Application of hypecoum erectum polysaccharide in preparation of anti-ulcerative colitis product

Abstract

The invention relates to application of hypecoum erectum polysaccharide in preparation of ulcerative colitis resisting products, and belongs to the technical field of ulcerative colitis resisting products. The hypecoum erectum polysaccharide disclosed by the invention can be used for remarkably relieving the shortening of colon of a mouse with colitis, restoring the completeness of colon wall, relieving inflammatory cell infiltration and playing a remarkable role in resisting ulcerative colitis by regulating intestinal flora, increasing the generation of short-chain fatty acid, regulating the level of inflammatory factors and the like, and has a remarkable intestinal protection function.

Description

Technical Field

[0001] This invention relates to the use of anise polysaccharide in the preparation of anti-ulcerative colitis products, belonging to the technical field of anti-ulcerative colitis products. Background Technology

[0002] Pectin, derived from medicinal plants, is gaining increasing recognition as a highly promising natural bioactive compound. It possesses various biological activities, including anti-inflammatory, immunomodulatory, antioxidant, antitumor, and gut microbiota-regulating effects. Pectin's unique biological activity, coupled with its low or non-toxicity, makes it a key focus of research in food science, nutritional supplements, and pharmaceuticals. Horned fennel (Hypecoum leptocarpum), known in Mongolian medicine as "Galun-Tabag," has the effects of removing "sticky" substances, clearing heat, and detoxifying, and is commonly used to treat influenza, jaundice, fatigue fever, and toxic heat. Modern research primarily focuses on the anti-inflammatory properties of the alkaloid components of horned fennel, with pharmacological activity studies demonstrating anti-inflammatory, antipyretic, analgesic, and hepatoprotective effects.

[0003] Ulcerative colitis (UC) is a chronic inflammatory bowel disease that severely impacts quality of life, making it a global health problem and imposing a significant economic burden. Ulcerative colitis also increases the risk of colorectal cancer, with a cumulative incidence rate of 18% over 30 years. Current treatments for ulcerative colitis aim to achieve and maintain remission, using medications including corticosteroids, mesalazine, infliximab, azathioprine, and methotrexate; however, these drugs have limited efficacy and significant side effects. Long-term use of corticosteroids can lead to drug dependence and serious complications, while immunosuppressants can damage the immune system. Biologics are expensive and often cause infections and allergic reactions. Therefore, finding an effective and affordable treatment for ulcerative colitis remains a challenge, highlighting the urgent need for newer, better-tolerated drugs.

[0004] Currently, there are no reports on the effects of crude polysaccharide from star anise on improving ulcerative colitis or its use in drugs for treating this disease. Furthermore, current research on star anise polysaccharide mainly focuses on the extraction process of its crude polysaccharide components. Further separation, purification, structural identification, and systematic research on the protective effects of star anise polysaccharide on the intestines are still insufficient. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide the use of anise polysaccharide in the preparation of anti-ulcerative colitis products.

[0006] In order to solve the above-mentioned technical problems, the present invention provides the following technical solution: the use of anise polysaccharide in the preparation of anti-ulcerative colitis products.

[0007] The anise polysaccharide of this invention can significantly reduce colonic shortening in mice with colitis, restore colonic wall integrity, reduce inflammatory cell infiltration, and exert significant anti-ulcerative colitis effects by regulating intestinal flora, increasing short-chain fatty acid production, and regulating inflammatory factor levels, thus exhibiting significant intestinal protective function.

[0008] In one specific embodiment, the anise polysaccharide is fine-fruited anise polysaccharide.

[0009] In one specific embodiment, the anise polysaccharide has a molecular weight of 19.7 kDa and has homogalacturonic acid (HG) and rhamnogalacturonic acid-I (RG-I) domains; the RG-I domain contains repeating disaccharide units composed of 1,4-α-galacturonic acid and 1,2-α-rhamnose, with arabinogalactan branches, and the galacturonic acid residues in the HG region are partially methylated.

[0010] In one specific embodiment, the anise polysaccharide is composed of rhamnose, galacturonic acid, galactose, arabinose and xylose, wherein the molar ratio of rhamnose, galacturonic acid, galactose, arabinose and xylose is (9.1):(83.4):(3.5):(2.4):(1.5).

[0011] According to an embodiment of the use of anise polysaccharide in the preparation of anti-ulcerative colitis products, the anise polysaccharide is obtained by the following method: the whole anise plant is cut into small pieces, ground into fine powder, and the fine powder is extracted with hot water; the supernatant is collected by centrifugation and concentrated; the concentrate is deproteinized using the Sevag method, precipitated with ethanol, centrifuged, freeze-dried, and crude anise polysaccharide is collected and labeled as HLP; the crude anise polysaccharide is further fractionated by DEAE cellulose column chromatography, eluted with sodium chloride, and the elution curve is tracked and detected by phenol-sulfuric acid method. The main peak product is collected according to the elution curve and labeled as HLP-4. The main peak product HLP-4 is then purified by gel column chromatography to obtain anise polysaccharide, labeled as HLP-4-2.

[0012] Specifically, the fine powder is subjected to two hot water extractions (water-to-powder ratio of 20:1 mL / g) at 60-100℃ for 2-5 hours, with a sodium chloride elution concentration of 0-2 mol / L. This invention establishes a three-stage purification system of "water extraction and alcohol precipitation-ion exchange chromatography-molecular sieve gel chromatography" for the extraction of anise polysaccharides, achieving significant results. Furthermore, this preparation method is simple, mild, efficient, and suitable for large-scale production.

