD), consisting of four crude herbs (Panax ginseng, Atractylodes macrocephala, Poria cocos and Glycyrrhiza uralensis) in the ratio of 3:3:3:2, has been widely applied for over one thousand years in the treatment of gastrointestinal diseases, such as stomach aches, rugitus, nausea, vomiting and diarrhea [1,2]. Modern pharmacological studies have further revealed its potential effects on the immune system activation and cancer treatment [1,3,4]. Due to the expensive price of Panax ginseng, Codonopsis pilosula, with similar biological activities, has been used as a substitute for more than hundreds of years in SJZD [5,6]. Flavonoids, coumarins, triterpenoid and polysaccharides were reported to be the main components of SJZD [7], and polysaccharides were considered as the most abundant and effective component [4].
The functions of polysaccharides from each herb of SJZD were widely reported. C. pilosula polysaccharides showed several bioactivities, such as enhancement of lymphocyte proliferation [8],

Fractionation of Pectic Polysaccharides from SJZDP
The crude water extracted SJZDP (200 mg) was subjected to anion exchange chromatography, whereby two acidic fractions, SJZDP-I (25 mg) and SJZDP-II (54 mg), were obtained. These two acidic fractions were further fractionated using gel filtration. After separation on a gel filtration column, SJZDP-I-I (8 mg) and SJZDP-I-II (13 mg) were obtained from SJZDP-I, and one fraction (SJZDP-II-I, 41 mg) was purified from SJZDP-II ( Figure 1). SJZDP-II-I was chosen for structure elucidation as the other two fractions showed no activity in promoting the antioxidant defenses of SW480 cells (data not shown).

Chemical Composition of Polysaccharide
The monosaccharide composition of the isolated polysaccharide fractions was determined by GC analysis after methanolysis and TMS-derivatization. As shown in Table 1, the monosaccharide compositions of the three isolated fractions were quite different. Fraction SJZDP-I-I was mainly composed of arabinose (Ara), galactose (Gal) and glucose (Glc), accounting for 86.0 mol % of the total carbohydrates, while the fraction SJZDP-I-II mainly consisted of Ara (18.9 mol %) and Glc (66.9 mol %). Compared to fraction SJZDP-I-I and SJZDP-I-II, the fraction SJZDP-II-I contained a higher amount of uronic acids. The fraction SJZDP-II-I was mainly composed of Ara, Gal and GalA, which accounted for 90.7 mol % of the total carbohydrates. The differences in monosaccharide composition between fractions SJZDP-I-I, SJZDP-I-II and SJZDP-II-I may explain the different activities in promoting the antioxidant defenses of SW480 cells. The obtained monosaccharide compositions of the polysaccharide fractions are different from those of previous reports, as SJZPS-Vb-1 and SJZPS-Vb-2 were mainly composed of Glc, Gal and mannose (Man) [23], and such differences may result from the different methods of extraction and purification. As SJZDP Fraction SJZDP-I-I was mainly composed of arabinose (Ara), galactose (Gal) and glucose (Glc), accounting for 86.0 mol % of the total carbohydrates, while the fraction SJZDP-I-II mainly consisted of Ara (18.9 mol %) and Glc (66.9 mol %). Compared to fraction SJZDP-I-I and SJZDP-I-II, the fraction SJZDP-II-I contained a higher amount of uronic acids. The fraction SJZDP-II-I was mainly composed of Ara, Gal and GalA, which accounted for 90.7 mol % of the total carbohydrates. The differences in monosaccharide composition between fractions SJZDP-I-I, SJZDP-I-II and SJZDP-II-I may explain the different activities in promoting the antioxidant defenses of SW480 cells. The obtained monosaccharide compositions of the polysaccharide fractions are different from those of previous reports, as SJZPS-Vb-1 and SJZPS-Vb-2 were mainly composed of Glc, Gal and mannose (Man) [23], and such differences may result from the different methods of extraction and purification. As SJZDP is a mixture of four herbs, the obtained monosaccharide compositions of polysaccharides may derive from different herbs. The monosaccharide Ara, Gal, Rha, Man and xylose (Xyl), may come from any of the four herbs, since the polysaccharides from all four of these herbs contained these monosaccharides [11,[24][25][26]. Additionally, a previous report on polysaccharides from P. cocos indicated that the Glc present in SJZDP came mainly from P. cocos [27].

