Radix astragali and radix angelicae sinensis homogeneous polysaccharide for resisting ulcerative colitis as well as preparation method and application of radix astragali and radix angelicae sinensis homogeneous polysaccharide

Abstract

The invention discloses an ulcerative colitis resisting astragalus membranaceus and angelica sinensis homogeneous polysaccharide and a preparation method thereof, and the homogeneous polysaccharide is composed of 1, 6-alpha-D-Glcp, T-alpha-D-Glcp, 1, 3-beta-D-Galp, 1, 4-alpha-D-Manp and 1, 2-alpha-D-Galp according to a molar ratio of 3.9: 1.2: 1.6: 1.0: 61.3. On the cellular level, it is found through ELISA detection that compared with a model group, AAPS-4a (10 mg. L <-1 >) remarkably inhibits LPS-induced TNF-alpha, IFN-beta and NO level rising. In an in-vivo experiment, compared with a normal group, the DAI score and spleen index of a model group mouse are remarkably improved, the colon length is remarkably shortened, and the levels of inflammatory factors TNF-alpha and IL-6 are remarkably improved. Compared with a model group, the AAPS-4a has the advantages that the DAI score, the spleen index, the TNF-alpha level and the IL-6 level are obviously reduced, and the colonic atrophy is improved. HE staining shows that AAPS-4a obviously inhibits infiltration of colitis sexual cells and relieves pathological damage of the colitis sexual cells. The separated AAPS-4a neutral homogeneous polysaccharide is a key active component playing an anti-UC role in the astragalus membranaceus-angelica sinensis medicine pair, and has potential clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine polysaccharide technology, specifically relating to a homogeneous polysaccharide of Astragalus and Angelica sinensis with anti-ulcerative colitis effect, its preparation method and application. Background Technology

[0002] Ulcerative colitis (UC) is a chronic, relapsing inflammatory disease affecting the colorectal mucosa, characterized by continuous, diffuse inflammatory infiltration and crypt abscess formation. Epidemiological data show that the annual incidence rate of UC in my country is increasing rapidly, with some patients progressing to severe UC, clinically manifested as persistent bloody stools, intestinal mucosal ulcers, and colonic shortening. Some cases may develop toxic megacolon or even cancer. Current treatment regimens for UC mainly consist of aminosalicylic acid preparations, glucocorticoids, and anti-tumor necrosis factor-α (TNF-α) biologics (such as infliximab), but these have limitations such as low treatment response rates (<60%), high relapse rates (annual relapse rate >30%), and the potential for opportunistic infections with long-term use. Therefore, there is an urgent need to further study the pathogenesis of UC and develop safer, more effective, and affordable UC treatment strategies.

[0003] Traditional Chinese medicine (TCM) has demonstrated unique advantages in the treatment of ulcerative colitis (UC), with its multi-target regulation of immune homeostasis and gut microbiota attracting significant attention. Formulas such as Shenling Baizhu San, Qingzi Zhitong Tang, Kuijie Yuan Tang, and Gegen Qinlian Tang have been found to regulate inflammation and repair damaged intestinal mucosa by modulating the Janus kinase-2 (JAK2) / signal transduction and transcriptional activator protein-3 (STAT3) and phosphatidylinositol 3-kinase (PI3K) / protein kinase B (Akt) pathways, thereby inhibiting the occurrence and development of UC. As a core carrier of TCM clinical practice, elucidating the pharmacodynamic material basis and mechanism of action of TCM compound formulas is a core issue for unlocking the clinical value of TCM and promoting its modernization. The Astragalus-Angelica pair is a classic combination for tonifying qi and nourishing blood, first recorded in Li Dongyuan's *Treatise on Differentiation of Internal and External Injuries*. The applicant's previous research, by integrating network pharmacology and in vivo experimental evidence, found that Danggui Buxue Tang can improve intestinal inflammation induced by dextran sulfate sodium (DSS) in UC mice by regulating the PI3K / Akt pathway, effectively maintaining the integrity of the intestinal epithelial barrier. Among them, small molecule components such as quercetin and isoflavone glycosides were identified as key active substances for their anti-UC effects. However, as an important water-soluble macromolecular component in the Astragalus-Angelica pair, the structural basis and mechanism of action of polysaccharides in anti-UC still lack systematic research.

[0004] Polysaccharides are among the most abundant water-soluble active substances in the decoction of Astragalus and Angelica sinensis, and their immunomodulatory effects have been widely confirmed in studies of Astragalus and Angelica sinensis alone. Astragalus polysaccharides can enhance the body's immune function and regulate immunity by modulating T cell proliferation and activation, regulating inflammation-related signaling pathways such as nuclear transcription factor-κB (NF-κB) and p38 mitogen-activated protein kinase (p38 and JNK), activating cytokines such as interleukin-2 (IL-2), interleukin-6 (IL-6), and interferon-γ (IFN-γ), and nitric oxide (NO). Meanwhile, Angelica sinensis polysaccharide APS-2I can bind to Toll-like receptor 4 (TLR4), inhibiting its formation of a complex with myeloid differentiation protein-2 (MD-2), thereby downregulating the expression of Toll / IL-1 receptor (TIR) ​​adaptor proteins and blocking lipopolysaccharide (LPS)-induced activation of downstream TLR4 signaling pathways.

[0005] Recent studies have found that polysaccharide remodeling may occur during the decoction process of compound traditional Chinese medicine, resulting in novel active sugar chains with synergistic effects. For example, the compound polysaccharide extracted from Sijunzi Decoction exhibits significantly better immunomodulatory activity than polysaccharides extracted from the four herbs individually and then combined. AAP-2A is a homogeneous polysaccharide isolated from Astragalus and Angelica sinensis in the early stages, with a relative molecular weight of 6.7 × 10⁵ Da. It is mainly composed of rhamnose, galactose, arabinose, and glucose, and has significant antioxidant activity. The applicant previously isolated and purified Astragalus and Angelica sinensis polysaccharides from the Astragalus and Angelica sinensis pair, finding that they have significant antitumor activity, effectively inhibiting tumor cell proliferation and inducing apoptosis. However, existing research mainly focuses on the pharmacological activity of polysaccharides from single herbs, and there is a lack of systematic research and reports on the structural basis and mechanism of action of polysaccharide components from the Astragalus and Angelica sinensis pair in anti-UC. Summary of the Invention

