Protective Effect of Alpinia oxyphylla Fructus Polysaccharides Extracted with Different Solvents on Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice

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Introduction
Ulcerative colitis (UC) is a chronic nonspecifc, noninfectious, infammatory bowel disease [1].Its clinical symptoms mainly manifested as hemorrhagic diarrhea, colic abdominal pain, and tenesmus.Te UC patients in 2023 were found to be about 5 million cases in the world, and the number will still increase in the future [2].In practice, the main therapeutic drugs for UC are 5-aminosalicylic acid (5-ASA), prednisone, azathioprine, and infiximab [3].However, most of them have certain side efects.For example, 5-ASA could induce severe myocarditis [4].Due to the drug resistance and prolonged treatment period of infiximab, it increases the risk of cancer for patients undergoing long-term treatment [5].Terefore, new and better ways to treat UC need to be found.
Te pathogenesis of UC is closely linked to the intestinal barrier, intestinal fora, and immunity.Te intestinal barrier composed of intestinal epithelial cells and tight junctions (TJ) is the frst defensive site for the invasion of toxoid and pathogenic bacteria, and if the intestinal epithelial cells and TJ are disrupted, excessive oxidative stress and infammatory reactions can be induced, leading to the deterioration of UC [6,7].Research has found that Agaricus blazei Murrill polysaccharides attenuate dextran sulfate sodium (DSS)induced colitis and protect the colon barrier from damage by decreasing MDA, TNF-α, IL-1β, and IL-6 levels and elevating SOD and IL-10 levels [8].Another research identifed the elevated level of IL-1β leading to increased intestinal TJ permeability, which is an important factor in promoting intestinal infammation [9].
Besides, disturbance of intestinal fora may lead to impaired intestinal barrier function and immune dysfunction [10].Te richness and diversity of the intestinal fora are signifcantly reduced in UC patients compared to healthy people, and the level of Proteobacteria often signifcantly increased [11].In spontaneous colitis mice induced by IL-10 defciency, the number of Proteobacteria in the intestine signifcantly increases with the development of colitis, indicating that the abundance of Proteobacteria is positively correlated with the severity of infammation [12,13].In particular, Lactobacillus may alleviate colitis by protecting the intestinal barrier and may also promote the recovery of dysbiosis of the intestinal fora and reduce the infammatory response caused by intestinal microbial invasion.Lactobacillus were found to signifcantly increase IL-10 levels, inhibit IL-1β, IL-6, and TNF-α levels, and restore TJ protein levels (including ZO-1, bcl-2, and claudin 1) [14].
Alpinia oxyphylla Miq. is a functional food mainly grown in the tropical region.A. oxyphylla has been consumed as a dietary supplement for more than 300 years in Yangjiang, China [15].In folk markets, the fresh fruit of A. oxyphylla was processed into various preserved fruits for consumption, such as Jiuzhi Yizhi, sour and sweet Yizhi, Tangsha Yizhi, candied Yizhi, and canned Tangshui Yizhi [16]. A. oxyphylla has great potential value in treating intestinal diseases.It is reported that stem and leaf extracts of A. oxyphylla can maintain the integrity of the duck's intestinal barrier and regulate intestinal fora [17].Protocatechuic acid from A. oxyphylla promotes the migration and proliferation of human adipose tissue-derived stromal cells (hADSCs), and hADSCs could regenerate the injured tissues, which helps repair the intestinal barrier [18].Meanwhile, A. oxyphylla may be an excellent alternative to steroids and nonsteroidal anti-infammatory drugs for the treatment of infammatory diseases [19].As the main component of A. oxyphylla fruit, polysaccharides have been found to possess multiple pharmacological efects.For instance, A. oxyphylla fruit polysaccharides (AOFPs) could improve learning and memory capacity in Alzheimer's disease (AD) mice by lowering NO, IL-1β, TNF-α, and PGE-2 levels in AD mice serum [20].AOFPs signifcantly could prevent the infection of porcine epidemic diarrhea virus in IPEC-J2 cells [21].AOFPs have signifcant immunomodulatory efects on macrophages polarizing [22].But still now, the research studies of AOFPs on the treatment of UC have not been reported.
Te extraction methods of polysaccharides are closely related to their activity.Diferent extraction methods have signifcant diferences in physicochemical properties and biological activity for polysaccharides.For example, the alkali-extracted Opuntia macrorhiza fruit peel polysaccharides have the best inhibitory activity on α-amylase, while the acid-extracted polysaccharides have better antioxidant and antiglycosylation activity [23].Terefore, the extraction method is an important factor for activity discovery.
In this study, four extraction methods for AOFPs (distilled water, 0.1M NaCl, 0.1M HCl, and 0.1M NaOH) were designed to explore the potential therapeutic efect and mechanism of AOFPs on DSS-induced UC in mice.Tis study will provide new ideas for the use of AOFPs and the treatment of UC.

