Allium schoenoprasum polysaccharide, and preparation method and application thereof

Abstract

This invention provides a polysaccharide from *Allium tuberosum*, its preparation method, and its application, belonging to the field of biomedical technology. Crude polysaccharides, AMBP 40, AMBP 60, and AMBP 80, were obtained from *Allium tuberosum* through extraction and purification. The polysaccharide component AMBP60-1 was prepared by separation, and then a homogeneous polysaccharide AMBP-1a was prepared. AMBP-1a mainly consists of galactose, glucose, and arabinose in a molar ratio of 0.836:0.158:0.006, and is a neutral heteropolysaccharide. The structural characteristics of polysaccharides are related to their activity; AMBP-1a can effectively improve the resistance to ApoE. ‑ / ‑ The pharmacological activities of the mouse model of atherosclerosis, such as blood lipid levels and atherosclerotic plaques, were investigated, and the intervention of atherosclerosis was achieved by regulating the gut microbiota and short-chain fatty acid content.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a polysaccharide from small garlic roots, its preparation method, and its applications. Background Technology

[0002] Cardiovascular disease is the leading cause of death among non-communicable diseases. According to WHO statistics in 2021, the global cardiovascular disease mortality rate has increased by 25% since 2000, reaching 17.9 million deaths in 2019, accounting for 32% of all deaths, making it the "number one killer" seriously threatening human health. Atherosclerosis (AS) is the main cause of the high incidence and mortality of cardiovascular disease. Clinical treatment of AS often involves drug therapy and surgery. While these can alleviate symptoms, they cannot reverse the disease. Long-term use inevitably leads to various adverse reactions such as decreased appetite, abdominal pain, liver damage, rhabdomyolysis, and nervous system damage. Surgical intervention can only temporarily improve blood flow and is prone to complications such as restenosis and thrombosis. Traditional Chinese medicine polysaccharides have good biocompatibility and rich pharmacological activities, including antioxidant, antitumor, immunomodulatory, and anti-inflammatory effects. The activity of polysaccharides is closely related to their structural characteristics, such as molecular weight, configuration, monosaccharide composition, and glycosidic bond type. Finding polysaccharides that can prevent and improve AS is of great significance for the clinical treatment of AS-related cardiovascular diseases.

[0003] Allium macrostemon Bunge or Allium chinense G. Don are the dried bulbs of plants belonging to the Allium genus of the Liliaceae family. Allium macrostemon Bunge has a pungent and bitter taste and is warm in nature. It has the effects of promoting yang and dispersing stagnation, soothing qi and relieving qi stagnation. It has long been a good medicine in traditional Chinese medicine for treating symptoms such as chest pain, abdominal pain, bloating and discomfort, and tenesmus after diarrhea.

[0004] Currently, the polysaccharide composition of *Allium tuberosum* is complex. While some homogeneous polysaccharides have been reported, they are mostly composed primarily of glucose or fructose, and it remains unclear whether these homogeneous polysaccharides can exert pharmacological activity in vivo to improve ankylosing spondylitis (AS). Finding homogeneous *Allium tuberosum* polysaccharides that can prevent or intervene in AS would provide a scientific basis for the development and application of *Allium tuberosum*, and would be of great significance to its development in the biopharmaceutical or health industry. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by proposing a small garlic polysaccharide, its preparation method, and its application.

[0006] To achieve the objectives of this invention, the following technical solution is adopted:

[0007] A method for preparing polysaccharides from small-rooted garlic includes the following steps:

[0008] S1. Crude polysaccharides were obtained from small garlic by extraction and purification, namely AMBP 40, AMBP 60 and AMBP80;

[0009] S2. Separation and preparation of polysaccharide component AMBP60-1;

[0010] S3. Preparation of homogeneous polysaccharide AMBP-1a from garlic root.

[0011] Preferably, in step S1, 200g of dried garlic root is accurately weighed and refluxed at 100°C for 1 hour with 95% ethanol at a material-to-liquid ratio of 1:20 (w / v). The mixture is then filtered through gauze to remove low-polarity impurities. The residue is collected and extracted with distilled water at a material-to-liquid ratio of 1:20 for 2 hours with boiling water. This process is repeated three times. After centrifugation at 4500 rpm for 10 minutes, the supernatants are combined and concentrated under reduced pressure to 1 / 4 of the original volume. 95% ethanol is slowly added to the concentrate while stirring until the final concentrations are 40%, 60%, and 80%, respectively. The mixture is then allowed to stand overnight. The precipitates from different alcohol-precipitated fractions are freeze-dried to obtain crude polysaccharides, which are named AMBP 40, AMBP 60, and AMBP80, respectively.

[0012] Preferably, in step S2, 400 mg AMBP 60 is dissolved in 10 mL of distilled water, centrifuged, and the supernatant is collected and purified by passing it through a DEAE Sepharose Fast Flow column (3.0 × 60 cm). Gradient elution is performed sequentially using sodium chloride solutions of different concentrations (0, 0.1 M, 0.3 M, 0.5 M) at a flow rate of 1.5 mL / min. The eluents from each gradient are collected using an automatic fraction collector. The sugar content of the eluent in each tube is determined at a wavelength of 490 nm using the phenol-sulfuric acid method. The elution curve of the polysaccharide is plotted with the number of collection tubes as the x-axis and the absorbance value as the y-axis. The polysaccharide fractions with the same collection are combined, concentrated, and freeze-dried to obtain a major polysaccharide component AMBP60-1.