[0013] The invention also includes the formulation of anise polysaccharide with acceptable edible excipients into tablets, granules, decoctions or capsules.

[0014] This invention has discovered that crude polysaccharide from aniseed possesses a significant ability to inhibit intestinal inflammation in mice with ulcerative colitis. This means that aniseed polysaccharide plays a crucial role in the anti-inflammatory efficacy of aniseed, similar to other anti-inflammatory components. This invention, starting from the extraction, separation, purification, and structural identification of aniseed polysaccharide, provides a method for its use in the preparation of anti-ulcerative colitis drugs, laying the foundation for quality control and in-depth research into its intestinal protective activity.

[0015] This invention is the first to demonstrate, through animal experiments, that HLP-4-2 significantly alleviates symptoms in a dose-dependent manner in a dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) mouse model, manifested as a decrease in disease activity index, improved colon length, and a reduction in spleen index. Mechanistically, HLP-4-2 enhances the intestinal barrier by increasing mucin secretion and protecting goblet cells, and reduces inflammation by decreasing pro-inflammatory cytokines such as tumor necrosis factor-α.

[0016] The HLP-4-2 of this invention can also improve the intestinal flora, increase beneficial short-chain fatty acid (SCFA) producing bacteria such as Prevotella, and reduce harmful bacteria such as Klebsiella. This change in flora increases the level of short-chain fatty acids (especially propionic acid and isobutyric acid) while decreasing the level of isohexanoic acid. Fecal microbiota transplantation (FMT) experiments have confirmed that these changes in microorganisms contribute to its therapeutic effect. These results support the traditional application of anise polysaccharides in the treatment of inflammatory diseases and provide theoretical basis and technical support for the development and quality control of anise polysaccharides in the preparation of products for treating ulcerative colitis. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the experimental effect of HLP-4-2 of the present invention in improving DSS-induced colitis in mice; (A) Schematic diagram of experimental design; (B) Representative images; (C) Changes in colon length; (D) Changes in body weight; (E) Disease Activity Index (DAI) score; (F) Spleen index (data are expressed as mean ± standard deviation, n=6); (GJ) Relative mRNA expression levels of key inflammatory cytokines: interleukin-6 (IL-6, G), interleukin-1β (IL-1β, H), tumor necrosis factor-α (TNF-α, I), cyclooxygenase-2 (COX-2, J) Figure 2 This is a schematic diagram illustrating the experimental effects of HLP-4-2 of the present invention on colonic histopathology; (A) Hematoxylin-eosin staining (H&E staining, magnification: ×100, ×400); lymphocytes (↑), fibroblasts (↑), and neutrophils (↑) indicate the corresponding cell types in the H&E staining image, respectively; (B) Colonic Alsin blue-periodic acid-Schiff staining (AB-PAS staining, magnification: ×100, ×400); (C) Histological scoring; (D) Detection of the number of Alsin blue positive areas (AB area) and goblet cells using ImageJ software. Figure 3 This is an experimental result diagram showing the effect of HLP-4-2 of the present invention on the intestinal flora composition of colitis mice; (A) phylum-level microbial composition; (B, C) relative abundance of Bacteroidetes and Firmicutes at the phylum level; (D) Bacteroidetes / Firmicutes ratio (relative abundance); (E) relative abundance of gut microbiota at the genus level (abbreviations: Erys. for Erysipelothrixceae, Staph. for Staphylococciaceae); (F) differences in the relative abundance of specific gut bacteria at the genus level among groups; (G, H) relative content of acetic acid, propionic acid, butyric acid, and total short-chain fatty acids (SCFAs). Figure 4 The AF is the nuclear magnetic resonance spectrum of HLP-4-2 in this invention; (A) 1H, (B) 13C, (C) COSY, (D) HSQC, (E) ROESY, (F) HMBC; Figure 5 This is an experimental spectrum for the structural identification of HLP-4-2 in this invention; (A) Ultraviolet full-spectrum scanning; (B) Fourier transform infrared spectroscopy (FT-IR spectroscopy); (C) High performance liquid chromatography analysis of monosaccharides; (D) Differential thermogravimetric analysis (DTG curve); (E) Thermogravimetric analysis curve (TGA curve); (F) Monosaccharide composition of HLP-4-2 after complete carboxyl reduction; (G) Congo red experiment; (H) Scanning electron microscope images (magnification: ×100, ×500, ×1000) Figure 6 This is a schematic diagram of the HLP-4-2 structure of the present invention; Figure 7 This is a schematic diagram of the experimental process of the present invention; Figure 8 This is a schematic diagram illustrating the experimental process and results of the purity evaluation of HLP-4-2 in this invention; Figure 9 This is the TIC chromatogram of HLP-4-2 methylation analysis according to the present invention; Figure 10 This is a mass spectrum of fragment ions representing the peak shown in this invention. Figure 11This is the TOCSY spectrum of the present invention. Figure 12 shows the distortion-free polarization transfer enhancement spectrum (DEPT135) of this invention. Detailed Implementation

[0018] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0019] Example 1 1. Preparation of Anise Polysaccharide: The whole plant of *Anise esculenta* was cut into small pieces and ground into a fine powder. The powder was extracted twice with hot water (water-to-material ratio of 20:1 mL / g) at 60-100℃ for 2-5 hours; the supernatant was collected by centrifugation. The solution was concentrated, deproteinized using the Sevag method, precipitated with ethanol, centrifuged, and freeze-dried to obtain crude polysaccharide HLP for further analysis; the crude polysaccharide was further fractionated using a DEAE cellulose column and labeled as HLP. It was eluted sequentially with 0-2 mol / L sodium chloride, and the elution curve was tracked using the phenol-sulfuric acid method. The main peak product was collected according to the elution curve and labeled as HLP-4. The main peak product (HLP-4) was purified by gel column chromatography to obtain anise polysaccharide, labeled as HLP-4-2.

