The chemical reagents were all of the analytical grades. R. chingii Hu was obtained from Guangxi province in China. The reference standards of glucose, galactose, rhamnose, xylose, arabinose, fucose, mannose, glucuronic acid, galacturonic acid, 1-phenyl-3-methyl-5-pyrazolone (PMP), trifluoroacetic acid (TFA), and lipopolysaccharide (LPS) were purchased from Sigma–Aldrich (St. Louis, CA, USA). Sulfasalazine (SSZ) and dextran sulfate sodium (DSS) were purchased from Aladdin (Shanghai, China). Mouse interleukin (IL)-1β and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Enzyme-linked Biotechnology Corporation (Shanghai, China).

After degreasing with 95% alcohol, fresh R. chingii Hu was extracted with ten-fold distilled water at 80°C for 4 h. Then, the extracts were concentrated. A four-fold amount of absolute ethyl alcohol was slowly added to the concentrated solution, stirred uniformly, and alcohol-precipitated overnight. The precipitate was collected and dialyzed (3,500 Da) with distilled water. The dialysate was further concentrated by a rotary evaporator and then freeze-dried to obtain crude polysaccharides of R. chingii Hu.

An appropriate amount of crude polysaccharides of R. chingii Hu need to be further purified by DEAE cellulose-52 column (6 × 50 cm) and gel column chromatography. Distilled water, 0.1 M NaCl, 0.2 M NaCl, and 0.5 M NaOH, were used as eluents. The same elution fraction was combined and concentrated to detect saccharide concentration with the phenol–H₂SO₄ method. The polysaccharides obtained using a DEAE cellulose-52 column were further purified by gel column chromatography. The fully swollen gel filler G-25 Sephadex and Sephacryl S300 were loaded into two slender chromatography columns (2.5 × 60 cm). The polysaccharide sample purified by DEAE-52 column chromatography was dissolved in 0.2 M NaCl, the sample was loaded slowly, and 0.2 M sodium chloride solution was then used for washing, after which homogeneous polysaccharides named RCHP-S were obtained after freeze-drying the fractions.

The high-performance gel permeation chromatography (HPGPC) was used to detect the purity and molecular weight of polysaccharide samples, which were connected to three serially linked ultrahydrogel columns (250, 1,000, and 2,000, 300 mm × 7.8 mm, 6 μm particles). The oven temperature was chosen as 40°C. T-Dextrans (5.2 × 10³, 48.6 × 10³, 668 × 10³ Da) were set as the standards. The standard curve is log (Mw) = 12.1317 – 0.1863T.

The neutral sugar content of polysaccharides was determined by the sulfuric acid phenol method, using glucose as the standard (14). The uronic acid content of polysaccharides was determined by the meta-hydroxybiphenyl method, and galacturonic acid was used as the standard (15). The protein content of polysaccharides was determined by the Coomassie brilliant blue, using globulin as the standard (16).

The monosaccharide composition of RCHP-S was analyzed with the 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization method by using high performance liquid chromatography (HPLC). The polysaccharide RCHP-S was hydrolyzed with 2 M TFA at 110°C for 4 h, then derivatized with PMP. The monosaccharide standards and the various monosaccharide compositions were examined via HPLC (NU3000, Hanbon Sci & Tech, China). The UV detection wavelength was set at 254 nm. A mobile phase with 80% of 0.05 M KH₂PO₄ (pH = 6.9) and 20% of acetonitrile mixture was used with a flow rate of 1.0 ml/min (17).

The 20 mg of the polysaccharide sample was dissolved in D₂O. After lyophilization, it will be re-dissolved in 500 μl of deuterated water. The ¹H NMR, ¹³C NMR, and two-dimensional (¹H-¹H COSY, ¹H-¹³C HSQC, ¹H-¹³C HMBC) scanning analysis of R. chingii Hu polysaccharides were completed using the Bruker AM 400 nuclear magnetic resonance (NMR) instrument at room temperature.

The methylation analysis was carried out according to the previously described method with slight modifications (18). The methylated fragments were hydrolyzed, reduced, and the final products were analyzed by gas chromatography–mass spectrometry (GC–MS).

Cytotoxicity assay was performed by using the MTT method as follows (19, 20). Different concentrations (50–400 μg/ml) of RCHP-S and mouse macrophages RAW 264.7 cells were treated for 48 h, after which the medium was discarded. A total of 0.5 mg/ml MTT was added to continue incubation in an incubator for another 4 h, after which the medium was discarded. The DMSO (100 μl) was added to dissolve blue-purple formazan in the cells. The absorbance was read at 570 nm with a microplate reader.

