Fructus amomi hainanensis polysaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the field of medicines, and particularly relates to fructus amomi hainanensis polysaccharide as well as a preparation method and application thereof. According to the method, the active ingredients of the polysaccharide are extracted by taking the fructus amomi hainanensis as a raw material, HPGPC, UV, FT-IR and monosaccharide composition analysis are adopted for structural analysis, the structural formula of the fructus amomi hainanensis polysaccharide is obtained, the structural characteristics of the fructus amomi hainanensis polysaccharide are defined, and a material foundation is laid for pharmacodynamic activity research of the fructus amomi hainanensis polysaccharide. After treatment with the fructus amomi hainanensis polysaccharide, the DAI score of a mouse with ulcerative colitis and the pathological damage degree of colon tissue are obviously reduced, the levels of proinflammatory factors IL-1beta, TNF-alpha and IL-6 are obviously reduced, the level of an anti-inflammatory factor IL-10 is obviously increased, the expression reduction of tight junction protein caused by DSS can be recovered, and the ulcerative colitis can be inhibited. Disorder of metabolites in mice with ulcerative colitis can be regulated, and reduction of diversity and abundance of intestinal microorganisms of the UC mice can be reversed.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals, specifically relating to a Hainan Amomum villosum polysaccharide, its preparation method, and its application. Background Technology

[0002] Amomum villosum Lour., Amomum villosum Lour. var. xanthioides TLWu et Sen-jen, or Amomum longiligulare T.L.Wu are all belonging to the genus Amomum in the ginger family (Zingiberaceae). Amomum longiligulare has a pungent and warm nature, and is used to dispel dampness, stimulate appetite, warm the spleen, stop diarrhea, regulate qi, and calm the fetus. In the Li ethnic region, people often use its fruit to make a decoction to treat indigestion. Amomum longiligulare is mainly produced in Chengmai and Lingshui counties of Hainan Province. It is a unique variety of Hainan and has a history of use for over 1300 years.

[0003] Polysaccharides are polymers composed of more than 10 monosaccharides linked by multiple glycosidic bonds, and possess a variety of biological activities such as anti-inflammatory, anti-tumor, anti-radiation, antibacterial, anti-fatigue, antioxidant, antiviral, hypoglycemic, and immunomodulatory effects.

[0004] Ulcerative colitis (UC) is a typical inflammatory bowel disease (IBD). Due to its increasing incidence, it has become the most common chronic intestinal disease worldwide, characterized by recurrent intestinal bleeding and mucosal inflammation from the rectum to the proximal colon. Clinically, UC is characterized by weight loss, abdominal pain, diarrhea, fatigue, and rectal bleeding. The pathogenesis of UC is complex and unclear, and is related to multiple factors including environment, genetic susceptibility, mucosal barrier defects, immune response dysregulation, congenital and acquired immunodeficiency, and gut microbiota dysbiosis. Many plant polysaccharides have demonstrated the ability to regulate the gut microbiota and have gained widespread attention worldwide due to their good pharmacological activity and few side effects. Therefore, the development of Amomum villosum polysaccharides with therapeutic effects on ulcerative colitis has a positive promoting effect on the treatment of ulcerative colitis. Summary of the Invention

[0005] This invention provides a Hainan Amomum villosum polysaccharide, its preparation method, and its application. The prepared Hainan Amomum villosum polysaccharide has a significant therapeutic effect on mice with ulcerative colitis.

[0006] The technical solution of this invention is implemented as follows:

[0007] A homogeneous polysaccharide from *Amomum villosum* (Hainan Amomum villosum) has a number-average molecular weight (Mn) of 35202 Da, a weight-average molecular weight (Mw) of 53895 Da, a peak molecular weight (Mp) of 42580 Da, and a calculated dispersion index (PDI) (Mw / Mn) of 1.53. This Hainan Amomum villosum polysaccharide is a typical pectin polysaccharide containing HG and RG-I domains. The main chain consists of →4)-α-D-GalpA-(1→4)-α-D-Galp-(1→2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→), and the side chains consist of →3)-β-D-Galp-(1→3)-β-D-Galp-(1→). The structural formula is shown below:

[0008]

[0009] The above-mentioned extraction method for Hainan Amomum villosum polysaccharides includes the following steps:

[0010] S1, the dried Hainan Amomum fruit is crushed and sieved to obtain Hainan Amomum powder;

[0011] S2, Hainan Amomum villosum powder is decolorized and degreased, and the decolorized and degreased powder is dried;

[0012] S3, add dried Hainan cardamom powder to water, extract in a water bath, filter to remove filter residue after cooling, and concentrate the extract to obtain concentrated solution.

[0013] S4. Add the concentrated solution to anhydrous ethanol, mix well, let stand to precipitate, filter to obtain the precipitate; after redissolving in distilled water, remove the pigment to obtain a polysaccharide solution.

[0014] S5, polysaccharide solution to remove protein, then dialyzed, molecular weight cutoff 3500 Da; after dialysis, polysaccharide solution concentrated, freeze-dried, to obtain Hainan Amomum villosum crude polysaccharide;

[0015] S6. Quantitatively weigh the crude polysaccharide from *Amomum villosum* (Hainan Amomum villosum), dissolve it in ultrapure water to prepare a 5 mg / mL crude polysaccharide solution, filter, and purify by anion exchange chromatography. The sample was loaded into a DEAE-52 cellulose column and eluted sequentially with 0, 0.1, 0.2, 0.3, and 0.4 mol / L NaCl solutions at a gradient elution rate of 2 mL / min. 10 mL of eluent was collected from each tube, and 80 tubes were collected for each gradient. The phenol-sulfuric acid method was used for monitoring and detection. Four eluent components were identified: ALP-A, ALP-B, ALP-C, and ALP-D. The ALP-D eluent was concentrated to a molecular weight cutoff of 3500 Da, dialyzed, and then lyophilized.

[0016] S7. Weigh out polysaccharide ALP-D quantitatively, add it to distilled water and dissolve it completely. Centrifuge and collect the supernatant to remove insoluble precipitate. Then filter the polysaccharide solution using a microporous membrane and collect the filtered polysaccharide solution, which means the polysaccharide solution processing is complete. Then add the polysaccharide solution to the pre-treated gel chromatography column. After the polysaccharide solution is loaded, use the eluent to elute and collect the fraction with an elution time of 119-138 min. Concentrate and then freeze-dry to obtain high-purity Hainan Amomum villosum polysaccharide after separation and purification.

[0017] Furthermore, step S1

[0018] Furthermore, in step S2, the powder is soaked in 95% ethanol for 24 hours to decolorize and degrease; the powder is then dried at 40°C.

[0019] Further, in step S3, the water bath temperature is 100℃ for 1 hour, and the extraction is repeated 3 times. The extracts from the 3 extractions are combined. The volume ratio of Hainan Amomum powder to water in the 3 extractions is 20:1, 15:1, and 10:1 respectively. The extract is then concentrated to 1 / 5 of its original volume.

[0020] Furthermore, in step S4, the volume ratio of the concentrated solution to anhydrous ethanol is 1:4, and the solution is allowed to stand at 4°C for 24 hours to precipitate; the pigment is removed by activated carbon adsorption.

[0021] Further, in step S5, the Sevag method is used to remove proteins: chloroform and n-butanol solutions are mixed at a volume ratio of 4:1 to prepare the Sevag reagent. The dissolved Hainan Amomum villosum polysaccharide solution is mixed with the Sevag reagent at a volume ratio of 4:1. The solution is shaken on a shaker for 30 minutes, resulting in three layers: a polysaccharide aqueous solution layer, a protein layer, and an organic reagent layer from top to bottom. After shaking, the solution is centrifuged at 4000 rpm for 10 minutes to remove the denatured proteins between the organic layer and the aqueous phase. This process is repeated three times.

[0022] Furthermore, in S6, the polysaccharide solution prepared has a mass concentration of 5 mg / mL, and the DEAE-52 cellulose column has a specification of 2.5 × 40 cm.

[0023] Further, in step S7, the sample is centrifuged at 6000 rpm for 8 min and filtered using a 0.45 μm microporous membrane; the silica gel column is a Chromdex 200PG gel chromatography column, and the sample loading volume of the silica gel column is 1% of the column volume; the distilled water flow rate is controlled at 2.0 mL / min, 5 mL is collected from each tube, and the total elution volume is the same as the column volume.