[0006] This invention focuses on the systematic screening of Astragalus and Angelica polysaccharides for their anti-ulcerative colitis (UC) activity, and conducts in-depth research on the structure and function of the screened active polysaccharides. The primary structures of the active polysaccharides were systematically characterized using high-performance size exclusion chromatography (HPSEC), Fourier transform infrared spectroscopy (FT-IR), gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance spectroscopy (NMR). The effects of Astragalus and Angelica polysaccharides on repairing the intestinal epithelial barrier and inhibiting inflammatory cell infiltration were evaluated using a DSS-induced acute UC model. This invention not only provides important experimental evidence for elucidating the pharmacodynamic material basis of Astragalus and Angelica polysaccharides in combating UC, but also provides theoretical support and technical reference for the development of functional foods or natural drugs based on Astragalus and Angelica polysaccharides.

[0007] A homogeneous polysaccharide of Astragalus membranaceus for treating ulcerative colitis, characterized in that the homogeneous polysaccharide is composed of 1,6-α-D-Glcp, T-α-D-Glcp, 1,3-β-D-Galp, 1,4-α-D-Manp and 1,2-α-D-Galp.

[0008] The sequence of glycosyl linkages in this homogeneous polysaccharide is as follows:

[0009] T-α-D-Glcp-(1→4)-α-D-Manp-(1→[6)-α-Glcp-(1] 30 →3)-β-D-Galp-(1→3)-α-D-Galp-1→2)-α-D-Galp-1→

[0010] The structural formula of the main repeating unit of the primary structure of this homogeneous polysaccharide is as follows:

[0011]

[0012] As a preferred embodiment, the above-mentioned homogeneous polysaccharide for treating ulcerative colitis is composed of 1,6-α-D-Glcp, T-α-D-Glcp, 1,3-β-D-Galp, 1,4-α-D-Manp, and 1,2-α-D-Galp in a molar ratio of 3.9:1.2:1.6:1.0:61.3.

[0013] As a more preferred option, the above-mentioned homogeneous polysaccharide for treating ulcerative colitis has a relative molecular mass of 7.6 × 10³ Da.

[0014] The preparation method of the homogeneous polysaccharide of Astragalus and Angelica sinensis according to the present invention includes the following steps:

[0015] (1) Mix Astragalus membranaceus and Angelica sinensis slices, add ethanol to reflux to defatt the oil, evaporate the solvent, add deionized water, decoct and extract, combine the extracts and concentrate, add 95% ethanol, let stand, centrifuge, collect the precipitate, and redissolve to obtain crude polysaccharide of Astragalus membranaceus and Angelica sinensis.

[0016] (2) The crude polysaccharide of Astragalus membranaceus from step (1) was subjected to repeated freeze-thaw cycles with Sevag reagent to remove proteins, and then decolorized with H2O2 solution; the reaction solution was filtered through a 0.45μm microporous membrane and then freeze-dried to obtain Astragalus membranaceus polysaccharide AAPS for later use.

[0017] (3) Isolation and purification of Astragalus and Angelica polysaccharide AAPS

[0018] AAPS was dissolved in ultrapure water and eluted in stages using a DEAE Sephadex A-25 column with a gradient elution of ultrapure water and NaCl to obtain four subfractions, AAPS-1 to AAPS-4. Each subfraction was further separated and purified using a Sephadex G-100 column with isocratic elution using NaCl solution. The main peak was collected by monitoring with the phenol-sulfuric acid method. The collected solution was purified by dialysis with a molecular weight cutoff of 1000 Da, concentrated under reduced pressure, and then lyophilized to obtain six refined polysaccharide fractions of Astragalus membranaceus, namely AAPS-1a, AAPS-1b, AAPS-2a, AAPS-2b, AAPS-3a, and AAPS-4a.

[0019] As a preferred embodiment, the preparation method of the above-mentioned homogeneous polysaccharide of Astragalus membranaceus includes the following steps:

[0020] (1) Take Astragalus membranaceus and Angelica sinensis slices at a mass ratio of 5:1, add 4 to 8 times the volume of anhydrous ethanol for defatting by reflux, evaporate the solvent, add 6 to 12 times the volume of deionized water, decoct and extract 1 to 3 times, each time for 0.5 to 3 hours, combine the extracts and concentrate, add 4 to 6 times the volume of 95% ethanol to the concentrate, let stand, centrifuge to collect the precipitate, and redissolve to obtain crude polysaccharide of Astragalus membranaceus and Angelica sinensis;

[0021] (2) The crude polysaccharide of Astragalus membranaceus from step (1) was subjected to repeated freeze-thaw cycles with Sevag reagent to remove proteins. Then, it was decolorized with a 3% H2O2 solution at a material-to-liquid ratio of 1:10 under pH 8.0 and in the dark. The reaction solution was filtered through a 0.45 μm microporous membrane and then freeze-dried to obtain Astragalus membranaceus polysaccharide AAPS for later use.

[0022] (3) Isolation and purification of Astragalus and Angelica polysaccharide AAPS

[0023] AAPS was dissolved in ultrapure water and eluted in stages using a DEAE Sephadex A-25 column with a gradient elution of ultrapure water, 0.5, 1.0, and 2.0 mol·L⁻¹ NaCl at flow rates of 1–1.5 mL·min⁻¹, yielding four subfractions: AAPS-1 to AAPS-4. Each subfraction was further purified by Sephadex G-100 column elution with isocratic elution using 0.1 mol·L⁻¹ NaCl solution. The main peak was collected using the phenol-sulfuric acid method. The collected solution was purified by dialysis with a 1000 Da molecular weight cutoff membrane for 24–48 h, concentrated under reduced pressure, and then lyophilized to obtain six purified polysaccharide fractions of Astragalus membranaceus: AAPS-1a, AAPS-1b, AAPS-2a, AAPS-2b, AAPS-3a, and AAPS-4a.