Determination of the Glycosidic Linkages
The purified fraction SJZDP-II-I was analyzed for the types of glycosidic linkages using GC-MS after conversion to partially methylated alditol acetates ( Table 2). As shown in the table, fraction SJZDP-II-I contained linkage units typical of pectic polysaccharides. The presence of 1,4-linked GalA indicates the presence of a homogalacturonan (HG) backbone, while 1,4-linked GalA with 1,2 and 1,2,4-linked Rha indicates a rhamnogalacturonan I region (RG-I) in the polymers [28]. The highly branched RG-I is known as the "hairy region" of the pectin, which normally consists of branching chains, such as arabinans, galactans and/or arabinogalactans (AG) [29]. Fraction SJZDP-II-I contained a high amount of 1,4-linked Gal units, suggesting that the polymers are rich in arabinogalactan type I (AG-I) structures, and the presence of 1,3-; 1,6-; and 1,3,6-linked Gal indicates the presence of arabinogalactan type II (AG-II) structures. The linkage units of fraction SJZDP-II-I were different from those of fractions SJZPS-Vb-1 and SJZPS-Vb-2 from SJZD, as they contain different monosaccharide compositions [23]. However, for the linkage types of Gal, fraction SJZDP-II-I contained similar linkage types with fractions SJZPS-Vb-1 and SJZPS-Vb-2, such as terminal linked, 1,6-linked and 1,3,6-linked Gal. The linkage type of fraction SJZDP-II-I was similar to those of polysaccharides from C. pilosula, but with a different ratio [11].

Structural Features
As shown in Figure 2, the FT-IR chromatogram of fraction SJZDP-II-I showed characteristic absorptions of polysaccharides [30]. The absence of absorption bands at 1735 cm −1 and 1250 cm −1 indicated that the GalA units of fraction SJZDP-II-I were not esterified [31,32]. These were different from the polysaccharides isolated from C. pilosula [11] and G. uralensis [26], while polysaccharides from C. pilosula contain bands at 1735 cm −1 and 1250 cm −1 , and polysaccharides from G. uralensis only contain a 1735 cm −1 band. pectin, which normally consists of branching chains, such as arabinans, galactans and/or arabinogalactans (AG) [29]. Fraction SJZDP-II-I contained a high amount of 1,4-linked Gal units, suggesting that the polymers are rich in arabinogalactan type I (AG-I) structures, and the presence of 1,3-; 1,6-; and 1,3,6-linked Gal indicates the presence of arabinogalactan type II (AG-II) structures. The linkage units of fraction SJZDP-II-I were different from those of fractions SJZPS-Vb-1 and SJZPS-Vb-2 from SJZD, as they contain different monosaccharide compositions [23]. However, for the linkage types of Gal, fraction SJZDP-II-I contained similar linkage types with fractions SJZPS-Vb-1 and SJZPS-Vb-2, such as terminal linked, 1,6-linked and 1,3,6-linked Gal. The linkage type of fraction SJZDP-II-I was similar to those of polysaccharides from C. pilosula, but with a different ratio [11].