[0024] This invention also provides the application of Hainan Amomum villosum polysaccharide in the preparation of anti-ulcerative colitis drugs.

[0025] This invention also provides the application of Hainan Amomum villosum polysaccharide in the preparation of formulations that reduce the DAI score and pathological damage of colon tissue in mice with ulcerative colitis.

[0026] This invention also provides the application of Hainan Amomum villosum polysaccharide in the preparation of formulations that reduce the levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6 and increase the level of anti-inflammatory factor IL-10.

[0027] This invention also provides the application of Hainan Amomum villosum polysaccharide in the preparation of formulations that restore the reduced expression of tight junction proteins caused by DSS, regulate the disorder of metabolites in mice with ulcerative colitis, and / or reverse the reduction in the diversity and abundance of intestinal microorganisms in UC mice.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] (1) This invention uses Hainan Amomum villosum as raw material to extract polysaccharide active ingredients. HPGPC, UV, FT-IR and monosaccharide composition analysis were used to preliminarily identify the chemical structure of the extracted Hainan Amomum villosum polysaccharide. At the same time, GC-MS and one-dimensional and two-dimensional NMR were combined to analyze the structure of Hainan Amomum villosum polysaccharide, obtain the structural formula of Hainan Amomum villosum polysaccharide, clarify the structural characteristics of Hainan Amomum villosum polysaccharide, and lay the material basis for the study of the pharmacological activity of Hainan Amomum villosum polysaccharide.

[0030] (2) The extraction method of Hainan Amomum villosum polysaccharide of the present invention adopts water extraction and alcohol precipitation method for extraction, and uses DEAE-52 cellulose and gel Chromdex 200PG column chromatography for separation and purification. The extracted Hainan Amomum villosum polysaccharide is a homogeneous polysaccharide, mainly containing one polysaccharide component. The extraction method of the present invention is simple, easy to operate and low in cost, and is a method that can be considered for large-scale production.

[0031] (3) This invention investigated the activity of Hainan Amomum villosum polysaccharide in the treatment of ulcerative colitis. It was found that treatment with Hainan Amomum villosum polysaccharide significantly reduced the DAI score and the degree of pathological damage in the colonic tissue of ulcerative colitis mice. Simultaneously, it was found that the levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6 were significantly reduced, while the level of anti-inflammatory factor IL-10 was significantly increased. Immunofluorescence results showed that Hainan Amomum villosum polysaccharide could restore the reduced expression of tight junction proteins caused by DSS. Non-targeted metabolic analysis showed that Hainan Amomum villosum polysaccharide could regulate the disorder of metabolites in ulcerative colitis mice. 16S rRNA detection results showed that ALP could reverse the reduction in intestinal microbial diversity and abundance in UC mice. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The results of the isolation and purification of Hainan Amomum villosum polysaccharide are as follows: (A) DEAE-52 elution curve; (B) Chromdex 200PG elution curve on gel filtration medium; (C) molecular weight of Hainan Amomum villosum polysaccharide; (D) Hainan Amomum villosum polysaccharide sample. Figure 2 The ultraviolet spectrum (left) and infrared spectrum analysis (right) of Hainan Amomum villosum polysaccharide.

[0034] Figure 3 For the monosaccharide composition analysis of Hainan Amomum villosum polysaccharide, (A) monosaccharide standard (1. mannose 2. glucosamine hydrochloride 3. rhamnose 4. glucuronic acid 5. galacturonic acid 6. glucosamine hydrochloride 7. glucose 8. galactose 9. xylose 10. arabinose 11. fucose); (B) monosaccharide composition of Hainan Amomum villosum polysaccharide.

[0035] Figure 4 This is a chromatogram of total methylated ions of Amomum villosum polysaccharide from Hainan.

[0036] Figure 5 The hydrogen NMR spectrum (top) and carbon NMR spectrum (bottom) of Hainan Amomum villosum polysaccharide.

[0037] Figure 6 The images show the HSQC spectrum (top) and COSY spectrum (bottom) of Hainan Amomum villosum polysaccharide.

[0038] Figure 7 The above image shows the HMBC (top) and NOESY (bottom) diagrams for Hainan Amomum villosum polysaccharide.

[0039] Figure 8 This study aimed to investigate the ameliorative effect of Hainan Amomum villosum polysaccharide (ALP) on DSS-induced colitis in mice. (A) Schematic diagram of the animal experiment process; (B) Colon images of each group; (C) DAI score; (D) Mouse body weight change; (E) Colon length; (F) Organ index of the spleen; (G) Histopathological scores of each group; (H) Histopathological sections of the colon tissue of each group. **P<0.01, ALP+DSS group and ALP group vs. DSS group. ## P<0.01, NC group _ vs DSS group.

[0040] Figure 9Effects of Hainan Amomum villosum polysaccharide on colonic tight junction protein expression and serum inflammatory factors in DSS-induced colitis mice. (A) Immunofluorescence staining of Claudin-1, Occludin, and ZO-1. (BD) Quantitative analysis of immunofluorescence signals of Claudin-1, Occludin, and ZO-1. (EH) IL-1β, IL-6, TNF-α, and IL-10; *P<0.05, **P<0.01, ALP+DSS group and ALP group vs DSS group; ## P<0.01, NC group _vs_DSS.

[0041] Figure 10 The Venn diagrams (top), non-metric multidimensional scaling (NMDS) analysis (bottom left), and principal coordinate analysis (PCoA) analysis (bottom right) of each group in the experiment on the effect of Hainan Amomum polysaccharide on the gut microbiota of DSS-induced UC mice are shown.

[0042] Figure 11 The figures below show the Ace (top left), Chao (top right), and Sobs indices (bottom) of Hainan Amomum villosum polysaccharide on the gut microbiota of DSS-induced UC mice.

[0043] Figure 12 The relative abundance of microbial composition at the phylum level (top figure) and the relative abundance of microbial composition at the genus level (bottom figure) in the experiment of Hainan Amomum polysaccharide on the gut microbiota of DSS-induced UC mice.

[0044] Figure 13 To investigate the differences in microbial groups among the groups, linear discriminant analysis (LDA) was used. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Example 1

[0047] In order to study and utilize Hainan Amomum villosum polysaccharide, this embodiment uses the traditional hot water soaking method to extract polysaccharide, and then obtains homogeneous Hainan Amomum villosum polysaccharide through alcohol precipitation, protein removal, dialysis and separation purification.

[0048] 1.1 Experimental Materials and Methods

[0049] 1.1.1 Instruments and Reagents

[0050] Table 1 Main Materials, Reagents and Instruments

[0051]

[0052]

[0053] 1.1.2 Extraction and purification of Amomum villosum polysaccharides from Hainan

[0054] Weigh out Hainan Amomum villosum, pulverize it using a pulverizer, and then soak it in 95% ethanol for 24 hours to decolorize and defatt it. Dry the defatted powder in a 40℃ oven. Extraction time: 1 hour; extraction temperature: 100℃; extraction frequency: 3 times; liquid-to-solid ratios for the three extractions: (20:1), (15:1), and (10:1) mL / g, respectively. After extraction, allow the extract to cool and filter to remove the residue. Combine the three extracts and concentrate them to 1 / 5 of their original volume using a rotary evaporator to obtain a concentrated solution. Add anhydrous ethanol to the concentrated solution at a volume ratio of 1:4, mix thoroughly, and allow to stand at 4℃ for 24 hours to precipitate. Filter to obtain the precipitate.

[0055] 1.1.2.1 Activated carbon adsorption method for pigment removal

[0056] This experiment used activated carbon adsorption to remove pigments. First, the obtained precipitate was redissolved with an appropriate amount of distilled water, and then 1% activated carbon (by mass of the solution) was added for decolorization. After decolorization for 40 minutes, the activated carbon was removed by centrifugation to obtain the decolorized Hainan Amomum villosum polysaccharide solution.

[0057] 1.1.2.2 Sevag method for protein removal

[0058] Protein removal using the Sevag method: Prepare the Sevag reagent by mixing chloroform and n-butanol solutions at a volume ratio of 4:1. Mix the decolorized Hainan Amomum villosum polysaccharide solution with the Sevag reagent at a volume ratio of 4:1. Shake on a shaker for 30 minutes. The solution will separate into three layers: a polysaccharide aqueous solution layer, a protein layer, and an organic reagent layer from top to bottom. After shaking, centrifuge at 4000 rpm for 10 minutes to remove the denatured protein between the organic layer and the aqueous phase, obtaining the polysaccharide aqueous solution layer. Repeat this process three times to obtain the crude Hainan Amomum villosum polysaccharide solution.