[0020] 2. HLP-4-2 Purity Evaluation Method The total sugar content of HLP-4-2 was determined using the phenol-sulfuric acid method with glucose (Glc) as the standard; the uronic acid content was determined using the sulfuric acid-carbazole method; and the protein content was determined using the Coomassie brilliant blue method. The average molecular weight (Mw) was determined by high-performance gel permeation chromatography (HPGPC) using a Shodex OH-pak SB-804 HQ column (7 μm, 8 × 300 mm, Shodex, Tokyo, Japan) equipped with a differential refractive index detector (RID).

[0021] like Figure 8 A shows the extraction and purification of HLP-4-2; firstly, HLP (10.67%, Figure 8B) was extracted from fine-fruited anise powder, and then the crude HLP was fractionated by DEAE-52 column to obtain HLP-4 (6.55%). Figure 4 B); HLP-4 was then purified by size exclusion chromatography to obtain the main component HLP-4-2 (55.79%). Figure 8 C). Based on molecular weight distribution, HLP-4-2 is the major polysaccharide of fennel. HLP-4-2 exhibits a single symmetrical elution peak in high performance gel permeation chromatography (HPGPC); its purity (homogeneous polysaccharide) was determined to be >99% by area normalization, and its estimated molecular weight (Mw) is 1.97 kDa.

[0022] 3. Structural Feature Analysis HLP-4-2 was analyzed by monosaccharide composition, Congo red assay, thermogravimetric analysis (TGA), scanning electron microscopy (SEM), methylation assay, and nuclear magnetic resonance (NMR). 3.1 Monosaccharide Composition Analysis The monosaccharide composition and molar ratio were determined using a modified, established method (Q. Yuan et al., 2024). Specifically, HLP-4-2 solution (5 mg / mL) was mixed with an equal volume of 4 mol / L trifluoroacetic acid (TFA) and hydrolyzed at 120 °C for 2 h, followed by cooling to room temperature. The hydrolysis product was derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) at 70 °C for 100 min. The PMP derivatives were analyzed by high-performance liquid chromatography (HPLC) using a Shimadzu Prominence LC-2030C 3D system equipped with a Zorbax Eclipse Plus C18 column (4.6 × 250 mm, Agilent).

[0023] In the experiment, the chemical composition of HLP-4-2 was quantified as a weight percentage (mean ± standard deviation).

[0024] Main components: carbohydrates (89.1±0.01%), uronic acid (82.81±1.6%); protein content is extremely low (0.9%, standard deviation = 0, consistent results in repeated experiments); the low protein content is consistent with the results of ultraviolet full-wavelength scanning.

[0025] As shown in Table 1 and Figure 5 As shown in Figure C, based on the retention time and peak area of ​​the monosaccharide standards, HLP-4-2 contains rhamnose (Rha), galacturonic acid (GalA), galactose (Gal), arabinose (Ara), and xylose (Xyl), with a molar ratio of 9.1:83.4:3.5:2.4:1.5. Based on its monosaccharide composition, it is inferred to be a mixed pectin, mainly composed of homogalacturonic acid (HG) and rhamnose-galacturonic acid polysaccharide-I (RG-I) domains. The ratio of (arabinose + galactose) to rhamnose reflects the number or length of the RG-I side chains.

[0026] Table 1. Monosaccharide composition analysis results of HLP-4-2

[0027] a) Calculated as: HG = GalA - Rha; b) Calculated as: RG-I = [GalA - HG] + Rha + Ara + Gal; Rha / GalA: Contribution of the RG-I region to the total pectin; (Ara + Gal) / Rha: Degree of RG-I branching. Experimental data showed high linearity: galacturonic acid / (galactose + arabinose + rhamnose + xylose) = 5.1, low branching index (0.65, (arabinose + galactose) / rhamnose), RG-I accounting for 24.2%, HG accounting for 74.3%, and a low rhamnose / galacturonic acid ratio (0.11). Consistent with the above results, this confirms that it is mainly composed of linear HG with limited RG-I content. It is noteworthy that the structural characteristics of HLP-4-2 (mainly linear HG with limited RG-I content) may be related to its biological activity.

[0028] Experimental studies have shown that the regulatory effect of HG pectin on ulcerative colitis (UC) is related to its galacturonic acid backbone, which can promote the growth of anti-inflammatory bacteria, reduce intestinal damage, and decrease inflammatory cell infiltration. In contrast, colonic fermentation of RG-I produces more butyrate, thereby regulating inflammation.

[0029] 3.2 Congo Red Experiment like Figure 5 As shown in G, the helical conformation of HLP-4-2 was elucidated using the Congo red assay, and the results showed its maximum absorption wavelength. A significant red shift was observed; in 0.1 mol / L sodium hydroxide solution, HLP-4-2 appeared to retain a stable helical structure and could bind effectively to Congo red, which supports its possible helical characteristics.