Structural Features
As shown in Figure 2, the FT-IR chromatogram of fraction SJZDP-II-I showed characteristic absorptions of polysaccharides [30]. The absence of absorption bands at 1735 cm −1 and 1250 cm −1 indicated that the GalA units of fraction SJZDP-II-I were not esterified [31,32]. These were different from the polysaccharides isolated from C. pilosula [11] and G. uralensis [26], while polysaccharides from C. pilosula contain bands at 1735 cm −1 and 1250 cm −1 , and polysaccharides from G. uralensis only contain a 1735 cm −1 band. The polysaccharide fraction SJZDP-II-I was also characterized by 1D NMR spectroscopy and the chemical shift values compared with data from the literature [11,[33][34][35][36]. Typically, the anomeric 1 H signals of α-pyranoside are found over 5 ppm while those of β-pyranoside are at less than 5 ppm [37]. As shown in Figure 3, the 1 H-NMR spectrum of polysaccharide fraction SJZDP-II-I contained five main anomeric protons at 5.11, 5.04, 5.02, 4.95 and 4.51 ppm, indicating the existence of both α-and β-configurations in the polysaccharide fraction SJZDP-II-I. The weak proton signal at 1.18 and 1.26 ppm indicated the presence of a methyl group of Rha in the fraction [35]. The 13 C signals at 175.74 ppm corresponded to carbonyl carbon of unesterified α-1, 4-GalpA, and the 103.14 ppm corresponded to C-1 of α-1, 4-GalpA. Other signals such as 107.29 ppm corresponded to α-L-Araf, while 106.80 ppm, 106.33 ppm and 104.29 ppm were corresponded to β-D-Galp [33]. The signals at 16.75 ppm represented the C-6 of α-L-Rha [36]. These results suggested that fraction SJZDP-II-I was The polysaccharide fraction SJZDP-II-I was also characterized by 1D NMR spectroscopy and the chemical shift values compared with data from the literature [11,[33][34][35][36]. Typically, the anomeric 1 H signals of α-pyranoside are found over 5 ppm while those of β-pyranoside are at less than 5 ppm [37]. As shown in Figure 3, the 1 H-NMR spectrum of polysaccharide fraction SJZDP-II-I contained five main anomeric protons at 5.11, 5.04, 5.02, 4.95 and 4.51 ppm, indicating the existence of both αand β-configurations in the polysaccharide fraction SJZDP-II-I. The weak proton signal at 1.18 and 1.26 ppm indicated the presence of a methyl group of Rha in the fraction [35]. The 13 C signals at 175.74 ppm corresponded to carbonyl carbon of unesterified α-1, 4-GalpA, and the 103.14 ppm corresponded to C-1 of α-1, 4-GalpA. Other signals such as 107.29 ppm corresponded to α-L-Araf, while 106.80 ppm, 106.33 ppm and 104.29 ppm were corresponded to β-D-Galp [33]. The signals at 16.75 ppm represented the C-6 of α-L-Rha [36]. These results suggested that fraction SJZDP-II-I was a typical pectic polysaccharide, with HG regions, RG-I regions and AG-I/AG-II side chains, which was consistent with the monosaccharide composition results.

SJZDP-II-I Treatment Didn't Affect the Proliferation of SW480 Cells
Total SJZDP was reported to enhance cell proliferation [15], but no such activity of was found in the fraction SJZDP-II-I of this study. SW480 cells, derived from colon adenocarcinoma, exhibited similar proliferation rates after SJZDP-II-I treatment under different doses. Additionally, SJZDP-II-I exhibited no cellular toxicity at the concentration up to 40 µg/mL ( Figure 4).

SJZDP-II-I Treatment Didn't Affect the Proliferation of SW480 Cells
Total SJZDP was reported to enhance cell proliferation [15], but no such activity of was found in the fraction SJZDP-II-I of this study. SW480 cells, derived from colon adenocarcinoma, exhibited similar proliferation rates after SJZDP-II-I treatment under different doses. Additionally, SJZDP-II-I exhibited no cellular toxicity at the concentration up to 40 µg/mL (Figure 4).

SJZDP-II-I Treatment Didn't Affect the Proliferation of SW480 Cells
Total SJZDP was reported to enhance cell proliferation [15], but no such activity of was found in the fraction SJZDP-II-I of this study. SW480 cells, derived from colon adenocarcinoma, exhibited similar proliferation rates after SJZDP-II-I treatment under different doses. Additionally, SJZDP-II-I exhibited no cellular toxicity at the concentration up to 40 µg/mL (Figure 4).