[0024] Beneficial Effects: This invention is the first to isolate a neutral homogeneous polysaccharide AAPS-4a with significant anti-UC activity from the Astragalus and Angelica sinensis herbal pair, and systematically analyzed its primary structure. The structural analysis showed that AAPS-4a is composed of 1,6-α-D-Glcp, T-Glcp, 1,3-β-D-Galp, 1,4-α-D-Manp, and 1,2-α-D-Galp. Based on this structural feature, a search in the GlyTouCan database revealed that its sugar residue type and ratio did not match in the GlyTouCan database, suggesting that it is an active polysaccharide with novel structural features. This finding is significantly different from previous studies: AAP-2A is a homogeneous polysaccharide of Astragalus and Angelica sinensis reported in 2016, with a relative molecular weight of 6.7 × 10⁵ Da, mainly composed of Rha, GalA, Ara, and Glc, which exhibits α-glucan-dominated antioxidant activity. The introduction of β-galactose (1,3-β-D-Galp) into AAPS-4a extracted and isolated in this invention may be closely related to its immunomodulatory function. AAPS-4a can dose-dependently inhibit the excessive secretion of TNF-α, IFN-β, and NO in LPS-induced RAW264.7 cells, and significantly reduce the DAI score, spleen index, and inflammatory factor levels (TNF-α, IL-6) in DSS-induced UC model mice, restore colonic shortening, and repair pathological damage to colonic tissue. In addition, the linear heteropolysaccharide structure of AAPS-4a may originate from the glycan remodeling of Astragalus and Angelica polysaccharides during the decoction process, which provides direct molecular-level evidence for the "synergistic effect of Astragalus and Angelica" hypothesis, filling the gap in the research on the compatibility mechanism of compound polysaccharides. Attached Figure Description

[0025] Figure 1 The HPSEC spectrum of AAPS-4a.

[0026] Figure 2 The image shows the FT-IR spectrum of AAPS-4a.

[0027] Figure 3 This is the HPLC spectrum of AAPS-4a.

[0028] Figure 4 The image shows the GC-MS analysis of AAPS-4a.

[0029] Figure 5 This is the 1D NMR spectrum of AAPS-4a.

[0030] Figure 6 The HSQC(A), COSY(B), HMBC(C), and NOESY(D) spectra of AAPS-4a are shown.

[0031] Figure 7The effect of AAPS-4a on body weight in DSS-induced UC model mice.

[0032] Figure 8 Effects of AAPS-4a on colonic histopathology in DSS-induced UC mice (HE, ×40, ×100). Detailed Implementation

[0033] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0034] Example 1

[0035] 1. Experimental Materials

[0036] TENSOR 27 infrared spectrometer (Bruker Technologies, Germany), Waters Alliance 2695 high performance liquid chromatograph (Waters Corporation, USA), Bruker Avance III nuclear magnetic resonance spectrometer (Bruker Technologies, Germany), TQ8050NX gas chromatograph-mass spectrometer (Shimadzu Corporation, Japan), MCO-50AICL-PC CO2 cell incubator (Shanghai Puhexi Health Medical Equipment Co., Ltd.), Synergy H4 Hybrid multi-functional microplate reader (Teng Instruments, USA), FD5-3 freeze dryer (Kingsmond Technologies, Germany).

[0037] Astragalus membranaceus slices (batch number 20220502) and Angelica sinensis slices (batch number 20220001) were purchased from Shaanxi Xingshengde Pharmaceutical Co., Ltd. They were identified by Professor Hu Benxiang of Shaanxi University of Traditional Chinese Medicine and met the relevant quality requirements of the 2025 edition of the *Pharmacopoeia of the People's Republic of China*. Mannose (Man, item number 04870942), rhamnose (Rha, item number 04871923), glucuronic acid (GlcA, item number 04869332), galacturonic acid (GalA, item number 04237345), glucose (Glc, item number 04931548), galactose (Gal, item number 04871138), arabinose (Ara, item number 04979621), and fucose (Fuc Reference standards (item number 04236851), and dextran molecular weight reference standards T-3 (item number SM9500), T-10 (item number SM9510), T-50 (item number SM9520), T-120 (item number SM9530), and T-150 (item number SM9430), LPS (batch number 818E035), and penicillin-streptomycin mixture (batch number 20200726) were purchased from Solarbio Biotechnology Co., Ltd. The reference standards were pure. All samples were ≥98% purity; blue dextran (Sigma-Aldrich Trading Co., Ltd., USA, catalog number S14096), sulfasalazine enteric-coated capsules (SASP, Guangdong Qiangji Pharmaceutical Co., Ltd., batch number H20051173), DMEM high-glucose basal culture medium (Wuhan Sewell Biotechnology Co., Ltd., batch number G4510), trypsin (containing EDTA) (Wuhan Sewell Biotechnology Co., Ltd., batch number CR2110038), fetal bovine serum (Gibco, USA, batch number 42G6185K), cell proliferation and viability assay kit (CCK-8) (Beyotime Biotechnology Co., Ltd., batch number 1755C459), TNF-α and interferon-β (IFN-β) enzyme-linked immunosorbent assay (ELISA) kit (Wuhan E-EL-M3063 and E-EL-M0033, respectively), mouse NO The ELISA kit (Shanghai Beyotime Biotechnology Co., Ltd., batch number S0021S) used methanol and acetonitrile of chromatographic grade, and all other reagents of analytical grade.

[0038] Mouse macrophages RAW264.7 were purchased from the Cell Bank of the Chinese Academy of Sciences (catalog number SCSP-5036). The initial purchase was at passage 4, and the cells used in the experiment were at passage 12.

[0039] Forty SPF-grade male C57BL / 6J mice, 6–8 weeks old and weighing 20–24 g, were provided by the Experimental Animal Center of Air Force Medical University (License No. SCXK(Shaanxi)2024-005). The mice were kept at (22±2)℃ and (55±5)% humidity for 7 days to allow them to acclimatize. The experimental protocol was approved by the Animal Ethics Committee of Air Force Medical University (License No. IACUC-20240076).