Extracting AOFPs Using Diferent Solvents. Te
A. oxyphylla fructus was ground into powder and sieved through the sieve with a pore diameter of 0.25 mm.Eight hundred grams of A. oxyphylla fructus powder was soaked in 95% ethanol at room temperature for 12 h to remove small molecular impurities.After extraction, the A. oxyphylla fructus powder was dried at 50 °C and used for subsequent experiments.
Te extraction method of AOFPs was referenced to that of blackberry polysaccharides [24].In brief, 200 g A. oxyphylla fructus powders were extracted twice with 4000 ml distilled water, 0.1M NaCl, 0.1M HCl, and 0.1M NaOH at 95 °C for 2 h, respectively.Te extracts were centrifuged at 8000 g for 10 min, the supernatant was collected, and their pH was adjusted to 7.0 with 0.1M HCl or NaOH.Subsequently, the extracts were concentrated to 1/10 of the original volume.Anhydrous ethanol was added to a fnal concentration of 80%, it was placed at 4 °C for 12 h, and the precipitates were dissolved in 400 ml of distilled water.Subsequently, the proteins were removed using Sevag reagent (1-butanol: chloroform, 1 : 4).Te supernatants were placed in dialysis membranes (MW: 7000) and dialyzed with distilled water for 48 h.In the end, the four extracts were freeze-dried to give AOFPs.Te four AOFPs were named DW, SC, AC, and Al, respectively.Figure 1 shows the extraction fow of AOFPs.

Chemical Composition Analysis.
Glucose was used as the standard to determine the total sugar contents at 490 nm according to the phenol-sulfuric acid method [25].Te content of uronic acid was determined using the mhydroxydiphenyl method at 526 nm with galacturonic acid as the standard [26].Te Coomassie brilliant blue 2 Journal of Food Biochemistry method was employed to determine the protein content at 595 nm with bovine serum albumin as the standard substance [27].

Monosaccharide Composition Analysis.
Te monosaccharide composition of AOFPs was analyzed on Waters e2695 liquid chromatography using 1-phenyl-3-methyl-5pyrazolone (PMP) precolumn derivatization with mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and arabinose as standards [24].Briefy, 10 mg DW, SC, AC, and Al were added to 2 mL of 4M trifuoroacetic acid (TFA) and hydrolyzed at 105 °C for 2 hours, respectively.1 ml of methanol was added and evaporated at 60 °C to remove residual TFA, and then the process was repeated three times.
Ten, 1 ml of ultrapure water was added to the hydrolysate to dissolve it.Ten, 500 μl of hydrolysate was taken and 500 μl of NaOH (0.3 M) and 500 μl of PMP solution (0.5 M) were added sequentially, mixed, and put into a water bath at 70 °C for 1 h.After cooling to room temperature, 500 μl HCl (0.3 M) was added and then extracted 3 times with chloroform.Te same derivatization steps were performed for mixtures of standard monosaccharides.A C18 column (4.6 × 250 mm) was used to analyze the AOFPs samples and standard solutions.Te 84% phosphate bufer solution (0.08 M, pH � 6.8) and 16% acetonitrile were used as mobile phases, and the fow rate was set to 1 ml/min.

Molecular Weight (MW) Determination.
Te molecular weight distribution of DW, SC, AC, and AL was determined on Shimadzu LC-2010 liquid chromatography.Te system was equipped with the RID-20A (SHIMADZU, Japan).TSK gel G4000PWXL (300 × 7.8 mm, 7 μm) column was used as a chromatographic column for molecular weight determination.Ultrapure water was added to the AOFPs to obtain 1 mg/ml polysaccharide solutions.Samples were fed in volumes of 10 μl.Ultrapure water was used as the mobile phase with a fow rate of 0.5 ml/min.Dextran standards were used to produce standard curves and calculate the molecular weight of AOFPs based on the standard curve [28].

Triple Helical Structure Analysis.
Te triple helix structure of AOFPs was determined using the Congo red test [29]. 1 mg/ml of DW, SC, AC, and AL solutions was prepared using ultrapure water.Each polysaccharide solution (2 ml) was added with 80 μm/L Congo red solution (2 ml).After 30 s of stirring, 1 mol/L NaOH was added sequentially to make the NaOH concentration of the mixed solutions 0, 0.  2(a)).On the eighth day, the mice were sacrifced through cervical dislocation and colons were collected, measured, and weighed and then stored at −80 °C.

Histopathological Analysis.
Te colons were taken and fxed with tissue fxative and embedded into parafn.Hematoxylin and eosin (H&E) were used to stain colon sections, and pathological damage in the colon was examined under a microscope.Te histological score was assessed according to the following criteria: 0 (normal colonic mucosa), 1 (loss of one-third of crypts), 2 (loss of two-thirds of crypts), 3 (lamina propria covered with a single layer of epithelial cell, with mild infammatory cell infltration), and 4 (erosions and marked infammatory cell).