SJZDP-II-I Promoted the Antioxidant Defense of SW480 Cells
SAs SJZD is widely used in gastrointestinal disorders, so it was reasonable to expect that SJZDP-II-I would affect the functions of these organs [1,2]. Total SJZD polysaccharides have been reported to promote the restoration of intestinal function by regulating intestinal homeostasis [2]. A growing body of evidence indicates that cellular oxidative stress and redox status are widely implicated in intestinal homeostasis, affecting cellular proliferation, apoptosis, metabolic processes, as well as the balance of intestinal microecology [21,38,39]. In this study, we found that SJZDP-II-I treatment could remarkably increase the total antioxidant capacity (TAC) of SW480 cells, but little antioxidant capacity of SJZDP-II-I itself was detected according to an in vitro assay ( Figure 5). Thus, the enhanced TAC may result from the increased levels of antioxidants induced by the enhancement of gene expression in SW480 cells themselves, but not SJZDP-II-I. In order to clarify this, the expressions of representative antioxidant enzymes were evaluated, showing a robust increase of these enzymes after SJZDP-II-I treatment, as well as the master regulator of antioxidant defense-PGC-1α ( Figure 5). These results indicated that SJZDP-II-I could protect SW480 cells from oxidative stress through the promotion of antioxidant enzymes' expression, and suggested that SJZDP-II-I may participate in the regulation of intestinal homeostasis through the redox status control of the intestine.

SJZDP-II-I Promoted the Antioxidant Defense of SW480 Cells
SAs SJZD is widely used in gastrointestinal disorders, so it was reasonable to expect that SJZDP-II-I would affect the functions of these organs [1,2]. Total SJZD polysaccharides have been reported to promote the restoration of intestinal function by regulating intestinal homeostasis [2]. A growing body of evidence indicates that cellular oxidative stress and redox status are widely implicated in intestinal homeostasis, affecting cellular proliferation, apoptosis, metabolic processes, as well as the balance of intestinal microecology [21,38,39]. In this study, we found that SJZDP-II-I treatment could remarkably increase the total antioxidant capacity (TAC) of SW480 cells, but little antioxidant capacity of SJZDP-II-I itself was detected according to an in vitro assay ( Figure 5). Thus, the enhanced TAC may result from the increased levels of antioxidants induced by the enhancement of gene expression in SW480 cells themselves, but not SJZDP-II-I. In order to clarify this, the expressions of representative antioxidant enzymes were evaluated, showing a robust increase of these enzymes after SJZDP-II-I treatment, as well as the master regulator of antioxidant defense-PGC-1α ( Figure 5). These results indicated that SJZDP-II-I could protect SW480 cells from oxidative stress through the promotion of antioxidant enzymes' expression, and suggested that SJZDP-II-I may participate in the regulation of intestinal homeostasis through the redox status control of the intestine.

Preparation of Polysaccharides
Sijunzi Decoction (SJZD) was derived from four herbs, C. pilosula, G. uralensis, P. cocos and A. macrocephala, in a ratio of 3:3:3:2 by weight. All prepared slices of these four herbs were purchased from Fushoutang Pharmacy (Chengdu, China). SJZD was used for the preparation of polysaccharides (SJZDP) as described previously [16]. Dried herbs (55 g) were soaked in distilled water (440 mL) for 30 min at room temperature and then extracted twice at 100 °C for 4 h. The extract fractions were combined and concentrated by rotary evaporation. The concentrated extracts were subjected for dialysis (3500 Da cut-off, Beijing Rui Da Heng Hui Science Technology Development Co. Ltd., Beijing, China) in running water for 2 days. After dialysis, the extracts were lyophilized, and named SJZDP.
The SJZDP was purified by anion exchange chromatography and gel filtration. First, the SJZDP (200 mg) was dissolved in distilled water (10 mL), filtered through a 0.45 µm filter and applied to a DEAE Sepharose (Fast Flow, FF) column (5 cm × 40 cm, Beijing Rui Da Heng Hui Science Technology Development Co. Ltd.). The neutral fraction was eluted with distilled water at 2 mL/min, while the acidic fractions were eluted with a linear NaCl gradient in water (0-1.5 M) at 2 mL/min. The elution profiles were monitored using the phenol-sulfuric acid assay [40]. The related fractions were pooled,