[0059] 1.1.2.3 Removal of small molecule impurities

[0060] The decolorized, defatted, and protein-removed crude polysaccharide solution was dialyzed using a dialysis bag (3500 Da) at 4°C for 24 hours, with the water changed three times during the process. After dialysis, the crude polysaccharide solution was concentrated using a rotary evaporator, and the concentrated polysaccharide solution was dried using a vacuum freeze dryer to obtain crude polysaccharide from Amomum villosum var. hainanense.

[0061] 1.1.3 Detection of protein content after purification of crude polysaccharide

[0062] Protein content was determined using the Coomassie Brilliant Blue assay. Following the instructions of the Coomassie Brilliant Blue kit, the protein content in the crude polysaccharide sample was detected. First, 1 mL of the protein standard solution was added to a standard tube containing 25 mg bovine serum albumin (BSA), and then the tube was vortexed to obtain a 25 mg / mL standard solution. The prepared protein standard was then stored at -20°C for long-term use. Before the protein detection experiment, an appropriate amount of the standard solution needed to be diluted using the same diluent as the sample diluent, resulting in a final protein concentration of 0.5 mg / mL. Subsequently, a standard curve was plotted according to the kit instructions. The standards were added to the 96-well plates at the following concentration gradients: 0, 1, 2, 4, 8, 12, 16, and 20 μL. If the volume of a well was less than 20 μL, it was brought to 20 μL with standard diluent. Simultaneously, the crude polysaccharide sample to be tested was diluted to an appropriate concentration with the diluent, and then 20 μL of the diluted solution was added to the 96-well plate. Subsequently, 200 μL of Coomassie Brilliant Blue solution was added to each well of a 96-well plate. After thorough mixing, the plate was incubated at room temperature for 3–5 minutes. The absorbance of each well was then measured at 595 nm and the data were recorded. The protein content in the sample could be calculated using a standard curve.

[0063] 1.1.4 DEAE-52 Isolation of Crude Polysaccharide from Amomum villosum var. hainanense

[0064] 1.1.4.1 DEAE-52 Fiber Chromatography Column Material Treatment

[0065] (1) Pretreatment of DEAE-52 cellulose: Weigh 200g of DEAE-52 cellulose, soak it in ultrapure water, and then filter it twice. Then, swell it in ultrapure water for 12 hours and filter it again. Next, soak the cellulose in 0.5mol / L NaOH solution for 30 minutes, and rinse the filtered cellulose with ultrapure water until it reaches neutrality. Treat it with 0.5mol / L HCl solution for 30 minutes, and then rinse the filtered cellulose with ultrapure water until it reaches neutrality. Finally, soak it again in 0.5mol / L NaOH solution for 30 minutes, and rinse the filtered cellulose with ultrapure water until it reaches neutrality. Finally, store the treated cellulose in a 4℃ refrigerator for later use.

[0066] (2) Regeneration of DEAE-52 cellulose: Pour the packing material into a beaker and soak it in a high-concentration NaCl solution (2 mol / L) for 1 hour, then rinse repeatedly with distilled water three times. Next, soak it in a 0.5 mol / L NaOH solution for 30 minutes, rinsing repeatedly with distilled water until the solution is neutral as determined by pH paper. Then, soak it in a 0.5 mol / L HCl solution for 30 minutes, rinsing repeatedly with distilled water until the solution is neutral as determined by pH paper. Finally, soak it again in a 0.5 mol / L NaOH solution for 30 minutes, rinsing repeatedly with distilled water until the solution is neutral as determined by pH paper. This completes the regeneration of DEAE-52 cellulose.

[0067] (3) Packing the DEAE-52 chromatography column: Pour the pretreated DEAE-52 cellulose into a beaker containing distilled water, suspending it in the water. Stir continuously with a glass rod until homogeneous. First, pour 1 / 3 of the distilled water into the chromatography column (2.5cm × 50cm). Then, slowly introduce the homogeneous packing material into the column using a glass rod, stirring continuously in the same direction to ensure uniform mixing of the packing material and distilled water, and to prevent the formation of air bubbles. After packing, equilibrate the column with distilled water at a flow rate of 2mL / min. After equilibration for 24 hours, the column is ready for use.

[0068] 1.1.4.2 Loading of crude polysaccharide solution and gradient elution

[0069] An appropriate amount of crude polysaccharide from *Amomum villosum* was weighed, dissolved in distilled water to prepare a 5 mg / mL polysaccharide solution, filtered, and purified by anion exchange chromatography. The sample was packed into a DEAE-52 cellulose column (2.5 cm × 40 cm), and eluted with different concentrations of NaCl solution, a total of 5 concentration gradients were set. First, elution was performed with distilled water without NaCl, followed by elution with 0.1, 0.2, 0.3, and 0.4 mol / L NaCl solutions sequentially. 80 tubes were collected for each elution gradient, with a flow rate controlled at 1–2 mL / min, and 10 mL per tube. The elution was then monitored using the phenol-sulfuric acid method. Four eluent components were identified: ALP-A, ALP-B, ALP-C, and ALP-D. ALP-D was the main purified component; its eluent was concentrated to one-fifth of its original volume, dialyzed (molecular weight cutoff 3500 Da), and lyophilized for further separation and purification.

[0070] 1.1.5 Gel separation of ALP-D

[0071] 1.1.5.1 Pretreatment of Gel Filtration Column Chromatography Column

[0072] (1) Pretreatment of Chromdex 200PG: First, according to the required amount of polysaccharide to be purified, weigh an appropriate amount of Chromdex 200PG and place it in a beaker. Add an appropriate amount of distilled water to the beaker to soak it. After the Chromdex 200PG is completely soaked, filter it with a vacuum filtration flask to remove the waste liquid after soaking. Then add 3 times the volume of distilled water and slowly stir and wash it with a glass rod. Filter to remove the waste liquid. Repeat this process several times until the Chromdex 200PG no longer emits an ethanol smell. Finally, add the same volume of distilled water as the Chromdex 200PG, stir well, and store it in a refrigerator at 4°C for later use.

[0073] (2) Column packing and equilibration: First, take the pretreated Chromdex 200PG out of the refrigerator and let it stand at room temperature. When the Chromdex 200PG is at the same temperature as the room temperature, stir it continuously with a glass rod to make it uniform. Then, slowly add Chromdex 200PG to the prepared chromatography column (φ2.6×100cm). After the chromatography column packing is completed, connect it to the chromatography system and set the flow rate to 30cm / h to press the column. When the gel surface of the chromatography column is stable, it indicates that the column packing is complete.

[0074] (3) Sample loading and elution: First, weigh an appropriate amount of polysaccharide ALP-D into a beaker, add an appropriate amount of distilled water to the beaker, and stir continuously with a glass rod until it is completely dissolved. Then, pour the polysaccharide solution into a centrifuge tube and centrifuge at 6000 rpm for 8 minutes. After centrifugation, take the supernatant and remove the insoluble precipitate. Then, filter the polysaccharide solution using a 0.45 μm microporous membrane and collect the filtered polysaccharide solution. The polysaccharide solution processing is now complete. The polysaccharide solution was then added to a pre-treated Chromdex 200PG gel chromatography column at a volume of 1% of the column volume. After loading, distilled water was used for elution at a flow rate of 2.0 mL / min, with 5 mL collected per tube. The total elution volume was the same as the column volume. A differential detector was used for online tracking, and the regions with higher peaks and better symmetry were collected. This process was repeated multiple times to complete the separation, purification, and enrichment of the polysaccharide. Finally, the fractions collected from the repeated elutions were concentrated under reduced pressure and then freeze-dried to obtain the purified high-purity Hainan Amomum villosum polysaccharide ALP-D1.

[0075] 1.1.5.2 Identification of polysaccharide purity and determination of molecular weight distribution

[0076] Accurately weigh 50 mg of dextran of different molecular sizes and place it in a 10 mL volumetric flask. Add a small amount of 0.05 mol / L NaCl solution. After the dextran is completely dissolved, add NaCl solution to bring the volume to the mark. This completes the preparation of a 5 mg / mL dextran standard. Filter the solution using a 0.22 μm microporous membrane before injection. Detect the dextran using high-performance gel permeation chromatography (HPLC). Then, plot the relative molecular weight on the ordinate and retention time on the abscissa, perform linear regression through data processing, and calculate the molecular weight calibration curve. To detect the molecular weight of sample ALP-D1, a test solution needs to be prepared, following the same preparation method as the dextran standard. The HPLC detection conditions are: mobile phase of 0.05 mol / L NaCl solution, flow rate of 0.65 mL / min, column temperature maintained at 40 °C, and injection volume of 30 μL.