Determination of MPO, SOD, and MDA in the Colons of Mice.
According to the instructions in the kits, the colon tissue sample was added to the corresponding solution, and then it was homogenized with a homogenizer and centrifuged at 5000 g for 10 min at 4 °C and the supernatant was collected for detection.and quantifed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA).Genes involving TNF-α, IL-1β, IL-6, and IL-10 were determined.To measure the mRNA expression level, β-actin was used as an endogenous control, and the relative expression level was calculated using the 2 −ΔΔCt method [30].Te primer sequences of mouse were designed and are listed in Table 1.
2.3.7.Western Blot.RIPA lysate and protease inhibitor were added to the colon tissue, homogenized, and placed at 4 °C for 1 h for lysis and then centrifuged at 12,000 g for 15 min, and the supernatant was taken as the protein sample.BCA protein assay kit (Biosharp, Hefei, China) was used to determine protein concentration in samples and subsequently adjusted with RIPA so that the concentrations of each group of samples were consistent.5 × DSD loading bufer was added to the samples and then boiled in boiling water at 100 °C for 5 min to denature the proteins.Groups of proteins were separated by electrophoresis and transferred to 0.45 μm polyvinylidene difuoride (PVDF) membranes (Millipore Corporation, MA, USA).PVDF membranes were blocked with 5% skim milk powder for 2 h, washed with Tris-bufered saline with Tween 20 (TBST), and incubated with primary antibody at 4 °C overnight and then washed again with TBST and incubated with secondary antibody for 1 h.Finally, 4 Journal of Food Biochemistry PVDF membranes were washed 3 times with TBST and then developed with a chemiluminescence kit (Beyotime Biotechnology, Shanghai, China).Te expression levels of proteins were quantifed and normalized to the relative expression levels of β-actin.

Intestinal Flora Analysis.
Collected fecal samples were used for intestinal fora analysis.DNA was extracted from the microbiome of mice feces.Te 16S rRNA gene in bacteria was amplifed by using TransStart ® FastPfu DNA Polymerase (Transgen, Beijing, China).PCR products were recovered and purifed with the AxyPrep DNA gel extraction kit (Axygen, USA).Te MiSeq library was built by using TruSeq ™ DNA Sample Prep Kit (San Diego, CA, USA).
Intestinal microbiota analysis was conducted by UPARSE (version 7.0.1090),all sequences were divided into OTUs based on diferent similarity levels, and bioinformatics analysis was performed on OTUs at 97% similarity levels.Based on the results of the OTU clustering analysis, the α-diversity analysis was calculated by Mothur (version 1.30.2).Te results of α-diversity analysis were tested for intergroup diferences using one-way ANOVA.β-Diversity analysis was performed by principal coordinates analysis (PCoA) and β-diversity was calculated by the Bray-Curtis distance.LDA efect size (LEfSe) analysis was performed using the nonparametric Kruskal-Wallis (KW) sum-rank test to detect the species richness diferences between diferent groups and obtain signifcantly diferent species, and then the Wilcoxon rank-sum test was used to test the consistency of diferences between diferent species in subgroups of diferent groups in the previous step, and fnally LDA (linear discriminant analysis) was used to estimate the impact of these diferent species on the diferences between groups.Te relationship between the microbes with infammation and intestinal barrier was analyzed using Spearman's rank correlation test.With the 16S rRNA gene amplicon sequencing results, PICRUST2 (version 2.2.0) was used for KEGG pathway analysis of the intestinal fora.

Data Analysis. Statistical signifcance among groups was analyzed using Student's t-test or one-way ANOVA in
GraphPad Prism version 9.4.1 (GraphPad Software, Inc., San Diego, CA, USA).All data were expressed as the mean-± standard error of the mean (SEM).Diferences of P values <0.05 were considered statistically signifcant.

Extraction Yields and Physicochemical Properties of
AOFPs.In this research, four water-soluble polysaccharides were extracted from the A. oxyphylla fructus by diferent extraction solvents.Extraction rates and physicochemical properties vary depending on the extraction solution.It can be seen from Table 2 that the yield of AOFPs varies according to the extraction solvent (about 0.86%-8.52%).
Among them, acid solution (AC, 8.52%) is the highest, which may be due to the fact that the glycosidic bonds in this AOF are more easily hydrolyzed by acid [31].Te yield of alkali solution (AL, 2.73%) was the second highest, and this may be due to the fact that alkaline solutions easily disrupt or hydrolyze cell walls, causing polysaccharides to difuse [32].Te study's results corroborate with those of Ulva intestinalis sulfated polysaccharides [33].Te extraction rate of water solution (DW, 1.01%) was not signifcantly diferent from that of salt solution (SC, 0.86%).Te highest total sugar content was found in AC (71.82%).It is possible that the glycosidic bonds were destroyed in the acidic environment at high temperatures [24].Te contents of uronic acid were more in DW (74.17%) and SC (63.15%) and less in AC (3.42%) and Al (8.72%), which means they are all acidic polysaccharides.Te protein content of DW, SC, and AC is relatively low (0.12-0.31%).Perhaps this is the result of solvent extraction reducing the isolated proteins in these AOFPs.However, the highest protein content was found in AL (2.32%).Tis may be due to the fact that hydrogen bonds were easily broken in alkaline environments, promoting protein production [34].