[0040] 2. Experimental Methods

[0041] 2.1 Extraction of Astragalus and Angelica Sinensis Total Polysaccharides (AAPS)

[0042] Astragalus root slices and Angelica sinensis root slices were mixed at a mass ratio of 5:1, and defatted by reflux with 4 times the volume of anhydrous ethanol. The solvent was evaporated, and 6 times the volume of deionized water was added. The mixture was decocted twice at 80℃ (1 hour each time). The extracts were combined and concentrated to 1 / 5 of the original volume. 4 times the volume of 95% ethanol was added, and the mixture was allowed to stand at 4℃ for 12 hours. The precipitate was collected by centrifugation and reconstituted to obtain crude polysaccharides of Astragalus and Angelica sinensis. The crude polysaccharides were subjected to repeated freeze-thaw cycles with Sevag reagent (chloroform-n-butanol 5:1) to remove proteins. Then, the polysaccharides were decolorized with 3% H₂O₂ solution (solid-to-liquid ratio 1:10) at pH 8.0 and 50℃ in the dark for 2 hours. The reaction solution was filtered through a 0.45 μm microporous membrane and then freeze-dried to obtain AAPS with a yield of 6.3%. The AAPS was then sealed and stored at -80℃ for later use.

[0043] 2.2 Isolation and purification of AAPS

[0044] AAPS was dissolved in ultrapure water and eluted in stages using a DEAE Sephadex A-25 column (100 cm × 5 cm, 40–120 μm) with ultrapure water, 0.5 mol·L⁻¹ NaCl, 1.0 mol·L⁻¹ NaCl, and 2.0 mol·L⁻¹ NaCl at a flow rate of 1.5 mL·min⁻¹, yielding four subfractions: AAPS-1 to AAPS-4. Each subfraction was further purified by separation using a Sephadex G-100 column (100 cm × 5 cm, 40–120 μm) with isocratic elution using 0.1 mol·L⁻¹ NaCl solution. The main peak was collected using the phenol-sulfuric acid method. The collected solution was purified by dialysis with a molecular weight cutoff of 1000 Da for 48 h, concentrated under reduced pressure, and then freeze-dried to obtain six purified polysaccharide fractions, namely AAPS-1a, AAPS-1b, AAPS-2a, AAPS-2b, AAPS-3a, and AAPS-4a, with yields of (20.3±1.8)%, (15.6±1.2)%, (21.5±2.1)%, (13.4±1.1)%, (12.7±1.2)%, and (13.5±1.4)% (based on the mass of AAPS), respectively.

[0045] 2.3 Determination of the homogeneity and relative molecular weight of AAPS-4a

[0046] The sample and dextran reference standards (T-3, T-10, T-50, T-120, T-150) were dissolved in water to prepare a solution with a mass concentration of 1 g·L⁻¹. -1 The solution was centrifuged, and 200 μL of the supernatant was collected for later use. A Waters Alliance 2695 high-performance liquid chromatograph, a Waters 2414 differential refractive index detector, and a TOSOH TSK-GEL G4000PWXL column (30.0 cm × 7.8 mm, 10 μm) were used with a mobile phase of 0.05 mol·L⁻¹. -1 Isocratic elution with Na2SO4 aqueous solution for 30 min, injection volume 10 μL, flow rate 0.5 mL·min -1 The column temperature was 25℃. Reference standards and samples were injected and analyzed under the above conditions. A standard curve was plotted with the logarithm of the retention time of the reference standard on the x-axis and the logarithm of the molecular weight on the y-axis. The sample peak retention time was then substituted into the regression equation to calculate the relative molecular weight.

[0047] 2.4 Determination of the functional group and glycosidic bond configuration of AAPS-4a

[0048] Take 2 mg of AAPS-4a, grind and mix it with potassium bromide, compress it into tablets, and measure the wavenumber range of 4000–400 cm⁻¹ using an infrared spectrometer. -1 The spectra were used to determine the major functional groups in the sugar molecule and the glycosidic bond configuration of the pyranose.

[0049] 2.5 HPLC determination of the monosaccharide composition of AAPS-4a

[0050] Dissolve 5.0 mg of each monosaccharide reference standard in 500 μL of distilled water, and then add 400 μL of the solution to each solution sequentially with 0.3 mol·L⁻¹ ... -1 200 μL sodium hydroxide solution, 0.5 mol·L⁻¹ -1 400 μL of 1-phenyl-3-methyl-5-pyrazolone (PMP) methanol solution was added, and the derivatization reaction was carried out at 70 °C for 30 min. Then, 0.3 mol·L⁻¹ of the solution was added. -1 Neutralize to pH 7.0 with HCl. Extract three times with chloroform to remove free PMP, combine the aqueous phases to obtain monosaccharide derivatives, and mix 200 μL of each derivative as a mixed control. Add 5 mg of AAPS-4a sample to 2 mol·L⁻¹ -1 4 mL of trifluoroacetic acid was hydrolyzed at 100 °C for 8 h, the acid was removed by nitrogen blowing, and the solution was reconstituted in ultrapure water before derivatization using the same method. A Waters Alliance 2695 high-performance liquid chromatograph with an Ultimate 3000 photodiode array detector and Acclimate was used. TMA 120C18 column (4.6 mm × 250 mm, 5 μm) was used, with ammonium acetate (100 mmol·L⁻¹)-tetrahydrofuran-acetonitrile (81:2:17) as the mobile phase, and elution was performed isocratically for 60 min at a flow rate of 1.0 mL·min⁻¹. -1 The column temperature was 25℃ and the injection volume was 10μL.

[0051] 2.6 GC-MS Analysis of AAPS-4a

[0052] 2.6.1 Sample Pretreatment

[0053] Take 20 mg of AAPS-4a polysaccharide sample, dissolve it in 4 mL of anhydrous dimethyl sulfoxide (DMSO), purge with nitrogen for 5 min to remove oxygen, and then stir for 12 h to activate. Add 1 mol·L⁻¹ -1 4 mL of NaOH-DMSO solution was added, and the mixture was stirred for 3 h under nitrogen protection. 3.6 mL of iodomethane was added dropwise in an ice bath, and the reaction was continued for 7 min in the dark, followed by 12 h at room temperature. The reaction solution was dialyzed against ultrapure water in a 1000 Da dialysis bag for 24 h (with 3 solution changes), and then lyophilized to obtain the fully methylated product. The product was then soluble in 90% formic acid (100 °C, 4 h) and 2 mol·L⁻¹. -1 Trifluoroacetic acid was hydrolyzed stepwise (100℃, 8h), and after acid removal by nitrogen blowing, it was dissolved in 2mL of ultrapure water. 60mg of sodium borodeuteride was added, and the mixture was reduced at room temperature for 24h. The product was then desalted using JK008 hydrogen-form resin and lyophilized. The dried product was then added to 1mL of pyridine-acetic anhydride (1:2), acetylated at 100℃ for 2h, and the residual reagents were removed by azeotropic mixing with ethanol. The mixture was then redissolved in chloroform and concentrated to 100μL.