[0077] 1.1.6 Structural characterization of polysaccharide ALP-D1

[0078] 1.1.6.1 Fourier Transform Infrared (FT-IR) and Ultraviolet-Vis (UV-vis) Spectroscopic Analysis of ALP-D1

[0079] First, weigh 1-2 mg of ALP-D1 and place it in a mortar. Then, weigh 200 mg of KBr and add it to the mortar. After thoroughly mixing the polysaccharide sample and KBr, use a tablet press to compress the mixture into transparent thin sheets. Finally, use an infrared spectrometer to scan and detect the sample, setting the wavelength range to 4000-4000 cm⁻¹. -1 And record the spectrum.

[0080] ALP-D1 was dissolved in distilled water (1 mg / mL), and the UV-Vis absorption spectrum of ALP-D1 was recorded in the range of 200–600 nm using a UV-Vis spectrophotometer (A590, AOE Instruments, Shanghai, China).

[0081] 2.1.6.2 Monosaccharide composition analysis of ALP-D1

[0082] Monosaccharide composition analysis of ALP-D1. The ALP-D1 sample was hydrolyzed with 2 mL of 3M TFA at 120 °C for 6 h. The TFA was then removed, and the hydrolysis product was dissolved in 0.8 mL of deionized water. 0.1 mL of 0.5 M PMP and 0.1 mL of 0.3 M sodium hydroxide solution were added, and the reaction was carried out at 70 °C for 30 min. After the reaction was complete, the solution was neutralized with 0.3 M hydrochloric acid and then extracted three times with chloroform. Finally, the sample solution was filtered through a 0.22 μm syringe filter and analyzed by HPLC (Thermo U3000 liquid chromatography system) using a ZORBAX Eclipse XDB-C18 column.

[0083] 1.1.6.3 Methylation analysis of ALP-D1

[0084] Accurately weigh 2–3 mg of polysaccharide sample and place it in a glass reaction flask. Add 1 mL of anhydrous DMSO and a methylation reagent, and allow the mixture to react completely to achieve full methylation. Then, add 2 mL of ultrapure water to terminate the reaction. The methylated monosaccharide is hydrolyzed in 1 mL of 2M trifluoroacetic acid (TFA) at 120 °C for 90 min, then reduced with sodium borohydride, and subsequently acetylated with acetic anhydride-pyridine. The product is dried to constant weight in a rotary evaporator. After methylation, the sample is hydrolyzed in 100 μL of 2M TFA at 120 °C for 90 min for further reduction and acetylation. Finally, the acetylated products are analyzed using gas chromatography-mass spectrometry (Agilent Technologies, USA).

[0085] 1.1.6.4 Nuclear Magnetic Resonance Analysis of ALP-D1

[0086] The lyophilized sample was dissolved in 0.5 mL of deuterated water (D2O) and then analyzed using a nuclear magnetic resonance (NMR) spectrometer. The detection range included one-dimensional and two-dimensional NMR. The calibration standards were: the hydrogen chemical shift (δH) of D2O was set to 4.70 ppm, and the carbon chemical shift (δC) of tetramethylsilane (TMS) was set to 0.00 ppm.

[0087] 1.2 Data Processing

[0088] All experiments were performed in triplicate, and results are expressed as mean ± standard deviation (±s). Data analysis and graph creation were performed using Origin and GraphPad Prism software, respectively.

[0089] 1.3 Results

[0090] 1.3.1 Separation and purification results

[0091] Hainan aleurone (ALP) was prepared using an optimized extraction process. After deproteinization with Sevage, depigmentation with activated charcoal, and removal of small molecule impurities by dialysis, the protein content was 0.66%. Figure 1 As shown in Figure A, four fractions of Hainan Amomum villosum polysaccharide (ALP) were separated using a DEAE-52 cellulose column and named ALP-A, ALP-B, ALP-C, and ALP-D, respectively. Their yields were 0.24%, 1.74%, 2.87%, and 9.45%, respectively. ALP-D had the highest content, therefore, further separation studies were conducted on ALP-D. Figure 1As shown in Figure B, the elution curve of the polysaccharide (ALP-D) obtained by gel filtration column chromatography was then analyzed by binding molecular weight. The fraction between 119 and 138 min was collected and named ALP-D1, with a yield of 10.8%. Figure 1 As shown in Table C and Table 2, the chromatogram of the purified polysaccharide fraction ALP-D1 shows a single symmetrical peak. The number-average molecular weight (Mn) of ALP-D1 is 35202 Da, the weight-average molecular weight (Mw) is 53895 Da, the peak molecular weight (Mp) is 42580 Da, and the calculated polydispersity index (PDI) (Mw / Mn) is 1.53. This indicates that ALP-D1 is a homogeneous polysaccharide; therefore, ALP-D1 was selected for further structural analysis. Figure 1 As shown in D, the homogeneous polysaccharide ALP-D1 sample obtained by separation is a white, fluffy powder.

[0092] Table 2 Molecular weight of ALP-D1

[0093]

[0094] 1.3.2 Structural characterization results of ALP-D1

[0095] 1.3.2.1 UV-vis analysis of ALP-D1

[0096] from Figure 2 The left figure shows that ALP-D1 does not produce absorption peaks at 260nm and 280nm in the ultraviolet absorption, indicating that ALP-D1 does not contain nucleic acids and proteins.

[0097] 1.3.2.2 FT-IR Analysis of ALP-D1

[0098] like Figure 2 As shown in the infrared spectrum on the right, ALP-D1 reaches a wavenumber of 3416.07 cm⁻¹. -1 There is a strong and broad peak, which indicates that there is OH stretching vibration between ALP-D1 molecules; ALP-D1 has a wavenumber of 2936.80 cm⁻¹. -1 A moderately intense peak indicates the presence of -CH3 and -CH2 stretching vibrations in ALP-D1; ALP-D1 at wavenumber 1733.13 cm⁻¹ -1 There is an absorption peak, which indicates that ALP-D1 exhibits C=O stretching vibration; 1611.94 cm⁻¹ -1 The absorption peak at 1450–1200 cm⁻¹ may be due to the stretching vibration of the carbonyl C=O group or the presence of water of crystallization; in addition, the absorption peak at 1450–1200 cm⁻¹ is also present. -1 Absorption peaks within the range (including 1413.62 cm⁻¹) -1 1328.33cm -1 and 1240.09cm-1 This corresponds to the variable-angle vibration and stretching vibration of CH. ALP-D1 at a wavenumber of 1146.44 cm⁻¹ -1 There is an absorption peak, which indicates that ALP-D1 has the stretching vibration of COC and also indicates that ALP-D1 has a pyranose structure; while 1150~1010cm -1 The two strong absorption peaks within the range are located at 1099.62 cm⁻¹. -1 and 1016.99cm -1 This further confirms the presence of pyranose structures in ALP-D1. (At 950.88 cm⁻¹) -1 The absorption peak at 893.04 cm⁻¹ reflects the vibrational characteristics of carbohydrate molecules. -1 The characteristic absorption peak at 835.20 cm⁻¹ is attributed to a β-pyranoside bond, suggesting the presence of a β-pyranose structure in the polysaccharide. -1 The absorption peak at 766.34 cm⁻¹ is related to the CH-angle vibration of the diastereomer of the α-terminal group of pyranose. Additionally, the peak at 766.34 cm⁻¹ is also relevant. -1 The absorption peak at that point reflects the symmetrical ring stretching vibration of the pyran ring.

[0099] 1.3.2.3 Monosaccharide composition analysis of ALP-D1

[0100] Depend on Figure 3 As shown, rhamnose (Rha), galacturonic acid (GalA), galactose (Gal), and arabinose (Ara) are the main components of ALP-D1, with a molar ratio of 16.05:52.83:9.68:8.32. Therefore, ALP-D1 is mainly an acidic polysaccharide with galacturonic acid (GalA) as its main monosaccharide component.