Monosaccharide Composition.
By comparing the retention time with the standard sample, the monosaccharides in the AOFPs were determined.As shown in Figure 3(a) and Table 2, AOFPs contain mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and arabinose.Te experimental results showed that all four AOFPs contained these eight monosaccharides, but there were diferences in their molar ratios.Diferent solution environments have different degrees of hydrogen bond disruption and partial degradation of polysaccharide molecules, resulting in diferent percentages of each monosaccharide in the four AOFPs.Te contents of glucuronic acid and galacturonic acid in AC and AL were lower, while the content of glucose was higher.Tis may be due to the degradation of uronic acid into small molecule sugars in an acidic or alkaline environment.Comparing the content of uronic acid measured data, the results were consistent [35].

Molecular Weight Determination. It was obvious from
Figure 3(b) that the MWs of the AOFPs were diferent based on the extraction method.Similar peaks were found in DW and SC, both with a single symmetry, and their MWs were 921.87 kDa and 748.35 kDa, respectively.In AC, two populations around molecular weights of 1675.7 kDa (3.21%) and 4.22 kDa (96.79%) were found.Te latter one indicates the presence of oligosaccharides, meaning that the polysaccharides were degraded.AL presented two main peaks with the MWs of 1099.86 kDa (97.08%) and 621.90 kDa (2.92%).

FT-IR Analysis.
Analysis of the distinctive functional groups of polysaccharides through FT-IR is frequent.Te FT-IR spectra of AOFPs revealed the typical features of polysaccharides, as illustrated in Figure 3(c).Te broadly stretched intense peaks at 3405 cm −1 represented the stretching of the -OH, and the absorption areas at 2950 cm −1 were caused by C-H stretching and bending vibrations [36].Te absorbance areas of 1744 cm −1 represented the symmetric stretching vibration of the C�O in acetyl groups, and it was observed only in DW and SC.Te absorbance areas of 1613 cm −1 represented the COO-deprotonated carboxyl group, implying that galacturonic acid was detected in the samples [24].Te absorption region at 1415 cm −1 was due to the stretching vibration of the C-O [37].Te absorption areas at 1099 cm −1 and 1010 cm −1 were due to the stretching vibration of the pyranose ring [36].Moreover, the peaks at 862 cm −1 represented the presence of a β-glycoside bond [38].

UV Analysis.
Ultraviolet-visible analysis can be used to determine the purity of extracted polysaccharides [39].In Figure 3(d), there are no obvious absorption peaks for DW, SC, AC, and AL at 260 nm and 280 nm, indicating that there are fewer nucleic acid substances or free proteins.Tese fndings agree with those of the chemical composition study.

Triple Helical Structure Analysis.
Te Congo red experiment is able to judge whether the AOFPs contain triple helix structures.If the sample has the triple helix structure, the λ max will be red-shifted [29].According to Figure 3(e), as the concentration of NaOH gradually magnifed, the λ max values of DW, SC, and AC were red-shifted, which suggested DW, SC, and AC had a triple helical conformation.On the contrary, the trend of the λ max of Al was similar to that of the control, so it was judged that AL did not have a triple helix structure.

Zeta Potential Analysis.
Zeta potential is a key indicator of the strength of the attractive and repulsive forces of macromolecules in solution and refects the stability of polysaccharide solutions.As the absolute value of zeta 6 Journal of Food Biochemistry potential increases, the stability of the polysaccharide solution gradually becomes stronger.Terefore, the zeta potentials of the four polysaccharides were determined [40,41].Te results in Figure 3(f ) show that the Zeta potentials of DW, SC, AC, and AL are −33.82mV, 30.65 mV, −35.78 mV, and −50.14 mV, indicating that they have good stability.DW, AC, and AL were negatively charged, implying that they are all anionic polysaccharides.However, SC was positively charged, indicating that the structure of polysaccharide may be modifed in NaCl solvent.

SEM Analysis.
SEM can be used to observe the surface morphology of polysaccharides.As shown in Figure 4, the four polysaccharides are all in lamellar structure, in which the DW surface is distributed with dense irregular caves.SC surface is rough, with diferent sizes of depressions.Te surface of AC and AL is smooth.Tis may be due to the diferent solutions used to extract polysaccharides, resulting in diferences in the indicated structures of AOFPs [42].shows that, during the experiment, the weight of the CON group mice continued to increase, while the weight of the MOD group signifcantly decreased.All fve treatment groups alleviated this trend.Figure 2(c) shows that POS, DW, SC, SC, and AL groups alleviated these phenomena and signifcantly improved the DAI score.Te results indicate that DSS-induced mice exhibited signifcant UC characteristics, such as weight loss, increased DAI score, decreased colon length, and histological changes.Tis is consistent with the report, indicating that the UC mouse model was fruitfully established [43].After treatment with AOFPs, these symptoms have been alleviated to varying degrees, with the most signifcant efcacy seen with AL.Te results indicate that AOFPs have therapeutic efects on UC and there are diferences in their efcacy.