[0030] 3.3 Thermogravimetric analysis (TGA) and scanning electron microscopy (SEM) analysis The thermal stability of HLP-4-2 was evaluated by thermogravimetric analysis, such as... Figure 5 As shown in D and E, differential heat Thermogravimetric (DTG) and thermogravimetric (TG) curves were obtained. Initial thermogravimetric analysis (25–130 °C) showed a slight weight loss of 17.5%, attributed to the evaporation of free / bound water. The differential thermogravimetric curves reflected the weight loss rate, thermal decomposition temperature range, and thermal stability, providing guidance for its application in temperature-sensitive environments.

[0031] like Figure 5As shown in H, the surface morphology of HLP-4-2 was observed using scanning electron microscopy at magnifications of ×100, ×500, and ×1000. The results showed that its surface was smooth, with a sheet-like structure and a large number of pores of 100–600 μm, which are typical pectin morphologies and may be related to its strong gelation properties.

[0032] 3.4 Methylation Experiment The carboxyl-reduced HLP-4-2 was methylated using sodium hydroxide powder in dimethyl sulfoxide (DMSO) and iodomethane (CH3I). The methylated sample was hydrolyzed in 2 mol / L TFA at 120 °C for 2 h, then reduced with NaBH4 and acetylated with acetic anhydride. The resulting partially methylated sugar alcohol acetate (PMAA) was redissolved in dichloromethane (CH2Cl2) and analyzed using an Agilent 6890–5973 gas chromatography-mass spectrometry (GC-MS) system equipped with an Rxi-5 Sil MS capillary column (30 m × 0.25 mm, 0.25 μm). The PMAA was identified by comparing its mass spectra with an online database (https: / / www.ccrc.uga.edu / specdb / ms / pmaa / pframe.html).

[0033] The results are shown in Table 2. Figure 9 , Figure 10 As shown, three arabinosyl glycosidic bond linkages were identified: terminal Araf, →5)-Araf-(1→ and →3,5)-Araf-(1→), and five galactosyl glycosidic bond linkages: terminal Galp, →4)-Galp-(1→, →6)-Galp-(1→, →3,4)-Galp-(1→ and →4,6)-Galp-(1→). Based on the monosaccharide composition results, most galactosyl linkage patterns in HLP-4-2 are presumably derived from the reduction of GalpA, indicating the presence of abundant linear HG or possibly partially branched HG. Furthermore, three rhamnosyl residue linkages were identified: terminal Rhap, →2)-Rhap-(1→ (14.1%) and →2,4)-Rhap-(1→ (1.3%), indicating the presence of a small amount of RG-I regions in HLP-4-2.

[0034] Table 2. Methylation Analysis Results

[0035] 3.5 Nuclear Magnetic Resonance (NMR) Analysis The sample was redissolved in 0.5 mL of heavy water. In the middle, freeze-dry, repeat the process three times, using Deuterium substitution for exchangeable protons. The sample was then redissolved in 0.5 mL. One-dimensional (¹H, ¹³C) and two-dimensional (¹H-¹H COSY, TOCSY, ROESY; ¹H-¹³C HSQC, HMBC) NMR spectra were acquired using a Bruker AVANCE NEO 600 MHz spectrometer equipped with a BBO cryogenic probe. Two-dimensional spectra were acquired according to Bruker's standard protocol. The ¹H / ¹³C NMR signals of polysaccharides were analyzed using the above two-dimensional isonuclear / heteronuclear experiments.

[0036] like Figure 4 As shown, the structural analysis of HLP-4-2 was further advanced using one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy techniques. For example... Figure 4 A shows ¹H NMR and Figure 4 Analysis of the ¹³C NMR spectrum shown in Figure B, combined with the methylation results, reveals 15 cross peaks (δ 5.25 / 112.3, 5.12 / 110.3, 5.10 / 110.3, 5.16 / 110.1, 4.65 / 106.8, 4.64 / 106.8, 4.52 / 105.8, 4.75 / 103.7, 4.98 / 103.1, 5.16 / 102.1, 5.12 / 102.1, 5.04 / 100.4, 5.29 / 101.3, 4.62 / 99.1, and 5.35 / 94.9 ppm) in the anterior carbon region of the heteronuclear single quantum coherence (HSQC) spectrum, as shown in Figure 4D. The results showed that HLP-4-2 contained at least 15 different sugar residues, which were ordered from high to low anodic carbon signal and the corresponding abbreviations are shown in Table 3.

[0037] The assignment of H1 to H5 signals of GA1,4, as shown in Figure 4C, was confirmed by the cross peaks observed in the correlation spectrum (COSY spectrum) (δ 5.12 / 3.75 (H1 / H2), δ 3.75 / 4.03 (H2 / H3), δ 4.03 / 4.47 (H3 / H4), and δ 4.47 / 4.69 (H4 / H5)). Subsequently, the C1 to C5 signals of GA1,4, as shown in Table 2, were confirmed by the corresponding coupling signals in the HSQC spectrum shown in Figure 3D. C6 of GA1,4 was identified by the cross peak of δ 177.6 / 4.69 (C6 / H5 of GA1,4) in the heteronuclear multibond correlation (HMBC) spectrum shown in Figure 3F.