NO contributes to decreasing enzyme activity, protein, and mRNA levels of distinct P450 enzymes during inflammation (21). In a 96-well plate, macrophages RAW 264.7 were cultivated with a phenol red-free medium. After cells were grown to approximately 60%, different concentrations (50, 100, and 200 μg/ml) of RCHP-S were added to act on the cells for 1 h, after which 2 μg/ml of LPS was added. After 24 h, the cell culture medium supernatant was aspirated to generate a new 96-well plate, and then, Griess A and Griess B reagents were added to detect NO levels with a microplate reader at 540 nm. PBS was taken as a blank control.

To detect changes in inflammatory factor expression in mouse macrophages RAW 264.7 under different polysaccharide concentrations, real-time fluorescent quantitative polymerase chain reaction (PCR) was used to determine the level of gene transcription. RAW 264.7 cells were cultured overnight in a 6-well plate, then combined with different RCHP-S concentrations which were 50 μg/ml, 100 μg/ml, and 200 μg/ml, pre-incubated for 1 h, then combined with 2-μg/ml LPS for 24 h. Cells were then gently rinsed three times with preheated PBS, and total RNA was extracted from the cells according to the TRIzol instructions of the TransGen Company (Beijing, China).

The fold change in relative gene expression levels was calculated according to the 2^−ΔΔCt equation. The RAW 264.7 cell anti-inflammatory level target gene-related primer sequence was as follows. The iNOS forward primer was 5′-ATGGAACAT CCCAAATACGA-3′, the reverse primer was 5′-GTCGTAGAGGACCAC TTT GT-3′, the TNF-α forward primer was 5′-CTTCTGTCTACTGAACTTCGGG-3′, the reverse primer was 5′-CAGGCTTGTCACTCGAATTTTG-3′, the IL-1β forward primer was 5′-ACG GACCCCAAAAGATGAAG-3′, the reverse primer was 5′-TTCTCCACAGCCACAATG AG-3′, the IL-6 forward primer was 5′-CAAAGCCAGAGTCCTTCAGAG-3′, and the reverse primer was 5′-GTCCTTAGCCACTCCTTCTG-3′. Reference gene for the GAPDH forward primer was 5′-GACATCAAGAAGGTGGTGAAGC-3′ and the reverse primer was 5′-GAAGGTGGA AGAGTGGGAGTT-3′.

Cytokine secretion was detected by ELISA. RAW264.7 cells (10⁴ cells/well) were seeded in 96-well plates, with a serum-free medium which was incubated overnight. Then, three concentrations of RCHP-S, which were 50 μg/ml, 100 μg/ml, and 200 μg/ml, were added and incubated for 12 h. Finally, the 2 μg/ml LPS was added to the model groups for another 2 h. The amount of IL-6 and TNF-α secretion in culture supernatants was determined by using an ELISA kit according to the manufacturer's instructions.

According to previous literature reports (22, 23), we have made some modifications to the protocols. Kunming female 5-week-old mice were provided by the Henan University of Chinese Medicine, and animal experiments were approved by the Animal Ethics Committee of the Henan University of Chinese Medicine. Mice were housed under a controlled room temperature of 22°C and humidity of 55% on a 12-h light–dark cycle with free access to diets and water. After a week of adaptation, the experiment was divided into 6 mice per group, which were respectively gavaged according to RCHP-S low-dose group 50 mg/kg, high-dose group 200 mg/kg, and 50 mg/kg positive drug sulfasalazine, in addition to controlling mice with purified water once a day. This was then changed with a 4% dextran sulfate aqueous solution (DSS, 40 kDa, Aladdin, Shanghai) for 4–8 days to drink freely for 5 days. The mice showed different clinical symptoms, such as diarrhea and blood in the stool, indicating that the model was successfully established. DSS was removed at 9–13 days, and all mice were switched to drink freely purified water. The experimental group continued to be gavaged until sacrificed on the 14th day.

Enzyme-linked immunosorbent assay determination (24) was performed as follows: 100 mg colon tissue and 200 μl PBS were homogenized. The homogenate buffer was collected by centrifugation at 12,000 r/min for 10 min. The supernatant was taken to detect levels of IL-6 and TNF-α which were then measured according to the instructions and detected at 450 nm with a microplate reader.

Histological examination (25) was as follows. The colons of each mouse group were taken to measure their lengths and calculate statistics. Part of the colon from mice in each group was taken and fixed in 4% paraformaldehyde solution for 2 days, after which hematoxylin-eosin staining (HE) sections were performed.

The DAI score was calculated according to the following formula (26) (Supplementary Table S1). DAI = weight loss index + stool formation index + stool blood index. From experimental analysis on the first day, mice were weighed at a fixed time every day to observe the color and characteristics of the mouse stool.