[0054] 2.6.2 Testing Conditions

[0055] Agilent DB-1 capillary column (30 m × 0.25 mm, 0.15 μm), helium as carrier gas, flow rate 1.0 mL / min. -1 Inlet temperature 220℃, split ratio 10:1, programmed temperature ramp (45℃ held for 5 min; then ramped at 10℃·min). -1 Heat to 140℃ and hold for 5 minutes; then increase the temperature by 0.5℃·min. -1 Heat to 170℃, hold for 1 minute, then reduce at 15℃·min. -1 Heating to 280℃ and holding for 5 min, electron impact source (EI), ion source temperature 280℃, collision energy 70eV, scan range m / z 50~600.

[0056] 2.7 NMR determination of AAPS-4a

[0057] The AAPS-4a polysaccharide sample was replaced with heavy water three times and placed in 500 μL of D2O. One-dimensional NMR spectroscopy (1D NMR), namely ¹H NMR and ¹³C NMR, and two-dimensional NMR spectroscopy (2D NMR), namely heteronuclear single quantum coherence (HSQC), hydrogen-hydrogen correlation (¹H-¹H COSY), heteronuclear multi-bond correlation (HMBC), and nuclear Overhauser effect (NOESY), were detected using a Bruker Avance III nuclear magnetic resonance spectrometer.

[0058] 2.8 Mouse RAW264.7 cell culture

[0059] Cells were seeded in T75 culture flasks (initial density 1×10⁶). 5 The cells (number per mL) were cultured in DMEM high-glucose medium containing 10% FBS and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator.

[0060] 2.9 CCK-8 assay for cell proliferation activity

[0061] RAW264.7 cells were grown at a rate of 5 × 10⁻⁶. 4 Inoculate 1 / mL of the sample into 96-well plates, allow to adhere overnight, and administer a final concentration of 0, 0.1–100 mg / mL. -1 AAPS-4a solution was used in 6 replicates per group. After incubation for 48 h, 10 μL of CCK-8 solution was added to each well, and the reaction was carried out at 37 °C for 2 h. The absorbance A at 490 nm was measured to calculate cell viability. Cell viability = (A experimental wells - A blank wells) / (A control wells - A blank wells) × 100%.

[0062] 2.10 ELISA was used to detect the levels of TNF-α, IFN-β, and NO in cells.

[0063] Cells at 2×10 5 LPS was seeded at 5 mg / mL in 6-well plates and allowed to adhere overnight. The control group received culture medium, while the LPS group received 5 mg / mL. -1 LPS was added to the sample groups, and polysaccharide solutions of corresponding concentrations were added. After 48 hours of incubation, the supernatant of each group was collected, and the levels of TNF-α, IFN-β, and NO in the supernatant were measured according to the ELISA kit instructions.

[0064] 2.11 Grouping, Model Establishment, and Drug Administration of UC Mice

[0065] Animals were randomly divided into a normal control group, a model group, a positive control group (SASP group, sulfasalazine enteric-coated capsules, Guangdong Qiangji Pharmaceutical Co., Ltd., batch number H20231173), and low- and high-dose polysaccharide AAPS-4a groups. The normal control group received routine feeding, while the other groups were given free access to 3.5% DSS solution in drinking water for 7 days to induce acute UC. After successful modeling, referring to literature reports, the positive control group was given SASP 75 mg / kg. -1 The low-dose and high-dose AAPS-4a groups were given 50 and 100 mg / kg of AAPS-4a, respectively. -1 The normal group and the model group were given an equal volume of physiological saline. All animals were administered the drug by gavage for 7 consecutive days, and the body weight of the mice was recorded. After the experiment, the mice were sacrificed, serum was collected, and the colon was harvested and its length was recorded. A portion of the colon was fixed with 4% paraformaldehyde, and the other portion was stored with the serum at -80°C for subsequent analysis. The spleen was harvested and the spleen index was calculated as spleen mass (mg) / body mass (g).

[0066] 2.12 General behavioral observation

[0067] Monitor changes in mouse body weight, fecal characteristics, and degree of blood in stool. Assess each group of mice with the Disease Activity Index (DAI) according to the reference scoring criteria.

[0068] 2.13 HE staining to observe pathological changes in colon tissue

[0069] Paraffin sections of colonic tissue were dewaxed and hydrated, then stained with hematoxylin for 5 min, differentiated with 1% hydrochloric acid and ethanol for 30 s, and counterstained with eosin for 2 min. The sections were then dehydrated in a gradient of ethanol, cleared with xylene, and mounted with neutral resin. The integrity of the colonic mucosa structure, inflammatory cell infiltration, and crypt morphological changes were observed using an optical microscope, and pathological features were recorded using a NIS-Elements D 4.30 image acquisition system.

[0070] 2.14 ELISA detection of serum TNF-α and IL-6 levels: ELISA kits were used to detect the differences in the expression of inflammatory factors TNF-α and IL-6 in the serum of mice in each group.

[0071] 2.15 Data Statistics and Analysis

[0072] SPSS 27.0 software was used for statistical analysis of the experimental data. Quantitative data were presented as follows: The comparison between two groups was performed using a t-test. For comparisons among multiple groups, if the groups conformed to a normal distribution and uniform variance, one-way ANOVA and Tukey's method for multiple comparisons were used. If the groups did not conform to a normal distribution or uniform variance, the Games-Howell test or Dunnett test was used. P < 0.05 indicated that the difference was statistically significant.