Efects of AOFPs on Colonic Length and Histopathology
in UC Mice.Colonic mucosal ulceration, infammatory cell infltration, crypt structure change, and colon shortening were frequently seen in the colon of UC mice [44].As seen in Figure 5(a), the colonic length of mice fed with 3% DSS solution was signifcantly shortened.POS, DW, SC, SC, and AL groups can alleviate this phenomenon, in which the AL group has the best efect.According to the H&E staining results of the colon in Figure 5(b), the muscularis, mucosa, and crypt structures of the colon in the CON group were normal, and a large number of goblet cells were visible.In the MOD group, the crypt structure was distorted, goblet cells were signifcantly reduced, mucosa edema and other tissue damage were observed, and there was obvious infammation in colon tissue.In addition, the POS, DW, SC, SC, and AL groups can reduce the pathological damage of  these tissues to varying degrees, in which the AL group has the best efect.Tese results suggest that AOFPs can effectively protect the colon from DSS-induced length shortening and infammation.

Efect of AOFPs on the Level of Oxidative Stress in the Colon of UC Mice.
To identify changes in colonic oxidative stress levels before and after treatment with AOFPs, SOD activity, MPO levels, and MDA levels were measured experimentally.As shown in Figure 6(a), DSS intervention resulted in a decrease in colon SOD levels, while POS, AC, and AL were able to signifcantly restore SOD levels (P < 0.05).As shown in Figure 6(b), DSS intervention induced an increase in MPO levels in the colon, but after POS, SC, AC, and AL treatment, MPO levels signifcantly decreased (P < 0.05).As shown in Figure 6(c), compared with the CON group, the level of MDA in the MOD group increased, but DW, SC, AC, and AL alleviated this change in varying degrees (P < 0.05).According to the experimental results, after treatment with AOFPs, the levels of SOD, MPO, and MDA have been restored, indicating that AOFPs can remove oxygen free radicals, repair oxidative damage to the mucosal barrier, and alleviate infammatory symptoms, with AL having the best antioxidative stress efect.

Efect of AOFPs on the Expression of Infammatory
Factors.Abnormal upregulation of proinfammatory cytokines is one of the hallmarks of UC [45].To obtain information about the changes in infammatory factor levels in the intestine of mice before and after treatment with AOFPs, we measured the expression of IL-1β, IL-6, IL-10, and TNF-α in colonic tissues by ELISA kits and real-time PCR.As depicted in Figure 7, the gene expression of IL-1β, IL-6, and TNF-α was signifcantly higher in the MOD group than in the CON group (P < 0.05).Nevertheless, this tendency was prominently reversed after treatment with POS, DW, SC, AC, and AL (P < 0.05).When compared to the CON group, IL-10 gene expression was dramatically reduced in the MOD group, while it prominently elevated following treatment with POS, DW, SC, AC, and AL (P < 0.05).

Efects of AOFPs on DSS-Induced Colon Barrier Integrity in UC Mice.
To determine whether AOFPs have an ameliorative efect on the intestinal barrier in UC mice, levels of E-cadherin and TJ protein (including claudin 1, occludin, and ZO-1) were measured in colonic tissue by Western blot.Te protein level in the MOD group was much lower than it was in the CON group, showing that the intestinal barrier had been compromised.As shown in Figure 8, DSS induces signifcant downregulation of claudin 1, occludin, ZO-1, and E-cadherin.DW signifcantly upregulated all four proteins.SC signifcantly upregulated occludin, ZO-1, and E-cadherin.AC significantly upregulated claudin 1 and ZO-1.AL only significantly upregulated ZO-1.