[0038] The presence of 1,4-α-6-O-methylgalacturonic acid (GA1,4Me) was confirmed by detecting the -OCH3 group signal at δ 55.8 / 3.82 and the characteristic cross peaks of C5 / H5 at δ 73.4 / (5.10 / 5.18) and C4 / H4 at δ 81.4 / 4.47 compared to GA1,4. Furthermore, in the HMBC spectrum (Figure 3F), [further details are needed]. of The signal is coupled to C6 of GA1,4Me at δ 173.8 / 3.82. The assignment of H1 to H4 signals of GA1,4Me is verified by the COSY spectrum (Figure 4C).

[0039] Based on the unique chemical shift patterns of residues R1,2 and R1,2,4, R1,2 was identified as 1,2-α-rhamnose (Rhap), and R1,2,4 as 1,2,4-α-rhamnose. The high-field H2 signal observed at approximately 4.0 ppm, the small shift of H6 (1.26 or 1.31 ppm), and the high-field C2 chemical shift at approximately 79 ppm played a crucial role in confirming these assignments.

[0040] Residues tAa, tAb, A1,5, and A1,3,5 were identified as α-arabinofuranose based on the relative high-field chemical shift of C1 (109.8–111.9 ppm). Starting from the anomeric proton signal (δ 5.25) of residue tAa, COSY spectrum ( Figure 4 C) shows cross-peaks of H1 / H2 (δ 5.25 / 4.22), H2 / H3 (δ 4.22 / 4.02), H3 / H4 (δ 4.02 / 4.14), and H4 / H5 (δ 4.14 / 3.74 and 4.14 / 3.83), a result obtained by... Figure 11 The fully correlated spectrum (TOCSY spectrum) shown was confirmed. Accordingly, the HSQC spectrum shown in Figure 3D exhibits cross peaks at H1 / C1 (δ 5.25 / 112.3), H2 / C2 (δ 4.22 / 85.0), H3 / C3 (δ 4.02 / 79.6), H4 / C4 (δ 4.14 / 86.5), and H5 / C5 (δ 3.74 / 63.8 and 3.83 / 63.8).

[0041] Based on the above NMR data, the distortion-free polarization transfer enhancement spectrum (DEPT135) results shown in Figure 12, and methylation analysis, residue tAa was ultimately identified as α-L-arabinofuranose-(1→); similarly, residue tAb was identified as α-L-arabinofuranose-(1→). Compared with the terminal residues tAa, tAb, and unsubstituted arabinofuranose residues, residues A1 and 5 exhibited high-field shifts at C5 (and H5), thus identifying them as →5)-α-arabinofuranose-(1→; similarly, residues A1,3, and 5 were identified as →3,5)-α-arabinofuranose-(1→).

[0042] Based on the relatively low field chemical shift of H2 (<3.7 ppm) and the relatively high field chemical shift of the substituted C4 position relative to the corresponding unsubstituted galactose (Galp), residues G1,4 and tG are classified as →4)-β-galactose-(1→ and β-galactose-(1→), respectively. In the HSQC spectrum, glucuronic acid (GlcA1,4) and methylglucuronic acid (tGlcAMe) both show a correlation between the ¹H signal at 4.52 ppm and the ¹³C signal at 105.8 ppm, and their C6 chemical shifts are 177.6 and 173.8 ppm, respectively. The signal of tGlcAMe shows the characteristic resonance of the carbonyl carbon, indicating that the uronic acid has undergone esterification. The cross-peaks at 4.52 / 105.8, 3.37 / 75.8, 3.58 / 77.2, and 3.59 / 82.0 correspond to the H1 / C1, H2 / C2, H3 / C3, and H4 / C4 positions of residue GlcA1,4, respectively. Although the H4 / C4 chemical shift signal of tGlcAMe is not obvious, it can be inferred from the comparison of chemical shift signals that methylation may have occurred at the C4 position of tGlcAMe.

[0043] As shown in Table 3, Figure 4 As shown in E and F, the glycosidic bonds between different residues were resolved based on the correlation signals observed in the Rotated Coordinate System Correlation Spectroscopy (ROESY) and Heteronuclear Multi-Bond Correlation Spectroscopy (HMBC). The coupling signals at δ 5.12 / 4.12 ppm (H1 of GA1,4a and H2 of R1,2,4) and δ 5.29 / 4.43 ppm (H1 of R1,2,4 / R1,2 and H4 of GA1,4b) indicate that there may be alternating linkages between →2)-α-rhamnose-(1→ and →4)-α-galacturonic acid-(1→). Furthermore, the cross-peaks at δ 5.29 / 80.0 ppm (H1 of R1,2,4 / R1,2 and C4 of GA1,4b) and δ (4.43 / 4.47) / 101.3 ppm (H4 of GA1,4a,b / GA1,4Me and C1 of R1,2,4 / R1,2) in the HMBC spectrum further confirm the linkage relationship between the residues.

[0044] The cross-peaks at δ 5.12 / 4.47 ppm (H1 of GA1,4a and H4 of GA1,4b) and δ 4.98 / 4.47 ppm (H1 of GA1,4Me and H4 of GA1,4a / GA1,4Me) in the ROESY spectrum indicate the presence of galacturonic acid polysaccharides linked by →4)-α-galacturonic acid-(1→). Combined with ROESY and HMBC spectral data, the classic rhamnose-galacturonic acid polysaccharide I (RG-I) backbone structure is clearly confirmed, characterized by an alternating linkage pattern of →4)-α-galacturonic acid-(1→2)-α-rhamnose-(1→).