[0073] 3. Experimental Results

[0074] 3.1 Comparative evaluation of the immunomodulatory effects of six isolated Astragalus and Angelica polysaccharides

[0075] Compared with the control group, LPS (5 mg L⁻¹) significantly promoted the secretion of TNF-α, IFN-β, and NO levels by RAW264.7 cells (P<0.01, P<0.01, P<0.01). Compared with the model group, AAPS-4a significantly inhibited LPS-induced secretion of TNF-α, IFN-β, and NO levels by macrophages in a dose-dependent manner (P<0.01, P<0.05, P<0.05). See Table 1.

[0076] Table 1. Effects of Astragalus polysaccharide on LPS-induced inflammatory factors and NO levels in RAW264.7 cells.

[0077]

[0078] Note: Compared with the normal group 1) P<0.05, 2) P<0.01; compared with the model group 3) P<0.05, 4) P<0.01

[0079] 3.2 Determination of the relative molecular weight of AAPS-4a

[0080] The chromatographic peaks of polysaccharide AAPS-4a exhibited a single symmetrical shape, indicating a uniform molecular weight distribution. A standard curve was established using dextran as a reference (y = -3.7768x + 6.8808, R0). 2 =0.999), its relative molecular mass is calculated to be 7.6 × 10³ Da, see Figure 1 .

[0081] 3.3 FT-IR Identification of AAPS-4a

[0082] The FT-IR spectrum of polysaccharide AAPS-4a shows typical polysaccharide characteristic absorption peaks. Among them, the peak at 3396.71 cm⁻¹ represents the stretching vibration signal of the OH bond. Peaks at 2934.55 cm⁻¹ and 1416.32 cm⁻¹ are also present. -1 The locations represent the symmetric and asymmetric tensile vibration signals of the CH bond, respectively. 1568.21 cm -1 The location represents the tensile vibration signal of the C=O bond. Furthermore, in the range of 916.2-1199 cm⁻¹... -1 A characteristic absorption band of the pyranose ring was observed in the region, with the main peak located at 1043.91 cm⁻¹. -1 (COC asymmetric stretching vibration), acromion length 1052.04 cm -1(COH bending vibration), the bimodal splitting pattern is consistent with the characteristics of α-pyranose reported in the literature. See Figure 2 .

[0083] 3.4 Determination of the composition of AAPS-4a monosaccharides

[0084] AAPS-4a was determined by HPLC to be composed of Man, Glc, and Gal. (See attached image) Figure 3 .

[0085] 3.5 Methylation and GC-MS

[0086] To elucidate the sugar residue linkages of AAPS-4a, its methylated derivatives were analyzed by GC-MS. GC analysis of AAPS-4a revealed five characteristic peaks, and the epimers were accurately distinguished by retention time differences (ΔRRT ≥ 0.2 min) and NMR cross-validation. All mass spectrometry data were double-validated using the CCRC standard database (https: / / www.ccrc.uga.edu / specdb / ms / ) and the internal retention time-fragment ion correspondence table. Based on the monosaccharide composition of AAPS-4a, the five sugar residues were identified as T-glucose (T-Glcp, 23.37 min, m / z 45, 118, 161, 162, 205), 1,2-galactose (1,2-Galp, 27.72 min, m / z 45, 161, 190, 234), 1,3-galactose (1,3-Galp, 34.25 min, m / z 45, 118, 161, 234, 277), 1,4-mannose (1,4-Manp, 34.87 min, m / z 45, 118, 162, 233), and 1,6-glucose (1,6-Glcp, 41.52 min, m / z 118, 162, 189, 233). (See [link to relevant documentation]). Figure 4 .

[0087] Table 2. GC-MS analysis of AAPS-4a sugar residue types and molar ratios

[0088]

[0089] Note: a. Ratio of peak retention time to T-Glcp peak retention time

[0090] 3.6 1D NMR Analysis of AAPS-4a

[0091] 1 In the 1H-NMR spectrum, the region from 4.30 to 5.50 ppm is the region of anomeric proton (H-1) signal for polysaccharides. The 1H NMR spectrum of AAPS-4a shows a strong H-1 signal at 4.91 ppm and three weak H-1 signals at 4.61, 5.10, and 5.26 ppm. (See...) Figure 5 -A. The H-1 characteristic signal at 4.61 ppm is that of β-configured sugar residues; the H-1 characteristic signal at 4.91 ppm is that of α-Glcp; and the H-1 characteristic signal at 5.26 ppm is that of α-Manp. The signal at 5.10 ppm may be Rhap or Galp. Based on the HPLC results, it is preliminarily identified as α-Galp. 13 In the C-NMR spectrum, the 95.0–110.0 ppm region represents the anomeric carbon (C-1) signal region of the polysaccharide. The 13C NMR spectrum of AAPS-4a shows one strong C-1 signal at 97.67 ppm and three weak C-1 signals at 95.68, 99.39, and 104.12 ppm. (See [reference needed]). Figure 5 -B. The C-1 characteristic signal of the β-configuration sugar residue is at 104.12 ppm, which corresponds to the H-1 signal at 4.61 ppm. The C-1 characteristic signal of α-Manp is at 99.39 ppm, and the C-1 characteristic signal of α-Glcp is at 97.67 ppm, corresponding to the C-1 signal at 4.91 ppm. These results suggest that the AAPS-4a structure mainly contains four types of sugar residues, with α-glucose residues (α-Glcp) being the predominant type.