[0101] 1.3.2.4 Methylation analysis of ALP-D1

[0102] Based on the gas chromatography-mass spectrometry (GC-MS) analysis results, and by comparing with standards and CCRC database information, the sugar residue types corresponding to each chromatographic peak were determined, and the relative content of each sugar residue was calculated using the peak area. The sugar residue composition analysis results of the ALP-D1 sample are summarized as follows: Figure 4 As shown in Table 3, the ALP-D1 methylation results contained 15 linkage modes. According to the relative molar ratio analysis, the main linkage modes of ALP-D1 were found to be 1,2-Rhap, t-GalpA and 1,4-GalpA, with a molar ratio of 7.005:7.030:60.089.

[0103] Table 3. Results of ALP-D1 methylation analysis

[0104]

[0105] 1.3.2.5 NMR results of ALP-D1

[0106] To further analyze the structure of ALP-D1, one-dimensional NMR technology was first used. 1 H-NMR, 13 C-NMR was used to analyze and detect the chemical shifts of H and C in ALP-D1, and then more advanced two-dimensional NMR was used to analyze the chemical shifts of H and C in ALP-D1. Two-dimensional NMR techniques include COSY, NOESY, HSQC, and HMBC. By using one-dimensional and two-dimensional NMR techniques, not only can the chemical shift information of H and C of most sugar residues in ALP-D1 be known, but the connection sequence of all sugar residues in ALP-D1 can also be inferred.

[0107] Analysis of ALP-D1 using 1H-NMR and 13C-NMR revealed that the H signals of glycosidic bonds in ALP-D1 are mostly concentrated in the δ 3.0–5.5 ppm range, with H-1 being an antecedent proton, its resonance range being δ 4.5–5.5 ppm. Studies have shown that 1H-NMR can detect signals from certain sugar residues; for example, methyl proton signals can be identified in the high-field region δ 0.8–1.4 ppm. In NMR studies of polysaccharides, C-1 is an antecedent carbon signal, with its chemical shift signal generally in the δ 90–110 ppm range. The C-1 signal for α-carbons is in the δ 95–103 ppm range, and the C-1 signal for β-carbons is above δ 101 ppm. For example, the chemical shift signals from C-2 to C-5 are generally in the range of δ65–85 ppm. Studies have shown that C-2 with substitution sites shifts the chemical shift signal to a lower field. Unsubstituted C-6 is generally in the range of δ60 ppm, but substituted C-6 signals shift to a lower field, typically to δ69 ppm. Research has also shown that in the 13C-NMR detection of polysaccharides, specific sugar residues can be detected. For instance, the methyl carbon signal for the deoxygenated sugar at position 6 can be found in the range of δ15–20 ppm, while the carboxyl group signals for uronic acid and acetyl groups in polysaccharides are in the range of δ170–180 ppm.

[0108] like Figure 5 The image above 1 As shown in the 1H-NMR, regarding the proton resonance signal, the H signal of ALP-D1 is mainly in the range of δ3.0-5.5 ppm, with significant signal overlap. The signal range of the anomeric hydrogen region is in the range of δ4.5-5.5 ppm. Further analysis of ALP-D1... 1¹H-NMR revealed multiple signals in the δ 4.5–5.5 ppm range, indicating that ALP-D1 contains various types of sugar residues. However, these signals were difficult to distinguish due to their high degree of overlap. Spectra analysis showed that, in addition to the aforementioned H signals, other H signals were mainly located in the δ 3.0–4.5 ppm range. These H signals were also difficult to distinguish due to significant overlap. Figure 5 The 13C-NMR spectrum in the image below shows that it is similar to ALP-D1. 1 Compared to H-NMR, it has relatively fewer spectral lines. The isocarbon head signal of polysaccharides is in the range of δ90–110 ppm. Multiple signals were found in this region of ALP-D1, indicating that ALP-D1 has multiple different types of sugar residues.

[0109] exist Figure 5 In the 1H-NMR and 13C-NMR spectra, ALP-D1 exhibited the following characteristic signal peaks: not only was the methyl proton H-6 of Rha found in the δ 1.10–1.30 ppm range, but a δ 16.51 ppm peak was also found in the 13C-NMR spectrum, indicating the presence of the C-6 of Rha in ALP-D1. This may be related to the deoxygenation structure in ALP-D1. Furthermore, through… Figure 6 E discovered a cross-peak at δ 1.15 / 16.51 ppm in the HSQC of ALP-D1, indicating the presence of a small amount of Rha sugar residues in ALP-D1. This result corroborates previous monosaccharide composition analysis. Previous monosaccharide composition analysis of ALP-D1 revealed a high content of galacturonic acid, with a relative content as high as 52.83%. Furthermore... 1 A strong absorption peak at δ174.94 ppm was found in 3C-NMR, which is a characteristic absorption peak of C-6 in galacturonic acid. Further 13C-NMR detection of ALP-D1 showed that ALP-D1 contains a large amount of galacturonic acid.

[0110] Monosaccharide composition and methylation analysis of ALP-D1 revealed a high content of galacturonic acid, along with small amounts of rhamnose, arabinose, and galactose. Methylation analysis further indicated that the sample primarily contained 1,4-galacturonic acid (GalpA) residues, along with 1,3,6-galactose (Galp), 1,3-galactose (Galp), terminal arabinose (T-Araf), 1,2-rhamnose (Rhap), and 1,2,4-rhamnose (Rhap). Based on this, it is speculated that the ALP-D1 sample may be rich in HG-type domains and contain small amounts of RG-I-type pectin domains. Analysis of the monosaccharide composition, methylation, and NMR results of ALP-D1 revealed multiple anodic signals. A review of existing polysaccharide literature revealed that most polysaccharide signals in the δ 98–102 ppm range were presumed to be anodic carbon signals of α-GalpA. The presence of this signal in ALP-D1 suggests the possible presence of α-GalpA within ALP-D1. Stronger signals in the δ 102–105 ppm region likely correspond to anodic carbon signals of the sugar residue β-Galp. Furthermore, signals were also observed in the δ 106–110 ppm range, presumed to be anodic carbon signals of the sugar residue α-Araf. Two reducing end-group signals appeared at δ 92.05 ppm and δ 96.04 ppm, as indicated in the HSQC spectrum. Figure 6 The cross peaks of these two signals (δ5.22 / 92.05ppm and δ4.52 / 96.04ppm) were found in the above figure. Then, the results were analyzed using COSY (…). Figure 6(See figure below) The cross-peaks in the spectrum are δ5.22 / 3.74ppm and δ4.52 / 3.41ppm, from which the H-2 chemical shifts of the two sugar residues are deduced to be δ3.74ppm and δ3.41ppm, respectively. By combining the methylation and monosaccharide composition results of ALP-D1, and based on reports in known literature, it is speculated that the C-1 signal peaks of Rα and Rβ may indicate the presence of α-GalpA and β-GalpA in ALP-D1. ALP-D1 contains several distinct anodic region peaks; therefore, these signals can be used to further analyze the structure of ALP-D1. The study of anodic signal peaks in ALP-D1 revealed anodic signals at δ4.97 / 98.86 ppm, δ4.40 / 102.61 ppm, δ4.55 / 103.79 ppm, δ5.15 / 108.95 ppm, and δ5.16 / 98.39 ppm. After classifying these anodic signals, two-dimensional and one-dimensional NMR spectroscopy experiments, combined with methylation and monosaccharide composition analysis, and further analysis based on similar results from relevant literature, labeled the sugar residue signals in the anodic regions of ALP-D1 as GA1,4, G1,3,6, G1,3, At, Rha1,2, and Rha1,2,4, respectively. The main 1H and 13C signals in ALP-D1 were assigned, and detailed assignments for 1H and 13C signals are shown in Table 4.

[0111] Table 4. Chemical shifts of 1H and 13C for each sugar residue in ALP-D1 samples.

[0112]

[0113] "--" indicates not determined or not detected.