Efect of AOFPs on Intestinal Flora of UC Mice
(1) Changes in the Diversity of the Intestinal Flora.To explore the changes in intestinal fora of UC mice before and after AOFP treatment, 16S rRNA gene sequencing analysis was used to compare the intestinal fora of seven groups.With the increase of sequencing number, it was observed that the sparse curve of the Shannon index gradually tends to be stable, and the coverage index exceeded 0.9979 (Figure 9(a)), demonstrating that the sequencing data were adequate and trustworthy for further study.Te overall number of OTUs and the number of distinct OTUs in each group can be represented visually using the Venn diagram [46], so that we used the Venn diagram (Figure 9(b)) to analyze the number of OTUs in the sample.Tere are 228 in each group, while the DW, SC, AC, and AL groups had 2, 1, 0, and 3 OTUs diferent from the other groups, respectively.
Te richness and diversity of intestinal fora were determined by α-diversity analysis [47].ACE and Chao refect the species richness of communities in a single sample.ACE is used to estimate the index of the number of OTUs in the community.Te Chao value rises as the overall number of species increases.Te Simpson index embodies the diversity of microorganisms.Te higher the Simpson index value, the better the community diversity.Sobs is the number of OTUs observed.It can be seen from Figure 9(c) that the ACE and Chao indexes of the MOD group are noticeably lower than those of the CON group.Te ACE and Chao indexes of the POS and DW groups were signifcantly greater than those of the MOD group, showing that DW can mitigate the decline in species richness brought on by DSS.Te Simpson index of the AL group was remarkably higher than that of the MOD group, indicating that AL can increase community diversity.Te Sobs index of the MOD group was lower than that of the CON group.Te Sobs index of the POS and DW groups dramatically rose in comparison with the MOD group.In β-analysis, PCoA is frequently used to identify diferences within and between groups of samples.In Figure 9(d), there was a clear distance between the MOD and CON groups, indicating that DSS can signifcantly alter the intestinal fora of mice.Meanwhile, there was a certain distance between the treatment groups and the MOD group, demonstrating that 5-ASA and AOFP treatment had altered the bacterial community structure in UC mice.
(2) Changes in Intestinal Flora Composition.According to Figure 10(a), the intestine microbiota of mice consisted mainly of Firmicutes, Verrucomicrobiota, Bacteroidota, Proteobacteria, and Actinobacteria.Of these, Firmicutes and Bacteroidota are dominant phyla in healthy individuals [48].According to Figures 10(b) and 10(c), in the CON group, the value of F/B was 2.26, and after DSS treatment, F/B increased to 5.64, while POS (F/B � 4.11), DW (F/B � 1.39), SC (F/ B � 1.23), AC (F/B � 1.68), and AL (F/B � 3.50) signifcantly suppressed this trend (P < 0.05).DSS led to a decrease in the relative abundance of Bacteroidota, while DW, SC, and AC signifcantly reversed this trend.In Figures 10(d CON group.However, DW, SC, AC, and AL signifcantly reduced the relative abundance of Actinobacteria.SC and AC signifcantly reduced the relative abundance of Proteobacteria. Te heat map analysis (Figure 11(a)) was carried out at the genus level.Figures 11(b)-11(g) display a few example species, and in the MOD group, the relative abundance of harmful bacteria signifcantly increased.After treatment with 5-ASA and AOFPs, the relative abundance of nor-ank_f__Muribaculaceae and norank_f__Lachnospiraceae increased signifcantly, the relative abundance of Escherichia-Shigella, Bifdobacterium, Romboutsia, Turicibacter, and Faecalibaculum in the MOD group signifcantly reduced.
In order to further identify the diferences between dominant and characteristic bacteria in each group, the linear discriminant analysis (LDA) of the efect amount (LEfSe) was performed.LEfSe is used to detect features with signifcant diferences in abundance and identify groups with signifcant diferences in abundance.Te LDA discrimination column chart counts the microbial groups with signifcant efects in multiple groups.Te LDA score was obtained through LDA analysis (linear regression analysis).Te higher the LDA score, the greater the species richness's impact on the difference efect.Figures 12(a) and 12(b) show the microbial communities with signifcant efects in each group, with LDA values greater than 3. Te results showed that, at the genus level, the species richness of Bifdobacterium, Romboutsia, Turicibacter, and Faecalibaculum in the MOD group had a greater impact on the diference efect.Te species with the highest LAD values in the POS, DW, SC, AC, and AL groups are norank_f__norank_o__Clostridia_UCG-014, Rikenella-ceae_RC9_gut_group, Akkermansia, and Escherichia-Shigella, respectively.
(3) Correlation Analysis of Intestinal Flora with Infammation and Intestinal Barrier.In order to better understand the relationship between the abundance of intestinal fora and colitis indicators in mice, a correlation analysis was conducted between microbe, infammatory factors, and intestinal barrier.As shown in Figure 13(a), Romboutsia, Turicibacter, Faecalibaculum, and Coriobacteriaceae_UGG-002 were positively and signifcantly correlated with TNF-α, IL-6, and IL-1β.

Lachnoclostridium, norank_f__Muribaculaceae, and
Eubacterium_fssicatena_group were negatively correlated with IL-1β and positively correlated with IL-10.In addition, Bacteroides, Alistipes, and Odoribacter were positively correlated with IL-6.As shown in Figure 13(b), norank_f__Muribaculaceae showed a positive correlation with E-cadherin and claudin 1 expression.Escherichia-Shigella showed a negative correlation with claudin 1 expression.Bacteroidetes showed a positive correlation with ZO-1 expression.Turicibacter showed a negative correlation with E-cadherin expression.Tese results suggest that infammation and intestinal barrier were closely related to the imbalance of intestinal fora in UC mice.
(4) Prediction of Changes in the Function of the Intestinal Flora.Te KEGG pathway of intestinal fora was analyzed by PICRUST 2 (version 2.2.0) based on the sequencing results of 16S rRNA gene amplifers.As shown in Figure 14, we found signifcant abnormalities in the metabolic microbiota of intestinal DSS, such as a decrease in carbohydrate metabolism, amino acid metabolism, energy metabolism, and an increase in membrane transport and signal transport.However, DW, SC, and AC treatment can signifcantly correct these abnormal changes and improve the dysfunction of intestinal fora in UC mice.However, no signifcant diference in pathway was found in the AL group.