Preparation of Polysaccharides
Sijunzi Decoction (SJZD) was derived from four herbs, C. pilosula, G. uralensis, P. cocos and A. macrocephala, in a ratio of 3:3:3:2 by weight. All prepared slices of these four herbs were purchased from Fushoutang Pharmacy (Chengdu, China). SJZD was used for the preparation of polysaccharides (SJZDP) as described previously [16]. Dried herbs (55 g) were soaked in distilled water (440 mL) for 30 min at room temperature and then extracted twice at 100 • C for 4 h. The extract fractions were combined and concentrated by rotary evaporation. The concentrated extracts were subjected for dialysis (3500 Da cut-off, Beijing Rui Da Heng Hui Science Technology Development Co. Ltd., Beijing, China) in running water for 2 days. After dialysis, the extracts were lyophilized, and named SJZDP.
The SJZDP was purified by anion exchange chromatography and gel filtration. First, the SJZDP (200 mg) was dissolved in distilled water (10 mL), filtered through a 0.45 µm filter and applied to a DEAE Sepharose (Fast Flow, FF) column (5 cm × 40 cm, Beijing Rui Da Heng Hui Science Technology Development Co. Ltd.). The neutral fraction was eluted with distilled water at 2 mL/min, while the acidic fractions were eluted with a linear NaCl gradient in water (0-1.5 M) at 2 mL/min. The elution profiles were monitored using the phenol-sulfuric acid assay [40]. The related fractions were pooled, dialyzed at cut-off 3500 Da against distilled water to remove of NaCl, and lyophilized. Second, the acidic fractions (50 mg) were dissolved in elution buffer (10 mM NaCl), filtered through a Millipore filter (0.45 µm), and subjected to gel filtration after application on a Sepharose 6FF column (2.5 cm × 100 cm, Beijing Rui Da Heng Hui Science Technology Development Co. Ltd., Beijing, China, and eluted with 10 mM NaCl at 1.0 mL/min. Fractions were pooled based on the elution profile, as determined by the phenol-sulfuric acid assay, dialyzed and lyophilized.

Chemical Compositions and Linkage Determination
The monosaccharide compositions of the fractions was determined by gas chromatography of the trimethylsilylated (TMS) derivatives of the methyl glycosides obtained after methanolysis with 3 M hydrochloric acid in anhydrous methanol for 24 h at 80 • C [41]. Mannitol was used as an internal standard. The TMS derivatives were analyzed by capillary gas chromatography on a Focus GC (Thermo Scientific, Milan, Italy). Glycosidic linkage elucidation was performed by methylation studies. Prior to methylation, the uronic acids at polymeric level were reduced with NaBD 4 to their corresponding neutral sugars [42]. After reduction of the polymers, methylation, hydrolysis, reduction and acetylation were carried out [43]. The derivatives were analyzed by GC-MS using a GCMS-QP2010 (Shimadzu, Kyoto, Japan) attached to a Restek Rxi-5MS column (30 m; 0.25 mm i.d.; 0.25 µm film). The injector temperature was 280 • C, the ion source temperature 200 • C and the interface temperature 300 • C. The column temperature was 80 • C when sample was injected, then increased 10 • C/min to 140 • C, followed by 4 • C/min to 210 • C and then 20 • C/min to 300 • C. Helium was the carrier gas (pressure control: 80 kPa). The compound corresponding to each peak was characterized by the interpretation of the retention times and the characteristic mass spectra. The estimation of the relative amounts of each linkage type was related to the total amount of each monosaccharide type as determined by methanolysis [44].

Molecular Weight Determination
The homogeneity and molecular weight of the native purified polysaccharide fractions were determined by size exclusion chromatography on a Hiload TM 16/60 Superdex TM 200 prep grade column (GE Healthcare, Uppsala, Sweden) combined with the Äkta system (FPLC, Pharmacia Äkta, Amersham Pharmacia Biotech, Uppsala, Sweden). Dextran polymers (Pharmacia) of 10, 40, 70, 500 and 2000 kDa were used as calibration standards [45].