[0045] In the ROESY spectrum, a cross-peak was observed at δ 5.12 / 4.43 ppm, corresponding to the H1 of tΔUMe and the H4 of GA1,4Me, indicating the presence of a structural fragment of galacturonic acid-6-methoxy-(1→4)-α-galacturonic acid-6-methoxy-(1→), suggesting possible enzyme-mediated modification in the plant. Cross-peaks at δ 4.65 / 4.18 ppm (H1 of G1,4 / tG and H4 of G1,4) and δ 3.71 / 80.2 ppm (H4 of R1,2,4 and C1 of G1,4) suggest the presence of glycosidic bonds such as →4)-β-galactose-(1→4)-β-galactose-(1→ and →4)-α-galactose-(1→2,4)-α-rhamnose-(1→). Further analysis of the ROESY spectrum showed that δ The cross peaks at 4.52 / 3.59 ppm (H1 and H4 of GlcA1,4) indicate the presence of →4)-β-glucuronic acid-(1→4)-β-glucuronic acid-(1→glycosidic bond).

[0046] Furthermore, the identification of linkage patterns such as →4)-β-galactose-(1→2,4)-α-rhamnose-(1→) highlights the significant structural heterogeneity of this polysaccharide. Simultaneously, the cross-peaks at δ 4.75 / 3.59 ppm (H1 of tR and H4 of GlcA1,4) suggest the presence of α-rhamnose-(1→4)-α-galacturonic acid-(1→) glycosidic bonds, indicating that HLP-4-2 may contain RG-I, homogalacturonic acid polysaccharide (HG), arabinogalactan-specific domains or linker segments, and may even contain other structural units.

[0047] In this invention, the C3 and C5 positions of the (A1,3,5)-α-arabinofuranose-(1→) residues are connected by terminal α-arabinofuranose-(1→) residues (tAa and tAb). This conclusion can be confirmed by the relevant signals at δ 5.16 / 3.97, 5.25 / 3.85, 4.10 / (3.81 / 3.90), and 3.85 / 110.1 ppm in the ROESY and HMBC spectra. The signals at δ 5.10 / 69.7 ppm (H1 of A1,5 and C5 of A1,5 / A1,3,5), δ 5.12 / (3.81 / 3.90) ppm (H1 of A1,3,5 and H5 of A1,5), and δ 4.10 / (3.81 / 3.90) ppm in the ROESY and HMBC spectra are also confirmed. The cross peaks at ppm (H3 and H5 of A1,3,5), δ 5.10 / 86.8 ppm (H1 and C3 of A1,3,5), and δ 5.10 / 3.71 ppm (H1 of A1,5 and H4 of R1,2,4) further confirm the glycosidic bonds of →5)-α-arabinofuranose-(1→5)-α-arabinofuranose-(1→, →3,5)-α-arabinofuranose-(1→5)-α-arabinofuranose-(1→ and →5)-α-arabinofuranose-(1→2,4)-α-rhamnose-(1→).

[0048] The complex linkage patterns observed in the RG-I region (especially the connections between terminal arabinose residues and →3,5)-α-arabinofuranose residues at positions C3 and C5), and the confirmation of these connections by the ROESY / HMBC signal, strongly indicate the presence of highly branched 1,5-α-arabinogalactan side chains in this polysaccharide. The structural characteristics of the HLP-4-2 pectin polysaccharide described above (including molecular weight, branching degree, degree of esterification, monosaccharide composition, and glycosidic bond sequence) hold promise for endowing it with unique physicochemical properties and biological activities.

[0049] Table 3. Distribution of ¹H / ¹³C signal in HLP-4-2

[0050] laboratory animals 4.1 Animals, Experimental Design, and Materials Male C57BL / 6 mice aged 6–8 weeks and weighing 20±2 g were housed in a specific pathogen-free (SPF) environment at the Southwest University Animal Center. Environmental conditions were controlled as follows: temperature 25±2℃, humidity 50±5%, and a 12-hour light / dark cycle. All animal experiments complied with the guidelines of the Southwest University Institutional Animal Care and Use Committee and the National Research Council's Guidelines for the Care and Use of Laboratory Animals (IACUC-20250113-03).

[0051] The water is purified water.

[0052] Mesalazine, The company produces it.

[0053] Sodium dextran sulfate, manufactured by MP Biomedicals.

[0054] 4.2 Experimental Methods 4.2.1 Establishment of an experimental ulcerative colitis model and HLP-4-2 treatment After 7 days of acclimatization, mice were randomly divided into 6 groups (n=6): a control group, a dextran sulfate sodium (DSS) induced model group, a mesalazine group, and 3 HLP-4-2 groups (low / medium / high dose). Acute ulcerative colitis (UC) was induced with 3% (w / v) DSS for 7 days. Figure 1 As shown in Figure A, from days 6 to 12, the HLP-4-2 group received doses of 50, 100, or 200 mg / kg / day, respectively; the control and DSS groups received distilled water; and the mesalazine group received a dose of 200 mg / kg / day. Mice were euthanized after treatment. The colon was removed, photographed, and its length measured. One segment of the colon was fixed in 4% paraformaldehyde for 24 hours and then embedded in paraffin for histopathological analysis; the remaining colon tissue and colon / cecal contents were preserved at -80°C for further analysis.