[0114] Sugar residue GA1,4: Anomalies of this sugar residue were identified at δ4.97 ppm (H-1) and δ98.86 ppm (C-1) using HSQC and COSY experiments, indicating its α-configuration. Based on relevant polysaccharide literature, it was speculated that GA1,4 might be an α-GalpA sugar residue. Furthermore, this study confirmed the H-1 chemical shift signal of α-GalpA at δ4.97 ppm using HSQC and COSY experiments. (In the COSY spectrum...) Figure 6 In the figure below, cross-peak analysis further deduced the chemical shifts of H-2, H-3, and H-4, with chemical shift signals of δ3.67 ppm, δ3.91 ppm, and δ4.33 ppm, respectively. After assigning hydrogens to the sugar ring, and as... Figure 6As shown in the figure above, this experiment also assigned chemical shifts to C-1, C-2, C-3, and C-4 on the sugar ring, which were δ98.86 ppm, δ67.88 ppm, δ68.58 ppm, and δ77.82 ppm, respectively. Furthermore, a cross-peak between H-5 and C-5 was observed in the HSQC spectrum, and their chemical shift cross-peak signals were found to be δ4.73 / 70.93 ppm. In addition to the above findings, it was also discovered that the signals of C-1 and C-4 in the sugar ring shifted to lower fields, indicating that C-1 and C-4 in the sugar ring have undergone substitution. Therefore, based on relevant literature research, this experiment hypothesizes that the sugar ring structure in ALP-D1 may be →4)-α-GalpA-(1→), and labels it as GA1,4, with its chemical shift signals shown in Table 1.4.

[0115] Following a similar method, and combining the results of monosaccharide composition determination, methylation analysis, and literature reports, by further deducing the hydrogen and carbon signals of other residues, it can be inferred that G1,3,6 corresponds to →3,6)-β-D-Galp-(1→), G1,3 corresponds to →3)-β-D-Galp-(1→), At is inferred to be α-L-Araf-(1→), Rha1,2 is →2)-α-L-Rhap-(1→), and Rha1,2,4 is →2,4)-α-L-Rhap-(1→). The chemical shifts of hydrogen (H) and carbon (C) of each major sugar residue in the polysaccharide samples are assigned in Table 1.4.

[0116] From ALP-D1 HMBC ( Figure 7 (Above image) and NOESY ( Figure 7The following coupling signals can be observed in the figure below: (1) In the NOESY spectrum, a significant spatial correlation signal is observed between the anterior proton with a chemical shift of 4.97 ppm and the H-4 proton at 4.33 ppm in the GA1,4 sugar unit. This phenomenon confirms the structural feature of the →4)-α-D-GalpA-(1→4)-α-D-GalpA-(1→ connection; (2) In the NOESY spectrum, a significant NOE correlation peak is detected between the H-1 proton with a δ value of 5.16 ppm in the Rha1,2 sugar unit and the H-4 proton (δ 4.33 ppm) in the GA1,4 sugar unit. This result supports the structural inference of the →2)-α-D-Rhap-(1→4)-α-D-GalpA-(1→ connection. (3) In the HMBC, it is found that the H-1 (δ 5.16 ppm) of Rha1,2,4 and the C-4 (δ 77.82 ppm) of GA1,4 are related. m) has a cross peak (Rha1,2,4H-1 / GA1,4C-4), indicating the presence of →2,4)-α-D-Rhap-(1→4)-α-D-GalpA-(1→ connection; (4) In the NOESY spectrum, the H-1 (δ4.55ppm) of sugar residue G1,3 and the H-3 (δ3.64ppm) of sugar residue G1,3,6 have a cross peak (G1,3H-1 / G1,3,6H-3), indicating that G1,3 has a →3)-β-D-Galp-(1→3,6)-β-D-Galp-(1→ connection mode, its The linking site is O-3; (5) In NOESY, the H-1 (δ4.40ppm) of G1,3,6 and the H-4 (δ3.64ppm) of sugar residue Rha1,2,4 have a cross peak (G1,3,6H-1 / Rha1,2,4H-4), indicating that there is a →3,6)-β-D-Galp-(1→2,4)-α-D-Rhap-(1→ link, the linking site is located at O-4; (6) In HMBC, the H-1 (δ5.15ppm) of At and the C-6 (δ68.58ppm) of G1,3,6 have a cross peak (At H-1 / G1,3,6C-6), indicating that At may have an α-L-Araf-(1→3,6)-β-D-Galp-(1→ link, the linking site is located at O-6.

[0117] Comprehensive structural analysis of ALP-D1 revealed it to be a complex polysaccharide, primarily composed of numerous HG domains and some RG-I domains with side chains. Its structural elements are shown below:

[0118]

[0119] Example 2

[0120] Given the important role of the gut microbiota in human health, the anti-inflammatory effect of polysaccharides from Amomum villosum Lour. var. xanthioides T. L. Wu & Senjen in alleviating DSS-induced ulcerative colitis in mice by regulating the gut microbiota will be studied. The polysaccharide component ALP-D1 obtained by separation and purification is used for the polysaccharides from Amomum villosum Lour. var. xanthioides T. L. Wu & Senjen, and ALP-D1 will be abbreviated as ALP in the following experiments.

[0121] 2.1 Experimental Materials and Methods

[0122] 2.1.1 Instruments and Reagents

[0123] Table 5 Main Reagents, Materials, and Instruments

[0124]

[0125]

[0126] 2.1.2 Establishment and Administration of Ulcerative Colitis Mouse Models

[0127] Male C57BL / 6J mice (weighing 18 - 22 g, 8 weeks old) were provided by Hunan Slack Experimental Animal Co., Ltd. (Certificate No.: SCXK(Xiang)2024 - 0002). All procedures related to the handling of experimental animals in this experiment were approved by the Animal Management Committee, Animal Care Review Committee, and Animal Use Committee of Hainan Medical University. Before the formal experiment began, the animals were housed in an environment with a temperature of 25 ± 0.5 °C and a humidity of 50 - 60%, allowed to drink water and eat freely, given free access to water and food for 3 days, and the light - dark cycle was 12 h.

[0128] The experimental grouping and administration methods are as Figure 8 shown in A. After 3 days of adaptive feeding of the mice, the mice were randomly divided into 4 groups, with 10 mice in each group, namely the normal group (NC), the model group (DSS), the administration group ALP (abbreviation of ALP-D1), and the administration treatment group (ALP + DSS). The entire experimental period was 14 days. On days 1 - 6, the mice in the ALP group and the ALP + DSS group were intragastrically administered polysaccharides from Amomum villosum Lour. var. xanthioides T. L. Wu & Senjen ALP (200 mg / kg), while the NC group and the DSS group were given the same volume of normal saline in the same way. On days 7 - 14, after intragastric administration to each group of mice, except for the NC group and the ALP group, all the mice in the remaining groups drank drinking water containing 2.5% DSS until the end of the experiment, and various indicators such as the body weight, mental state, and fecal formation degree of each group of mice were recorded on days 7 - 14. On day 14, the mice were sacrificed by cervical dislocation, and blood, spleen, colonic contents, and colonic tissues were collected. After quick-freezing in a liquid nitrogen tank, they were finally stored in a -80 °C refrigerator.

[0129] 2.1.3 Spleen Organ Index and Disease Activity Index (DAI)

[0130] After the mice were sacrificed, the blood on the surface of the spleen tissue was dried with filter paper, and then the tissue was weighed. The organ index of the spleen tissue was calculated using the following formula:

[0131] Organ index = Organ weight (mg) / Body weight (g)

[0132] Disease Activity Index (DAI). During the 7–14 day administration period, the mice's body weight change, fecal viscosity, and degree of rectal bleeding were observed, recorded, and scored daily. The formula for calculating the body weight change of each group of mice was: Body weight change (%) = [1 + (current body weight of each group of mice - original body weight of each group of mice) / original body weight of each group of mice] × 100%; the specific scoring criteria were: 0 = no weight loss; 1 = 1–5%; 2 = 5–10%; 3 = 10–15%; 4 ≥ 15%. Fecal viscosity was defined as: 0 = granular stool; 2 = loose, pasty stool; 4 = watery stool. The specific scoring criteria for rectal bleeding were: 0 = no occult blood; 1 = positive occult blood; 4 = significant bleeding.

[0133] 2.1.4 Histopathological evaluation

[0134] Colonic tissue was fixed in 4% paraformaldehyde solution and embedded in paraffin. Colon sections were then prepared at 4 μm thickness and stained with hematoxylin and eosin (H&E). Pathological scoring was performed on each group of colonic tissues as follows: 0 = no inflammation; 1 = mild inflammation with mononuclear cell infiltration; 2 = moderate inflammation; 3 = high-level inflammation with significantly thickened cell walls and increased vascular density; 4 = maximum inflammation with transmural leukocyte infiltration and goblet cell loss.

[0135] 2.1.5 Detection of inflammatory factors

[0136] After the experiment, blood was collected from the eyeballs. After the blood was left at room temperature for 3 hours, it was centrifuged in a centrifuge at 4°C for 10 minutes at 3000 rpm. Serum was collected, and inflammatory factors IL-6, IL-1β, TNF-α, and IL-10 were detected according to the instructions on the ELISA kit.