Discussion
Te etiology of UC is complex, and multiple studies have suggested that it may be related to epithelial barrier dysfunction, infammation, and intestinal fora [11,49].A. oxyphylla is benefcial for intestinal health and can repair the intestinal barrier and regulate intestinal bacteria [17].In this research, we used deionized water, NaCl, HCl, and NaOH solutions to obtain A. oxyphylla polysaccharides and investigated the therapeutic efects of AOFPs on DSSinduced UC mice.Te results showed that there are signifcant diferences in the monosaccharide composition, molecular weight, FT-IR spectra, surface morphology, triple helix structure, and Zeta potential of polysaccharides obtained by diferent extraction methods.Te polysaccharides obtained from diferent extraction solvents were evaluated at Data are presented as the mean ± SD (n � 6).### P < 0.001compared with the control group.* P < 0.05 and * * * P < 0.001 compared with the model group.12 Journal of Food Biochemistry the same dose, to explore the diferences in the therapeutic efects of AOFPs on UC.DSS can impair the integrity of the colon barrier and downregulate the expression of TJ protein, improve the permeability of the intestinal mucosa to endotoxin and antigens, and induce a ferce infammatory response [45,50].TJ proteins in the intestinal epithelium, including claudin 1, occludin, and ZO-1, are important factors for the infammation relief and mucosal repair process in ulcerative colitis [51].E-cadherin is a cell adhesion protein that plays a crucial role in maintaining the shape of intestinal epithelial cells, villus morphology, and barrier function.Te loss of Ecadherin could lead to the destruction of intestinal tissue structure, which is related to the damage of the location and function of goblet cells and Paneth cells, the reduction of the expression of antibacterial factors, and the insufcient clearance of intestinal pathogens [52].In addition, in DSSinduced UC mice and E-cadherin-defcient mice exhibited more severe weight loss, dehydration, and bloody stool symptoms, which were signifcantly associated with more severe acute infammation [52].In this experiment, it can be seen that DSS can signifcantly reduce the levels of claudin 1, occludin, ZO-1, and E-cadherin in colon tissue, while the AOFPs restore their expression to varying degrees, indicating that AOFPs have a protective efect on the intestinal barrier.
Te balance between anti-infammatory (IL-10) and proinfammatory (IL-1β, IL-6, and TNF-α) cytokines is an important factor in maintaining intestinal health.TNF-α can promote the increase of IL-6 and IL-1β, while large amounts of TNF-α, IL-6, and IL-1β may lead to increased intestinal infammation, thereby disrupting the intestinal mucosal barrier [53,54].IL-10 can inhibit the production of proinfammatory cytokines.Loss of IL-10 receptor expression can disrupt the regulatory role of macrophages and promote the spontaneous development of severe colitis [55].In our study, AOFPs reduced the level of TNF-α, IL-6, and IL-1β and increased the level of IL-10, which indicated that infammation in the colon has been alleviated after AOFP treatment.
When UC occurs, it is often accompanied by oxidative stress reactions, and previous studies have shown that excessive oxygen free radicals could promote the development of colitis and barrier damage [54,56].SOD is a metal ion-rich protein with powerful antioxidant efects, which converts superoxide anion radicals into oxygen and hydrogen peroxide.Terefore, SOD plays a crucial role in antioxidant activity and is also an important indicator for evaluating antioxidant activity [46].MPO is a peroxidase with pro-oxidant and proinfammatory efects.Its activity in the colon is closely related to neutrophil infltration.Terefore, its level can refect the degree of mucosal damage [54,57].MDA refects the intensity of lipid peroxidation in the body and indirectly shows the extent of tissue   peroxidative damage [58].Trough this research, we found AOFPs could increase the level of SOD and decreased the levels of MPO and MDA.Tese results indicated that AOFPs have a good protective efect on DSS-induced colonic oxidative damage.
Intestinal fora is closely related to the development of UC.Previous research has confrmed that DSS could lead to a decrease in the diversity and richness of intestinal fora in mice [59].At the phylum level, the ratio of Firmicutes to Bacteroidota (F/B) is a crucial marker for assessing these alterations.Disturbance of intestinal fora can lead to an increase in F/B values [60].Te research report proved that Octadecanol-H treatment can reduce the F/B ratio of mice, which may be related to the anti-infammatory efect of Bacteroidota [61,62].In our study, AOFPs also presented a reduced F/B ratio and increased the relative abundance of Bacteroidota.Tis indicated that the regulatory efect of polysaccharides at the phylum level may be one of the reasons for their anti-infammatory efects.Proteobacteria is a biomarker for microbial imbalance and the risk of UC [63].Te intestinal infammation deteriorated with the increase of the relative abundance of Proteobacteria [12].SC and AC could signifcantly reduce the relative abundance of Proteobacteria, which is benefcial for the stability of intestinal fora.
Research has found that the balance of intestinal fora levels is closely related to intestinal permeability, such as increasing the relative abundance of Bacteroides, Faecalibaculum, and Turicibacter and reducing the relative abundance of Desulfovibrio, Lactobacillus, Lachnospiraceae, Alistipes, and Enterorhabdus, which will lead to an increase in intestinal mucosal permeability [47].Te Bifdobacterium bifdum signifcantly enhances the intestinal TJ barrier in mice in a Toll-like receptor-2-dependent manner and protects the permeability of the mouse colon from DSS damage [64].Another study found that the relative abundance of norank_f__Muribaculaceae was downregulated, while the relative abundance of Streptococcus, Escherichia-Shigella, Romboutsia, Turicibacter, and Faecalibaculum is upregulated, which is related to the alteration of intestinal infammation [65].After treatment with AOFPs, we also found that intestinal fora was reversed at the genus level, such as an increase in the relative abundance of norank_f__Muribaculaceae and norank_f__Lachnospiraceae and a decrease in the relative abundance of Escherichia-Shigella, Romboutsia, Turicibacter, and Faecalibaculum, indicating that AOFPs can restore the imbalance of gut microbiota caused by DSS.
In order to discover the contribution of intestinal fora to colitis, we conducted a correlation analysis between differential microbiota and infammation and permeability indicators.Trough correlation analysis, we found Romboutsia, Turicibacter, Faecalibaculum, and Coriobacteriaceae_UCG-002 were signifcantly positively correlated with TNF-α, IL-6, and IL-1β.Te results indicated an increase in the proportion of these microbiota promotes the deterioration of intestinal infammation.Te correlation analysis results between microbiota and intestinal permeability indicators show that benefcial bacteria such as norank_f_Muribaculaceae were positively correlated with claudin 1 and E-cadherin, while Streptococcus was signifcantly negatively correlated with claudin 1, occludin, and E-  cadherin.Tese results suggested that polysaccharides may improve intestinal permeability by upregulating the nor-ank_f_Muribaculaceae and downregulating Streptococcus.Due to the inconsistent regulation of the intestinal fora by the four types of AOFPs, their anti-infammatory efects and barrier repair capabilities vary.For example, there is a signifcant correlation between Turicibacter, Faecalibaculum, and anti-infammatory factors.In the AL group, the relative abundance of Turicibacter and Faecalibaculum is closest to that of the CON group, and AL exhibits the best anti-infammatory efect in ELISA and real-time PCR results.However, the regulatory efect of AL on other bacterial communities is not ideal, and its ability to repair the intestinal barrier is not as good as other AOFPs.Tere is a signifcant correlation between norank_f__Muribaculaceae, norank_f__Lachnospiraceae, and claudin 1. DW has the strongest upregulation ability towards them.In western blot results, DW had the strongest recovery efect on claudin 1.
In addition, the overall function of the intestinal fora also varies with the composition of the intestinal fora proportion.Previous research found that intestinal fora in the DSS group can enhance the signal transduction and membrane transport capacity of bacteria and reduce the biosynthesis of other secondary metabolites and glycan biosynthesis and metabolism pathways [66].Our research results found that oral administration with DW, SC, and AC could signifcantly increase the biosynthesis of other secondary metabolites and reduce membrane transport.Tis indicates that AOFPs may contribute to the recovery of intestinal fora function.
Trough the gut microbiota analysis of AOFPs, we found that they play a comprehensive role in regulating gut infammation and barrier function by regulating the levels of gut microbiota.Among them, AL has a strong regulatory efect on the bacterial community of intestinal infammation, while DW has a strong regulatory efect on intestinal permeability.