[0055] 4.2.2 Establishment of an experimental antibiotic (ABX) model and HLP-4-2 treatment After acclimatization, mice were randomly divided into three groups (n=6): control group, DSS model group, and HLP-4-2 treatment group. Intestinal flora clearance was achieved via gavage (twice daily for 7 days). The gavage medications included 2.5 mg ampicillin, 2.5 mg neomycin sulfate, 2.5 mg metronidazole, and 1.25 mg vancomycin hydrochloride (all purchased from Maclean's). The experimental procedure is as follows: Figure 1 As shown in Figure A: From day 1 to day 7, all groups were given antibiotics to clear the bacterial flora; from day 8 to day 14, the DSS model group and the HLP-4-2 treatment group were given 3% (w / v) DSS, while the control group was given distilled water; from day 13 to day 19, all groups received fecal transplantation from the corresponding donor.

[0056] 4.2.3 Fecal microbiota transplantation After acclimatization, mice were randomly divided into three groups (n=12): control group, DSS group, and HLP-4-2 group. From day 0 to 7, the HLP-4-2 group and DSS group received 3% (w / v) DSS; the control group received physiological saline (Figure 3A). From day 6 to 12, the HLP-4-2 group received 200 mg / kg HLP-4-2; the control group and DSS group continued to receive physiological saline. These groups served as donor groups. Fecal sample processing followed the method described in the literature (S. Zhang et al., 2025): 200 mg of fresh feces was collected, mixed with 1 mL of sterile physiological saline containing 10% glycerol, homogenized, centrifuged (1500 rpm, 5 minutes, 4℃), filtered, and the supernatant was collected.

[0057] 4.2.4 Disease Activity Index (DAI) Assessment The Disease Activity Index (DAI) was assessed daily based on three symptoms of ulcerative colitis (weight loss, stool characteristics, and rectal bleeding). The scoring criteria are shown in Table 4. The total DAI score is the sum of the scores for the three symptoms.

[0058] Table 4. DAI Scoring Analysis Table

[0059] 4.2.5 Histopathological analysis and mucus staining of colon tissue Colon samples were paraffin-embedded and cut into 4 μm thick sections, which were then stained with hematoxylin and eosin (H&E) and Alsin-periodic acid-Schiff (AB-PAS). The H&E-stained sections were observed under an optical microscope to assess the degree of colonic tissue damage, and the histopathological scoring method was based on the literature (Gong et al., 2024). The AB-PAS-stained sections were observed under an optical microscope to count the number of goblet cells and assess the amount of colonic mucus secretion.

[0060] 4.2.6 Gut microbiota analysis Colon samples were rapidly frozen at -80°C, and bacterial DNA was extracted from the cecal contents using the DNeasy PowerSoil kit. DNA quality was assessed using a NanoDrop spectrophotometer and agarose gel electrophoresis. PCR amplification of the V3-V4 region of the 16S rRNA gene was performed using universal primers. Sequencing was performed on an Illumina NovaSeq 6000 platform, with 250 bp paired-end reads. 16S rRNA gene sequencing and analysis were performed by Shanghai Paisenuo Biotechnology Co., Ltd. (Shanghai, China).

[0061] 4.2.7 Determination of Short-Chain Fatty Acids (SCFA) Content Based on Gas Chromatography-Mass Spectrometry (GC-MS) The contents of the cecum were mixed with deionized water at a ratio of 1:9 (w / v) and centrifuged twice (5500×g, 10 min, 4℃). The supernatant was filtered through a 0.22 μm filter and analyzed by GC-MS. Standard curves (eight concentration gradients) were plotted by adding known concentrations of analytes to blank samples: acetic acid (1.5–80 mmol / L), propionic acid (0.75–48 mmol / L), n-butyric acid (0.5–36 mmol / L), and isobutyric acid, n-valeric acid, and isovaleric acid (0.125–8 mmol / L).

[0062] All data are expressed as mean ± standard deviation (X ± SD). Statistical analysis was performed using GraphPad Prism 7 software. One-way ANOVA combined with the Bonferroni test was used. *p < 0.05 was considered statistically significant.

[0063] 4.3 HLP-4-2 improves DSS-induced colitis in mice A mouse model of colitis induced by DSS can effectively mimic the pathological features of human ulcerative colitis (UC) (Dou et al., 2025). The experimental protocol is shown in Figure 1A. Mice given 3% DSS developed typical symptoms of colitis, including weight loss, increased disease activity index (DAI), and colonic shortening. Results showed that, compared with the control group, the DSS group exhibited significantly more pronounced colitis symptoms, specifically: a significant decrease in body weight (…). Figure 1 D), DAI score ( Figure 1 E) and spleen index ( Figure 1 F) significantly increased, colon length significantly shortened (Figure 1B, C), and inflammatory marker levels significantly increased ( Figure 1 GJ).

[0064] HLP-4-2 treatment (especially the high-dose group) significantly alleviated these DSS-induced symptoms. These results indicate that, similar to the mesalazine group, HLP-4-2 effectively improves DSS-induced colitis in mice.

[0065] 4.4 HLP-4-2 reduces DSS-induced histopathological damage to the colon. Ulcerative colitis (UC) disrupts the histological integrity of the colon, leading to epithelial damage, mucosal edema, and inflammatory infiltration; the mucus layer works synergistically with tight junction (TJ) proteins to form a key barrier that maintains intestinal stability.