All of the results are expressed as the mean ± SD. Statistical analysis was determined by using one-way ANOVA with GraphPad Prism 5.0 software. Statistical significance was chosen as ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.

The five sugar residues were easy to describe as residues A–I in Table 1. Methylation analysis was an indispensable experimental method to study different glycosidic bonds in polysaccharides and their linkage methods. In this study, we performed methylation analysis on RCHP-S combined with nuclear magnetic resonance spectroscopy to jointly analyze the connection between monosaccharides in RCHP-S. As shown in Table 1 and Supplementary Figure S1, RCHP-S was mainly composed of nine derivatives, namely, 2,3,6-Me₃-Gal, 2,3,4,6-Me₄-Gal, 2,3-Me₂-Gal, 3,4-Me₂-Rha, 3-Me-Rha, 2,3-Me₂-Ara, 2,3,5-Me₃-Ara, 2,3,6-Me₃-Glc, and 2,3,6-Me₃-Man.

The chemical shifts of carbon and hydrogen on RCHP-S were assigned by HSQC and ¹H-¹H COSY. The results are summarized in Supplementary Table S3.

There are strong signals at position δ3.78 in the ¹H NMR spectrum of RCHP-S (Figure 3A), indicating that the sugar residue GalA was methylated. According to the ratio of the integral hydrogen spectrum of methyl ester and the integral ratio of anomeric carbon points, the proportion of sugar residue GalA methyl ester was ~74.67%. The signal at δ1.98 indicates the 2-position hydroxyl group part of the sugar residue GalA was acetylated. The signal at δ1.25 represents the methyl group at position 6 of the sugar residue Rha.

In the ¹³C NMR spectrum of RCHP-S shown in Figure 3B, the signal appeared in the sugar anomeric carbon region combined with the result of methylation analysis to give a signal of 100.4 ppm for C-1 of residue A. In addition, 170.8 ppm and 175.2 ppm were signals for C-6 and acetyl groups of residue A, which also verified that most 6-position carboxyl groups of GalA in RCHP-S were methylated and part of the 2-position hydroxyl groups were acetylated. C-6 signals of residues D and E appeared in the 16.5 ppm region, which is the typical methyl signal peak for rhamnose.

The chemical shifts of carbon and hydrogen for RCHP-S were assigned by ¹H-¹H COSY (Figure 4A) and HSQC (Figure 4B). In the HMBC (Figure 4C) spectrogram, the significant cross-peaks of (A-C4, A-H1) and (A-C1 and A-H4) are shown, indicating that most of the residue A was self-phased by the O-4 link. The appearance of cross-peaks (C-H1 and E-C4) indicates residue C is linked to residue E via O-4. Results show RCHP-S was mixed pectin composed of HG-type and RG-I (Figure 5). Combined with the results of the methylation analysis, we could draw the following conclusions. Polysaccharide RCHP-S was a highly methyl esterified pectin. The main chain of polysaccharide RCHP-S was composed of 1,4-Gal A and 1,2-Rha. Furthermore, this pectin contains two structural regions, namely, the HG type of the smooth zone and the RG1 type of the rough zone. Based on the above information, the inferred structure of the polysaccharide is shown in Figure 5.

In recent years, pectin polysaccharides had been an area of interest for many researchers, as they possess many activities, such as good biocompatibility with human cells, low toxic side effects, and so on. As shown in Supplementary Figure S2, RCHP-S has no obvious cytotoxic effect on RAW 264.7 cells when the concentration range is between 50 and 400 μg/ml, which indicates that the polysaccharide RCHP-S has good biocompatibility.

The functions of endothelial cells (such as vascular endothelial cell) will be changed in inflammation, with nitric oxide production changing, pro-inflammatory chemokines and cytokines increasing, resulting in the recruitment of immune cells into inflamed sites (27). Nitric oxide (NO) plays the role of an inflammatory indicator where it would accumulate as the hepatocytes injuries (28). When compared with the blank control, the 2 μg/ml LPS in the positive control group significantly increased NO secretion from RAW264.7 cells after 24 h. Cells were pre-treated with polysaccharides for 1 h and then incubated with LPS. For high concentration (200 μg/mL) p < 0.01, medium concentration (100 μg/mL) p < 0.01 and low concentration (50 μg/mL) p < 0.05, different dose groups could reduce cellular NO secretion (Supplementary Figure S3).