[0137] 2.1.6 Immunofluorescence analysis

[0138] The pretreatment for immunofluorescence analysis was similar to that described in "2.1.4". Paraffin-embedded colon sections, 4 μm thick and fixed in 4% paraformaldehyde (PFA), were dehydrated in xylene and ethanol, and then the antigen was extracted using sodium citrate buffer. After blocking with 5% bovine serum albumin (BSA) solution for 60 min, the colon tissue sections were incubated overnight at 4°C with the designated primary antibody, followed by incubation with the corresponding secondary antibody at room temperature for 1 h. The sections were then reverse-stained with DAPI to label cell nuclei. Finally, the results were observed and photographed using a fluorescence microscope.

[0139] 2.1.7 Non-targeted metabolomics analysis of colon tissue

[0140] Colon tissue from "2.1.2" was harvested, and an appropriate amount of colon tissue was cut off. The colon contents were cleaned with physiological saline, and after cleaning, the tissue was placed in a cryovial and immediately placed in a liquid nitrogen tank for freezing. After several hours of freezing, the colon samples were stored in a -80°C freezer. The colon tissues from each group of mice were sent to Shanghai Meiji Biotechnology Co., Ltd. for non-targeted metabolomics analysis, and six biological replicates were established for each group.

[0141] 2.1.8 Analysis of Gut Microbial Composition Based on 16S rRNA Sequencing

[0142] use Total microbial genomic DNA was extracted from colon contents samples of mice from each group using a soil DNA kit (Omega Bio-tek, Nocross, GA, USA). Subsequently, the V1-V2 regions of the bacterial 16S rRNA gene were amplified from the DNA of the colon samples from each group of mice using a pair of universal primers 27F (AGRGTTYGATYMTGGCTCAG) and 1492R (RGYTACCTTGTTACGACTT). Sequencing was then performed on the Nextseq2000 platform, and the raw sequences were imported into a database.

[0143] Bioinformatics analysis of the colonic gut microbiota was performed using the Majorbio cloud platform (https: / / cloud.majorbio.com). The Vegan v2.5-3 software package was used, and principal coordinate analysis (PCoA) was employed to determine the similarity between microbial communities in different samples. Linear discriminant analysis (LDA) effect size (LEfSe) (http: / / huttenhower.sph.harvard.edu / LEfSe) was used to identify significantly abundant bacterial taxa (from phylum to genus) among different groups.

[0144] 2.2 Data Processing

[0145] Experimental data were processed using GraphPad Prism 9.5.1 software for statistical analysis. All quantitative data were expressed as mean ± standard deviation. Plotting analysis was performed using Origin 9.0 software (OriginLab, USA). Pearson correlation analysis was used to analyze the correlation between dose and serum biochemical indicators and gut microbiota (BAs). P < 0.05 was considered statistically significant.

[0146] 2.3 Results

[0147] 2.3.1 ALP alleviates symptoms and histopathological damage in UC mice

[0148] like Figure 8 As shown in Figure D, during the DSS modeling period, the body weight of mice in each group remained relatively constant from day 6 to day 11. However, starting from day 12, the body weight of the DSS group showed a decreasing trend, especially on day 14, when the decrease in body weight in the DSS group was significantly greater than that in the NC group, indicating that UC modeling was successful. Figure 8 As shown in Figure C, using the DAI value as an indicator of UC severity, the DAI value of the DSS group was significantly increased compared to the NC group from day 7 to day 14. Compared to the DSS group, the DAI value of the ALP+DSS group was significantly lower in the ALP+DSS group. The DAI values ​​of the ALP group and the NC group were basically the same, indicating that ALP did not harm the mouse colon. In particular, on the last day of the experiment, day 14, the DAI of the DSS group was significantly higher than that of the NC group, while the DAI of the ALP+DSS group was significantly lower than that of the DSS group, indicating that ALP significantly alleviated the progression of UC.

[0149] One characteristic of DSS-induced colitis in mice is the shortening of the colon. After euthanizing the mice, the colon length of each group was calculated and photographed for recording. Figure 8 As shown in B and 8E. Compare the changes in the mouse colon between groups. Figure 8 As shown in Figure B, the colon of normal mice (NC) is longer and thinner, and the intestinal contents are granular. In contrast, mice in the DSS-induced group showed varying degrees of colonic pathological changes, including edema and congestion, and the intestinal contents became irregular, further confirming the successful establishment of the ulcerative colitis (UC) model. The ALP+DSS group had a lower level of colonic damage, a relatively longer colon, no obvious edema, and granular intestinal contents. The colon length of the DSS group was significantly shorter than that of the NC group (P<0.01). Figure 8 E). The colon length in the ALP+DSS group was significantly longer than that in the DSS group (P<0.01). Figure 8 E). Studies have shown that DSS leads to an increase in the spleen index in mice. Therefore, the organ index of the mouse spleen was statistically analyzed, and the results are as follows: Figure 8 As shown in Figure F. The results showed that the spleen index of mice in the DSS group was significantly higher than that in the NC group and the ALP group (P<0.01), while the spleen index of mice in the ALP+DSS group was significantly lower than that in the DSS group (P<0.01). In conclusion, ALP inhibited the phenotypic changes induced by DSS in UC mice and improved UC symptoms.

[0150] HE-stained colon tissue sections, as shown Figure 8 As shown in (H), the histopathological scores of each group are as follows: Figure 8As shown in (F). The NC and ALP groups showed normal morphology: dense columnar epithelial mucosa and submucosa were clearly separated, intestinal crypts were deep and narrow, and goblet cells were abundant. The DSS group exhibited a significant inflammatory response, especially the DSS group, whose pathological sections showed severe epithelial surface damage, submucosal edema, loss of numerous crypts accompanied by severe structural changes (such as crypt tortuosity, upward displacement, and surface irregularity), basal lymphocyte aggregation, and extensive inflammatory cell infiltration. The ALP+DSS group showed significantly lower levels of pathological damage, with no obvious edema observed, but a small amount of inflammatory cell infiltration and mild inflammatory response were present. These results indicate that the ulcerative colitis (UC) mouse model was successfully established, and that ALP has a significant protective effect against DSS-induced colonic injury.

[0151] 2.3.2 ALP alleviates colonic barrier disruption in DSS-induced colitis mice

[0152] This experiment used immunofluorescence to detect the expression levels of tight junction proteins (ZO-1, Occludin, and Claudin-1) in colon tissue. The results are as follows: Figure 9 As shown in (AD), compared with the NC group, the expression of tight junction proteins ZO-1, Occludin, and Claudin-1 was significantly decreased in the DSS group (P<0.01), consistent with the fluorescence results. Compared with the DSS group, the expression of tight junction proteins (ZO-1, Occludin, and Claudin-1) in the ALP+DSS group was significantly higher than that in the DSS group (P<0.01), indicating that ALP can restore the decrease in tight junction protein expression caused by DSS and maintain the integrity of the intestinal barrier.

[0153] 2.3.3 Results of the determination of inflammatory factors in mouse serum

[0154] To assess the effect of ALP on systemic inflammation in DSS-induced colitis mice, the serum levels of inflammatory factors IL-1β, TNF-α, IL-6, and IL-10 were measured in each group of mice. Serum pro-inflammatory factors (IL-1β, TNF-α, and IL-6) and anti-inflammatory factor (IL-10) were detected by ELISA. Figure 9 As shown in (EG), the levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6 in the DSS group were significantly higher than those in the NC group (P<0.01); ALP treatment reversed DSS-induced inflammation-related factors, as the serum levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6 in the ALP+DSS group were significantly lower than those in the DSS group (P<0.01). Figure 9 As shown in Figure H, the level of the anti-inflammatory factor IL-10 in the DSS group was significantly lower than that in the NC group (P<0.01), while the level of the anti-inflammatory factor IL-10 in the ALP+DSS group was significantly higher than that in the DSS group (P<0.05).