[0092] 3.7 2D NMR Analysis of AAPS-4a

[0093] To clarify the linkage mode and configuration of sugar residues in AAPS-4a, 2D NMR (HSQC, 2D NMR) was performed. 1 H- 1 HCOSY, HMBC, and NOESY analyses were performed. In the HSQC spectrum, δ4.91 / 97.67 was assigned to the H-1 / C-1 signal of α-Glcp, see [reference needed]. Figure 6 -A. Based on the four relevant signals δ4.91 / 3.51, δ3.51 / 3.65, δ3.65 / 3.45, and δ3.45 / 3.84 in COSY, the H-2, H-3, H-4, and H-5 of this residue were determined. See [link to COSY]. Figure 6 -B. Further analysis using HSQC revealed that the C-2, C-3, C-4, and C-5 of this residue were located at 71.43, 73.34, 69.54, and 70.13 ppm, respectively. HMBC showed long-range C-H correlation signals separated by 3–4 chemical bonds. The δ4.91 / 65.47 signal in the HMBC spectrum indicated a long-range triple bond correlation between H-1 (δ4.91) and C-6 (δ65.47) of this residue, confirming that the C-6 of this α-Glcp was located at 65.47. (See [link to HMBC spectrum]). Figure 6 -C, Figure 6-D. Methylation analysis (1,6-Glcp accounting for 88.8%) and 13C NMR data (C-6, δ 65.47) completely matched the characteristic signals of 1,6-α-D-Glcp reported in the literature. Furthermore, four cross signals at δ 3.51 / 3.65, δ 3.65 / 3.37, δ 3.37 / 3.91, and δ 3.77 / 3.91 in COSY indicated the presence of another Glcp. Combining five signals at δ 3.65 / 71.38, δ 3.37 / 69.48, δ 3.91 / 70.28, δ 3.71 / 60.47, and δ 3.77 / 60.47 in HSQC and the δ 3.37 / 60.47 signal in HMBC, the H-1 / C-1 to H-6 / C-6 of this α-Glcp were determined, ultimately identifying this residue as T-α-D-Glcp.

[0094] δ4.61 / 104.12 represents the H-1 / C-1 signal of β-Galp. In COSY, δ4.61 / 3.18, δ3.18 / 3.43, and δ3.43 / 3.643 signals represent H-1 / H-2, H-2 / H3, and H-3 / H4 correlations. Based on three signals in HSQC (δ3.18 / 74.04, δ3.43 / 75.88, and δ3.64 / 69.51), the C-2, C-3, and C-4 of this residue were determined (see [reference]). Figure 6-B. The C-3 of this residue shows a significant lower field shift, and the signals of the remaining C-6 in the HSQC spectrum are all below 63.0 ppm, thus identifying this residue as 1,3-β-D-Galp. Furthermore, δ5.26 / 99.39 is a characteristic H-1 / C-1 signal of α-Manp. Based on δ5.26 / 3.51, δ3.51 / 3.96, δ3.96 / 3.76, and δ3.51 / 73.03, δ3.96 / 69.99, δ3.76 / 80.56, the H-2 / C-2 to H-3 / C-4 of this residue were obtained. The C-4 of this residue shows a significant lower field shift, thus identifying this residue as 1,4-α-D-Manp. Combining HPLC and 1D NMR results, δ5.10 / 95.68 was assigned to the H-1 / C-1 signal of α-D-Galp. In COSY, δ5.10 / 3.37, δ3.37 / 3.93, and δ3.93 / 3.71 represent the H-1 / H-2, H-2 / H-3, and H-3 / H-4 correlations of this residue, respectively. Combined with δ3.37 / 79.69, δ3.93 / 69.71, and δ3.71 / 69.79 in HSQC, the C-2 to C-4 positions of this residue were determined. Its C-2 chemical shift significantly shifts to a lower field, thus identifying this residue as 1,2-α-D-Galp. Combining 1D and 2D NMR, the five sugar residues in AAPS-4a were finally identified as 1,6-α-D-Glcp, α-TD-Glcp, 1,3-β-D-Galp, 1,4-α-D-Manp, and 1,2-α-D-Galp, as shown in Table 3.

[0095] HMBC spectroscopy was used to further analyze the sugar residue linkage sequence of AAPS-4a. Signals at δ 5.26 / 65.47 indicated a correlation between H-1 of 1,4-α-D-Manp and C-6 of 1,6-α-D-Glcp; at δ 4.91 / 65.47, a correlation between H-1 and C-6 of 1,6-α-D-Glcp; and at δ 4.91 / 75.88, a correlation between H-1 of 1,6-α-D-Glcp and C-3 of 1,3-β-D-Galp. Furthermore, a signal at δ 4.61 / 79.69 indicated a correlation between H-1 of 1,3-β-D-Galp and C-2 of 1,2-α-D-Galp. Based on the combined GC-MS and NMR results, the main repeating units in AAPS-4a are inferred as follows: T-α-D-Glcp-(1→4)-α-D-Manp-(1→[6)-α-Glcp-(1) 30 →3)-β-D-Galp-(1→3)-α-D-Galp-1→2)-α-D-Galp-1→.

[0096] Table 3. NMR signal attribution of AAPS-4a

[0097]

[0098] 3.8 Evaluation of the in vivo anti-UC effect of AAPS-4a

[0099] Compared with the normal group, the model group mice showed significantly reduced body weight (P<0.01), significantly increased DAI score and spleen index (P<0.01, P<0.01), significantly shortened colon length (P<0.01), and significantly increased serum TNF-α and IL-6 levels (P<0.01, P<0.01). Compared with the model group, the high-dose AAPS-4a group showed significantly restored body weight (P<0.01), significantly reduced DAI score (P<0.05) and spleen index (P<0.05), significantly restored colon length (P<0.01), and significantly decreased serum TNF-α and IL-6 levels. See Figure 7 Table 4.

[0100] Table 4. In vivo intervention effects of AAPS-4a on DSS-induced UC mice

[0101] Grouping <![CDATA[Dose / mg·kg -1 > DAI rating Colon length / cm Spleen Index <![CDATA[TNF-α / ng·L -1 ]]> <![CDATA[IL-6 / ng·L -1 ]]> normal group 0.24±0.19 7.85±0.32 0.45±0.01 56.14±11.17 8.14±1.9 Model group <![CDATA[3.45±0.31 2) ]]> <![CDATA[5.45±0.43 2) ]]> <![CDATA[0.57±0.01 2) ]]> <![CDATA[216.69±25.5 2) ]]> <![CDATA[57.17±6.78 2) ]]> AAPS-4a-L 50 2.76±0.42 5.99±0.6 0.49±0.05 193.78±14.35 53.36±4.28 AAPS-4a-H 100 <![CDATA[1.5±0.27 4) ]]> <![CDATA[6.78±0.36 3) ]]> <![CDATA[0.46±0.02 3) ]]> <![CDATA[155.5±15.44 4) ]]> <![CDATA[41.16±2.13 4) ]]> SASP 75 <![CDATA[1.11±0.36 4 )]]> <![CDATA[7.33±0.5 4 )]]> <![CDATA[0.41±0.03 4 )]]> <![CDATA[163.36±14.28 4 )]]> <![CDATA[39.03±2.9 3 )]]>