Conclusions
In this study, we obtained four diferent polysaccharides through deionized water, NaCl, HCl, and NaOH extraction.At the same time, their physicochemical properties and structural characteristics were analyzed.Animal experiments have verifed the therapeutic efect of AOFPs on colitis induced by DSS.According to the results, we speculate that AOFPs have treated UC by regulating the intestinal fora to repair the intestinal barrier and inhibit intestinal infammation.

Figure 2 :
Figure 2: Experimental design and the efect of AOFPs on DSS-induced UC mice.(a) Modelling and treatment plans in animal experiments.(b) Body weight change.(c) DAI scores.Data are represented as mean ± SD (n � 10).### P < 0.001 compared with the control group.*P < 0.05, * * * P < 0.001 compared with the model group.
AOFPs on DSS-Induced UC Mice 3.3.1.Efects of AOFPs on Body Weight and DAI Scores in UC Mice.In order to study whether AOFPs have potential therapeutic efects on UC, this study fully evaluated the clinical symptoms of the mouse UC model induced by DSS, such as weight loss, diarrhea, and bloody stool.Figure 2(b)

Figure 5 :
Figure 5: Efects of AOFPs on colon length and histomorphology in DSS-induced colitis mice.(a) Colon length and images of each group.(b) Histopathological score and representative H&E staining images.Data are represented as mean ± SD (n � 6).### P < 0.001 compared with the control group.* P < 0.05, * * P < 0.01, and * * * P < 0.001 compared with the model group.

Figure 10 :
Figure 10: AOFPs alters microbial composition on the phylum level.(a) Compositions of the microbial community on the phylum level.(b) Te ratio of Firmicutes to Bacteroidota.(c) Te relative abundance of Bacteroidota.(d) Te relative abundance of Proteobacteria.(e) Te relative abundance of Actinobacteria.Data are presented as the mean ± SD (n � 6).### P < 0.001compared with the control group.* P < 0.05 and * * * P < 0.001 compared with the model group.

Figure 14 :
Figure 14: Analysis of diferences in KEGG metabolic pathways between groups.(a) MOD vs CON.(b) MOD vs DW.(c) MOD vs SC.(d) MOD vs AC.

Figure 1 :
Te processes of polysaccharide extracted from Alpinia oxyphylla fructus with diferent extraction solvents.

Table 2 :
Extraction yields, chemical properties, and compositions of AOFPs from A. oxyphylla by using four extraction solvents.