FT-IR and NMR Spectroscopy
Approximately 1 mg of polysaccharide sample was mixed with 150 mg of dried KBr powder, and pressed into a 1 mm thick disk for the analysis using a PerkinElmer FT-IR spectrophotometer (PerkinElmer, Waltham, MA, USA). The IR spectra were recorded in the range of 4000-400 cm −1 [46]. 1 H-NMR and 13 C-NMR spectra of two main polysaccharides fractions were recorded in D 2 O solution at 25 • C on an AV600 spectrometer (600 MHz, Bruker, Rheinstetten, Germany) after deuterium exchange three times by freeze-drying in D 2 O.

Cell Culture
SW480 cells were routinely cultured in DMEM (Gibco, Waltham, MA, USA), supplemented with 10% FBS in an incubator under an atmosphere of 5% CO 2 at 37 • C. Cells were plated in 6-well cell plates (1 × 10 6 cells per well); 12 h after the polysaccharide supplement, cells were collected for gene expression detection and ABTS Radical Scavenging Activity Assay. Meanwhile, cell viability was assayed 12 h after the polysaccharide treatment in 96-well cell plate (5 × 10 4 cells per well), using CCK-8 kit.

Cell Viability Assay
Cell viability was measured by Cell Counting Kit-8 (CCK-8) system (Dojindo, CK04-11, Minato-ku, Tokyo, Japan) according to the manufacturer's instructions. Briefly, CCK-8 solution (10 µL per 100 µL of medium in each well) was added, and the plates were then incubated at 37 • C for 1 h. The absorbance of each well was read at 450 nm using a microplate reader (Thermo, Waltham, MA, USA).

Quantitative Realtime PCR
RNA extraction from the intestine and real time PCR for antioxidant genes detection were performed as previously reported [47]. Briefly, SW480 cells were lysed with Trizol Regent (Invitrogen, Waltham, MA, USA), and the total RNA was extracted from the tissues according to the manufacturer's instructions. The quality of RNA was assessed by agarose gel and the concentration was measured with a spectrophotometer (NanoDrop 2000, Thermo Scientific, Shanghai, China). Total RNA was subjected to reverse transcription with a reverse transcriptase, according to the manufacturer's instructions (Fermentas, Waltham, MA, USA). Quantitative real-time PCR was performed using the Bio-Rad CFX96 system, and the relative gene expression was normalized to the internal control actin. Primer sequences for SYBR Green probes of the target genes are described in Table 3. Table 3. qRT-PCR primers for antioxidant defense genes.

ABTS Radical Scavenging Activity Assay
The total antioxidant capacity was evaluated using the ABTS method (Beyotime, Shanghai, China). ABTS radical cation (ABTS + ) solution was produced by reacting ABTS stock solution with 2.45 mmol/L potassium persulfate in the dark at room temperature for 12-16 h before use, then it was diluted with 80% ethanol to adjust the absorbance to 0.70 ± 0.05 at 734 nm. After that, 10 µL of sample or Trolox standard was added into 200 µL of diluted ABTS + solution. The absorbance at 734 nm was measured after 2-6 min in the dark at room temperature. Trolox, a water-soluble analogue of vitamin E, was used as the reference standard to prepare a calibration curve with a concentration range of 0.15-1.5 mM. Results were expressed as mmol/g Trolox equivalent antioxidant capacity.

Statistical Analysis
Data represent the means and SD. One-way ANOVA and post hoc tests were performed for all statistical significance analysis using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). * p < 0.05, ** p < 0.01, *** p < 0.001.

Conclusions
In summary, a purified polysaccharide from SJZD was obtained by using ion exchange chromatography and gel filtration. Structural analysis by FT-IR and NMR identified SJZDP-II-I as a typical pectic polysaccharide. Moreover, this SJZDP-II-I was shown to effectively promote the antioxidant defense capability in SW480 cells, which will be valuable for further research and applications.