[0102] Note: Compared with the normal group 1 P<0.05, 2) P<0.01; compared with the model group 3 P<0.05, 4) P<0.01 Figure 8 HE staining of the colon showed that the normal control group had an intact and continuous intestinal mucosal structure, normal crypt morphology, and no inflammatory cell infiltration. Compared with the normal group, the model group showed extensive exfoliation of intestinal epithelial cells, extensive infiltration of neutrophils and lymphocytes in the lamina propria, severe destruction of crypt structure, and complete disappearance of crypts in some areas. Compared with the model group, the low-dose AAPS-4a group showed partial repair of intestinal epithelium and reduced inflammatory cell infiltration, while the high-dose AAPS-4a group showed near-complete restoration of intestinal epithelial continuity, significantly reduced inflammatory cell infiltration, and intact crypt structure. The improvement of colonic injury by AAPS-4a in UC was dose-dependent, with the high-dose group showing effects similar to the SASP-positive control group.

[0103] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A homogeneous polysaccharide of Astragalus and Angelica sinensis for treating ulcerative colitis, characterized in that, This homogeneous polysaccharide is composed of 1,6-α-D-Glcp, T-α-D-Glcp, 1,3-β-D-Galp, 1,4-α-D-Manp and 1,2-α-D-Galp. The sugar linkage order is: T-α-D-Glcp-(1→4)-α-D-Manp-(1→[6)-α-Glcp-(1) 30 →3)-β-D-Galp-(1→3)-α-D-Galp-1→2)-α-D-Galp-1→.

2. The Astragalus-Angelica Homogeneous Polysaccharide for Anti-ulcerative Colitis according to claim 1, characterized in that, This homogeneous polysaccharide is composed of 1,6-α-D-Glcp, T-α-D-Glcp, 1,3-β-D-Galp, 1,4-α-D-Manp and 1,2-α-D-Galp in a molar ratio of 3.9:1.2:1.6:1.0:61.

3.

3. The Astragalus and Angelica homogeneous polysaccharide for treating ulcerative colitis according to claim 2, characterized in that, The relative molecular mass of this homogeneous polysaccharide is 7.6 × 10³ Da.

4. The method for preparing the homogeneous polysaccharide of Astragalus and Angelica as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Mix Astragalus membranaceus and Angelica sinensis slices, add ethanol to reflux to defatt the oil, evaporate the solvent, add deionized water, decoct and extract, combine the extracts and concentrate, add 95% ethanol, let stand, centrifuge, collect the precipitate, and redissolve to obtain crude polysaccharide of Astragalus membranaceus and Angelica sinensis. (2) The crude polysaccharide of Astragalus membranaceus from step (1) was subjected to repeated freeze-thaw cycles with Sevag reagent to remove proteins, and then decolorized with H2O2 solution; the reaction solution was filtered through a 0.45μm microporous membrane and then freeze-dried to obtain Astragalus membranaceus polysaccharide AAPS for later use. (3) Isolation and purification of Astragalus and Angelica polysaccharide AAPS AAPS was dissolved in ultrapure water and eluted in stages using a DEAE Sephadex A-25 column with a gradient elution of ultrapure water and NaCl to obtain four subfractions, AAPS-1 to AAPS-4. Each subfraction was further separated and purified using a Sephadex G-100 column with isocratic elution using NaCl solution. The main peak was collected by monitoring with the phenol-sulfuric acid method. The collected solution was purified by dialysis with a molecular weight cutoff of 1000 Da, concentrated under reduced pressure, and then lyophilized to obtain six refined polysaccharide fractions of Astragalus membranaceus, namely AAPS-1a, AAPS-1b, AAPS-2a, AAPS-2b, AAPS-3a, and AAPS-4a.

5. The method for preparing Astragalus and Angelica homogeneous polysaccharide according to claim 4, characterized in that, Includes the following steps: (1) Take Astragalus membranaceus and Angelica sinensis slices at a mass ratio of 5:1, add 4 to 8 times the volume of anhydrous ethanol for defatting by reflux, evaporate the solvent, add 6 to 12 times the volume of deionized water, decoct and extract 1 to 3 times, each time for 0.5 to 3 hours, combine the extracts and concentrate, add 4 to 6 times the volume of 95% ethanol to the concentrate, let stand, centrifuge to collect the precipitate, and redissolve to obtain crude polysaccharide of Astragalus membranaceus and Angelica sinensis; (2) The crude polysaccharide of Astragalus membranaceus from step (1) was subjected to repeated freeze-thaw cycles with Sevag reagent to remove proteins. Then, it was decolorized with a 3% H2O2 solution at a material-to-liquid ratio of 1:10 under pH 8.0 and in the dark. The reaction solution was filtered through a 0.45 μm microporous membrane and then freeze-dried to obtain Astragalus membranaceus polysaccharide AAPS for later use. (3) Isolation and purification of Astragalus and Angelica polysaccharide AAPS AAPS was dissolved in ultrapure water and eluted in stages using a DEAE Sephadex A-25 column, with ultrapure water, 0.5, 1.0, and 2.0 mol·L⁻¹ being used sequentially. -1 NaCl gradient elution, flow rate 1–1.5 mL / min -1 Four subfractions, AAPS-1 to AAPS-4, were obtained. Each subfraction was further separated and purified using a Sephadex G-100 column at 0.1 mol·L⁻¹. -1 Isocratic elution with NaCl solution, collection of the main peak by monitoring with phenol-sulfuric acid method, purification of the collected solution by dialysis membrane with a molecular weight cutoff of 1000 Da for 24-48 h, concentration under reduced pressure and freeze drying, finally obtaining 6 refined polysaccharide fractions of Astragalus membranaceus, namely AAPS-1a, AAPS-1b, AAPS-2a, AAPS-2b, AAPS-3a and AAPS-4a.

6. The use of the Astragalus-Ginseng Polysaccharide according to any one of claims 1 to 3 in the preparation of pharmaceuticals or health products for treating ulcerative colitis.

7. The application according to claim 6, characterized in that, Astragalus and Angelica homogenate polysaccharides are prepared into oral liquids, tablets, pills, granules, capsules, extracts, or mixtures using pharmaceutically acceptable carriers.

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