[0066] As shown in Figures 2A and 2C, hematoxylin and eosin (H&E) staining results revealed that, compared with the control group, the DSS group exhibited extensive necrosis of the colonic mucosa, with inflammatory cells infiltrating into the muscularis propria and adventitia. Notably, these pathological symptoms significantly improved after HLP-4-2 intervention, particularly the loss of colonic crypts and inflammatory infiltration.

[0067] As shown in Figures 2B, D, and E, Alsin-Periodic Acid Schiff (AB-PAS) staining revealed that DSS treatment significantly reduced colonic mucus volume and decreased goblet cell count; conversely, HLP-4-2 restored goblet cell count and increased mucin levels. These results confirm that HLP-4-2 restores intestinal barrier function by protecting the colonic mucosa from DSS-induced damage.

[0068] 4.5 HLP-4-2 regulates gut microbiota diversity and short-chain fatty acid (SCFA) levels in DSS-treated mice Gut microbiota dysbiosis and its interaction with the mucosal immune system play a crucial role in the pathogenesis of colitis. Therefore, this invention employs 16S rRNA gene sequencing technology to investigate the effects of HLP-4-2 on the gut microbiota of ulcerative colitis (UC) mice, such as... Figure 3 As shown in A and E, the bacterial community structure was analyzed at the phylum and genus levels.

[0069] Compared with the control group, such as Figure 3 As shown in B, C, and D, the Bacteroidetes phylum in the DSS group ( Bacteroidetes ) and Firmicutes ( Firmicutes The abundance and proportion of pathogenic bacteria were significantly reduced; however, this trend was significantly improved after HLP-4-2 treatment. At the genus level, compared with the control group, the pathogenic bacteria genus (Clostridium) in the DSS group were significantly reduced. Clostridium , Turicibacter Genus, Sartorius Sutterella Klebsiella spp. Klebsiella Increased abundance of Prevostia spp. Prevotella Abundance decreases; HLP-4-2 can reverse these changes.

[0070] The abundance of *Prevotella* was negatively correlated with the incidence of ulcerative colitis; conversely, the abundance of *Clostridium* was negatively correlated with the incidence of ulcerative colitis. Turicibacter Bacteria such as *S.*, *S.*, and *Klebsiella* have been shown to exacerbate intestinal inflammation. Therefore, HLP-4-2 may partially improve DSS-induced gut microbiota dysbiosis by inhibiting the proliferation of pathogens associated with inflammatory bowel disease. Furthermore, among the bacteria whose abundance is promoted by HLP-4-2, the increased abundance of *Prevotella* can alleviate DSS-induced chronic colitis by inhibiting the IRE1α-JNK and NF-κB signaling pathways.

[0071] Short-chain fatty acids (SCFAs) are metabolites produced by the gut microbiota and are known to enhance tight junction (TJ) protein expression, protect the intestinal epithelial barrier, and reduce inflammation. Compared with the control group, such as Figure 3 As shown in G and H, the concentrations of propionic acid and total short-chain fatty acids in the cecal contents of the DSS group decreased, while the concentration of isohexanoic acid increased; after HLP-4-2 treatment, the level of short-chain fatty acids gradually approached that of the control group.

[0072] The main physiologically significant products of pectin and the degradation of indigestible carbohydrates are propionic acid and butyric acid, two short-chain fatty acids. Furthermore, studies have found that isohexanoic acid is a byproduct of Clostridium difficile (Clostridium difficile). Clostridioides difficile The specific fermentation product of HLP-4-2 can serve as a biomarker for Clostridium difficile infection in children with inflammatory bowel disease (IBD). In summary, the protective effect of HLP-4-2 on mice with ulcerative colitis may be related to its regulation of gut microbiota and short-chain fatty acids.

[0073] 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. Use of anise polysaccharide in the preparation of anti-ulcerative colitis products.

2. The use of the anise polysaccharide according to claim 1 in the preparation of anti-ulcerative colitis products, characterized in that, The anise polysaccharide is a type of anise polysaccharide.

3. The use of the anise polysaccharide according to claim 1 in the preparation of anti-ulcerative colitis products, characterized in that, The molecular weight of the anise polysaccharide is 19.7 kDa.

4. The use of the anise polysaccharide according to claim 1 in the preparation of anti-ulcerative colitis products, characterized in that, The anise polysaccharide is composed of rhamnose, galacturonic acid, galactose, arabinose and xylose, and the molar ratio of rhamnose, galacturonic acid, galactose, arabinose and xylose is (9.1): (83.4): (3.5): (2.4): (1.5).

5. The use of the anise polysaccharide according to claim 1 in the preparation of anti-ulcerative colitis products, characterized in that, The anise polysaccharide was obtained by the following method: the whole anise plant was cut into small pieces, ground into a fine powder, and the fine powder was extracted with hot water; the supernatant was collected by centrifugation and concentrated; the concentrate was deproteinized using the Sevag method, precipitated with ethanol, centrifuged, freeze-dried, and crude anise polysaccharide was collected; the crude anise polysaccharide was further fractionated by DEAE cellulose column chromatography, eluted with sodium chloride, and the elution curve was tracked and detected by phenol-sulfuric acid method. The main peak product was collected according to the elution curve, and the main peak product was then purified by gel column chromatography to obtain anise polysaccharide.

6. The use of the anise polysaccharide according to claim 1 in the preparation of anti-ulcerative colitis products, characterized in that, This includes formulating anise polysaccharides with acceptable edible excipients into tablets, granules, decoctions, or capsules.

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