Experimental results showed nitric oxide synthase iNOS and common inflammatory factors (IL-1β, IL-6, TNF-α) play a key role in inflammation development (29, 30). Following LPS-stimulated macrophages, the expression of iNOS, IL-1β, IL-6, and TNF-α related genes increased significantly. In the polysaccharide experimental group, cells were pre-treated with different polysaccharide concentrations, showing a significant inhibitory effect on iNOS and IL-1β genes (p < 0.05). Similarly, IL-6 and TNF-α expression showed more effective results (p < 0.01; Figure 6). We calculated the expression of IL-6 and TNF-α detected by ELISA in RAW264.7 cells. The expression of IL-6 and TNF-α was significantly inhibited after RCHP-S was added. The results of cytokine expression level and RT-PCR were consistent (Supplementary Figure S4).

As shown in Figure 7A, the mice received intragastric administration according to the procedure described. Blank control mice had normal diets and drinking water, with no significant weight loss or blood in the stool. For the DSS group, after drinking DSS, mice showed obvious clinical symptoms after the third day (Figure 7B). The main manifestations were rough coat color, weight loss, loose stools, and bloody stools, but no deaths. Through the administration of polysaccharides and anti-inflammatory drugs, we found scores for the high-dose polysaccharide and DSS groups were significantly different (p < 0.05). DAI score results are shown in Figure 7C, indicating that the high-dose polysaccharide group can effectively alleviate colitis symptoms and protect the colon.

Based on anatomy, the DSS-induced mouse colon was significantly shorter than the normal group (Figure 7D). For the RCHP-S high-dose group, the mouse colon length (n = 6) was significantly different from the model group (p < 0.01; Figure 7E), indicating that RCHP-S could effectively reduce colon inflammation and be used as a natural anti-inflammatory drug.

Inflammatory factors play a key role in inducing enteritis. We detected IL-6 and TNF-α in colon tissue and found that the IL-6 and TNF-α content in the model group was significantly higher than in the blank control group (Figure 8). In comparison to the DSS group, the high dose of the polysaccharide group is significantly reduced. In the colon tissue, IL-6 plays a very important role in regulating inflammatory bowel disease. Chand et al. reported that STAT3 signaling via the IL-6ST/gp130 cytokine receptor promotes epithelial integrity and intestinal barrier function during DSS-induced colitis (31).

Ulcerative colitis (UC) is a chronic nonspecific inflammatory disease, which exhibited abdominal pain, diarrhea, and so on (32). Ulcerative colitis may be that the two conditions result from an inappropriate and exaggerated mucosal immune response to constituents of the intestinal flora in a genetically susceptible host (33). But there is no definitive cure for UC (34), except for steroids and non-steroidal anti-inflammatory drugs, which have serious side effects with long-term use, including hyperlipidemia, systemic infection, and herpes zoster infection (35).

Pectin, majorly comprised of galacturonic acid, is a non-cellulosic polysaccharide, which is intricately connected with the cellulose and hemicellulose units (36) but had some differences in composition, structure, and molecular weight (37). The pectin that has a high number of neutral sugar side chains ameliorates colonic tissue damage and decreases colonic IL-1β and IL-6 levels to attenuate DSS- and 2,4,6-TNBS-induced experimental colitis. In addition, the protective effect of pectin against experimental colitis is mediated in a side chain-dependent manner (38). Many studies show that Tremella polysaccharide has a molecular weight of 5.17 × 10⁴ Da, which is mainly composed of mannose, glucose, and a small amount of galactose, glucuronic acid, xylose, and arabinose. It can effectively improve the clinical symptoms of colitis. The length of the colon is 13.85% longer than that of the model group, and the spleen index is 11.95% lower than that of the model group (39).

Our results show that RCHP-S can effectively inhibit cytokines, thereby improving enteritis symptoms. Detection of tumor necrosis factor TNF-α showed that the low-dose group of RCHP-S can inhibit its secretion (Figure 8B). The results indicate that polysaccharides can improve intestinal inflammation by inhibiting inflammatory factors, which were similar to cell experiments.

Using HE staining and colonic pathology observations, we found that mucosal damage, basal inflammatory cell infiltration, abnormal crypt structure, and epithelial villi defect occurred in the DSS group compared to the negative control group. In the low-dose RCHP-S group, some crypts were missing (arrow 3), but in the high-dose group, the colonic mucosa was basically intact, and epithelium and crypt morphology (arrow 4) remained essentially unchanged (Figure 9).

In summary, we evaluated the anti-inflammatory effects of RCHP-S using cell and animal experiments. The colitis model was established, and RCHP-S was used for treatment. This study showed that RCHP-S can reduce DSS-induced enteritis to produce inflammatory factors and show RCHP-S can alleviate inflammatory responses of DSS in the intestinal tract.