[0155] 2.3.4 Effects of ALP on gut microbiota dysbiosis in UC mice

[0156] To investigate the effects of ALP on the gut microbiota of mice with DSS-induced colitis, this study used 16S rRNA technology to detect the gut microbiota in feces from the colon of each group of mice (NC, DSS, ALP, ALP+DSS). Figure 10 As shown in the Venn diagram, each group collectively possesses 123 OTUs, with 4, 5, 3, and 9 unique OUTs identified in the NC, DSS, ALP, and ALP+DSS groups, respectively. Figure 10 As shown, principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS) revealed significant differences between the NC and ALP groups and other groups. ALP intervention led to a shift in gut microbiota, with the ALP+DSS group showing a significant difference from the DSS group. We used the Ace, Chao, and Sobs indices to assess gut microbiota diversity and abundance, and the results are as follows: Figure 11 As shown, the results indicate that DSS leads to a decrease in the abundance and diversity of gut microbiota in mice, while ALP treatment partially reversed the decrease in gut microbiota diversity and abundance in UC mice. Furthermore, the Ace, Chao, and Sobs indices of the ALP-only group were higher than those of the NC group, suggesting that ALP can act as a prebiotic to increase the diversity and abundance of gut microbiota.

[0157] To more specifically assess the changes in gut microbiota structure in each experimental group, the relative abundance of colonic microbiota in each group of mice was examined at the phylum and genus levels. The results are as follows: Figure 12At the phylum level, compared with the NC group, the DSS group showed a decrease in the relative abundance of Bacillota and Actinomycetota, while the relative abundance of Bacteroidetes remained basically unchanged. Among them, the relative abundance of Pseudomonas, a harmful intestinal bacterium, increased significantly. Compared with the DSS group, the ALP group showed an increase in the relative abundance of Bacillota, Actinomycetota, and Bacteroidetes, while the relative abundance of Pseudomonas decreased. Compared with the DSS group, the ALP+DSS group showed an increase in the relative abundance of Bacillota, Actinomycetota, and Thermodesulfobacteriota, while the relative abundance of Bacteroidetes and Pseudomonas decreased. At the genus level, compared with the NC group, the relative abundance of Staphylococcus and Ligilactobacillus decreased in the DSS group, while the relative abundance of Escherichia increased significantly. Compared with the DSS group, the relative abundance of Staphylococcus and Ligilactobacillus increased significantly in the ALP group, while the relative abundance of Escherichia decreased significantly. Compared with the DSS group, the relative abundance of Staphylococcus and Ligilactobacillus increased in the ALP+DSS group, while the relative abundance of Escherichia decreased significantly.

[0158] To identify species with significant differences in abundance at different levels, the microbial communities of the four experimental groups were compared using LEfSe analysis. The results are shown below. Figure 13 Screening at different taxonomic levels revealed the following dominant bacteria in the NC group: o-Bacillales and f-Staphylococcaceae. In the DSS group, p-Pseudomonadota, c-Gammaproteobacteria, and g-Escherichia were dominant. In the ALP group, f-Lactobacillaceae and g-Lactobacillus were dominant. In the ALP+DSS group, p-Actinomycetota, g-Faecalibaculum, and f-Bifidobacteriaceae were dominant.

[0159] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A polysaccharide from Hainan Amomum villosum, characterized in that, The Hainan Amomum villosum polysaccharide has a number-average molecular weight of 35202 Da, a weight-average molecular weight of 53895 Da, a peak molecular weight of 42580 Da, and a calculated dispersion index of 1.

53. The main chain consists of →4)-α-D-GalpA-(1→4)-α-D-Galp-(1→2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→2)-α-L-Rhap-(1→4)-α-D-GalpA-(1→), and the side chains consist of →3)-β-D-Galp-(1→3)-β-D-Galp-(1→). The structural formula is shown below:

2. The method for extracting Hainan Amomum villosum polysaccharide as described in claim 1, characterized in that, Includes the following steps: S1, the dried Hainan Amomum fruit is crushed and sieved to obtain Hainan Amomum powder; S2, Hainan Amomum villosum powder is decolorized and degreased, and the decolorized and degreased powder is dried; S3. Add the dried Hainan Amomum powder to water and extract it in a water bath. After cooling, filter to remove the residue to obtain the extract. Concentrate the extract to obtain the concentrate. S4. Add the concentrated solution to anhydrous ethanol, mix well, let stand to precipitate, filter to obtain the precipitate; after redissolving in distilled water, remove the pigment to obtain a polysaccharide solution. S5, polysaccharide solution to remove protein, then dialyzed, molecular weight cutoff 3500 Da; after dialysis, polysaccharide solution concentrated, freeze-dried, to obtain Hainan Amomum villosum crude polysaccharide; S6. Quantitatively weigh the crude polysaccharide from *Amomum villosum* (Hainan Amomum villosum), dissolve it in ultrapure water to prepare a 5 mg / mL crude polysaccharide solution, filter, and purify by anion exchange chromatography. The sample was loaded into a DE AE-52 cellulose column and eluted sequentially with 0, 0.1, 0.2, 0.3, and 0.4 mol / L NaCl solutions at a gradient elution rate of 1–2 mL / min. 10 mL of eluent was collected from each tube, and 80 tubes were collected for each gradient. The phenol-sulfuric acid method was used for monitoring and detection. Four eluent components were identified: ALP-A, ALP-B, ALP-C, and ALP-D. The ALP-D eluent was concentrated to a molecular weight cutoff of 3500 Da, dialyzed, and then lyophilized. S7. Weigh out polysaccharide ALP-D quantitatively, add it to distilled water and dissolve it completely. Centrifuge and collect the supernatant to remove insoluble precipitate. Then filter the polysaccharide solution using a microporous membrane, collect the filtered polysaccharide solution, and add it to a pre-treated gel chromatography column. After the polysaccharide solution is loaded, elute with eluent and collect the fraction with an elution time of 119-138 min. Concentrate the fraction and then freeze-dry it to obtain high-purity Hainan Amomum villosum polysaccharide after separation and purification.

3. The extraction method as described in claim 2, characterized in that, In step S1, the powder is pulverized; in step S2, it is soaked in 95% ethanol for 24 hours to decolorize and degrease; the powder is then dried at 40°C.

4. The extraction method as described in claim 2, characterized in that, In step S3, the water bath temperature is 100℃ for 1 hour, and the extraction is repeated 3 times. The extracts from the 3 extractions are combined. The volume ratio of Hainan Amomum powder to water in the 3 extractions is 20:1, 15:1, and 10:1, respectively. The extract is concentrated to 1 / 5 of its original volume. In step S4, the volume ratio of the concentrated liquid to anhydrous ethanol is 1:4, and the mixture is allowed to stand at 4℃ for 24 hours to precipitate. The pigments are removed by activated carbon adsorption.

5. The extraction method as described in claim 2, characterized in that, In step S5, the Sevag method is used to remove proteins: chloroform and n-butanol solutions are mixed at a volume ratio of 4:1 to prepare the Sevag reagent. The dissolved Hainan Amomum villosum polysaccharide solution is then mixed with the Sevag reagent at a volume ratio of 4:

1. The solution is shaken on a shaker for 30 minutes, resulting in three layers: a polysaccharide aqueous solution layer, a protein layer, and an organic reagent layer from top to bottom. After shaking, the solution is centrifuged at 4000 rpm for 10 minutes to remove the denatured proteins between the organic layer and the aqueous phase. This process is repeated three times.

6. The extraction method as described in claim 2, characterized in that, In step S6, the prepared polysaccharide solution has a mass concentration of 5 mg / mL, and the DEAE-52 cellulose column has a specification of 2.5 × 40 cm. In step S7, the sample is centrifuged at 6000 rpm for 8 min and filtered using a 0.45 μm microporous membrane. The gel chromatography column is a Chromdex 200PG gel chromatography column, and the sample loading volume is 1% of the column volume. The distilled water flow rate is controlled at 2.0 mL / min, and 5 mL is collected from each tube, with the total elution volume being the same as the column volume.

7. The use of the Hainan Amomum villosum polysaccharide as described in claim 1 in the preparation of anti-ulcerative colitis drugs.

8. The application of the Hainan Amomum polysaccharide as described in claim 1 in the preparation of a formulation that reduces the DAI score and pathological damage of colonic tissue in mice with ulcerative colitis.

9. The application of the Hainan Amomum polysaccharide as described in claim 1 in the preparation of formulations that reduce the levels of pro-inflammatory factors IL-1β, TNF-α, and IL-6 and increase the level of anti-inflammatory factor IL-10.

10. The application of the Hainan Amomum villosum polysaccharide as described in claim 1 in the preparation of a formulation for restoring the reduced expression of tight junction proteins caused by DSS, regulating the disorder of metabolites in mice with ulcerative colitis and / or reversing the reduction in the diversity and abundance of intestinal microorganisms in UC mice.

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