[0013] Preferably, in step S3, the polysaccharide is purified using a Sephadex G-50 column (20.0×1200cm), eluted with ultrapure water at a flow rate of 0.5mL / min, and an elution curve is plotted using the phenol-sulfuric acid method. The same polysaccharide fractions are combined, concentrated, and freeze-dried to obtain homogeneous polysaccharide of Allium tuberosum, named AMBP-1a.

[0014] Preferably, AMBP-1a is mainly composed of galactose, glucose and arabinose in a molar ratio of 0.836:0.158:0.006, and is a neutral heteropolysaccharide.

[0015] More preferably, the main framework of AMBP-1a is composed of repeating units of →4)-β-D-Glcp-(1→4)-β-D-Galp-(1→4)-β-D-Galp-(1→4)-β-D-Galp-(1→4)-α-D-Galp-(1→5)-α-L-Araf-(1→), with branch structures of β-D-Galp-(1→3)-β-D-Glcp-(1→) and β-D-Galp-(1→, connected to C-6 of →4,6)-β-D-Glcp-(1→) and C-2 of →2,4)-α-D-Galp-(1→).

[0016] Another technical solution of the present invention: the application of a small-root polysaccharide in the preparation of drugs for preventing and treating atherosclerosis or improving blood lipid levels, wherein the small-root polysaccharide is AMBP-1a.

[0017] Preferably, the application of the allium polysaccharide in the preparation of drugs for preventing and treating atherosclerosis or improving blood lipid levels, wherein AMBP-1a regulates intestinal flora and short-chain fatty acids to improve atherosclerosis.

[0018] More preferably, in the application of the allium polysaccharide in the preparation of drugs for preventing and treating atherosclerosis or improving blood lipid levels, the dosage form of the drug is any one of capsules, tablets, pellets, powders or solutions.

[0019] In a further preferred embodiment, the drug also includes pharmaceutically acceptable excipients.

[0020] Beneficial effects:

[0021] Compared to the homogeneous polysaccharide structures reported in *Allium tuberosum*, the AMBP-1a structure isolated by this method is more novel. Its monosaccharide composition is predominantly galactose, with small amounts of glucose and arabinose. Furthermore, its glycosidic bond linkage is primarily →4)-Galp-(1→), accounting for a high proportion of 72.57%, a structural characteristic not found in previously reported homogeneous polysaccharides from *Allium tuberosum*. The structural characteristics of polysaccharides are related to their activity; AMBP-1a can effectively improve the response to ApoE. - / - The pharmacological activities of this study, including blood lipid levels and atherosclerotic plaques in mice with atherosclerosis, are demonstrated by regulating gut microbiota composition and short-chain fatty acids to intervene in atherosclerosis. However, no similar activity has been observed with homogeneous polysaccharides of garlic previously reported. Attached Figure Description

[0022] For ease of explanation, the present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

[0023] Figure 1 This is a schematic diagram of the extraction, separation, and structural characterization of AMBP-1a.

[0024] Figure 2 The image shows the infrared spectrum and methylation results of AMBP-1a.

[0025] Figure 3 The image shows the nuclear magnetic resonance (NMR) results of AMBP-1a.

[0026] Figure 4 The structure of AMBP-1a is shown in the diagram.

[0027] Figure 5 AMBP-1a effectively improves ApoE - / - The efficacy results of the drug in a mouse model of atherosclerosis.

[0028] Figure 6 AMBP-1a effectively improves ApoE - / - Figure showing the composition of gut microbiota (α-diversity) in a mouse model of atherosclerosis.

[0029] Figure 7 AMBP-1a effectively improves ApoE - / - Figure showing the composition of the gut microbiota (phylum and genus level) in a mouse model of atherosclerosis.

[0030] Figure 8 For AMBP-1a to effectively regulate ApoE - / - Figure showing the results of short-chain fatty acid analysis in a mouse model of atherosclerosis. Detailed Implementation

[0031] The following are specific embodiments of the present invention, described in conjunction with the accompanying drawings, to further illustrate the technical solutions of the present invention. However, the present invention is not limited to these embodiments. Specific details, such as particular configurations, are provided in the following description merely to aid in a comprehensive understanding of the embodiments of the present invention. Therefore, those skilled in the art should understand that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present invention.

[0032] It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0033] Unless otherwise specified, the materials, practices, and experimental equipment involved in the embodiments of this invention are all commercially available products in the relevant chemical and biotechnology fields.

[0034] Example 1: Method for extracting and separating polysaccharides from small garlic roots:

[0035] (1) Extraction and purification:

[0036] Accurately weigh 200g of dried garlic root, add 95% ethanol at a solid-liquid ratio of 1:20 (w / v), heat to reflux at 100℃ for 1 hour, repeat 3 times to remove low-polarity impurities, filter through gauze and collect the residue, add distilled water at a solid-liquid ratio of 1:20, extract with boiling water for 2 hours, repeat 3 times, centrifuge at 4500 rpm for 10 minutes, combine the supernatants, concentrate under reduced pressure to 1 / 4 of the original volume, slowly add 95% ethanol to the concentrate while stirring to the final concentrations of 40%, 60% and 80%, respectively, and let stand overnight. Freeze-dry the precipitates from different alcohol precipitation fractions to obtain crude polysaccharides, named AMBP 40, AMBP 60 and AMBP 80, respectively.

[0037] (2) Separation and preparation:

[0038] 400 mg AMBP 60 was dissolved in 10 mL of distilled water, centrifuged, and the supernatant was purified by passing it through a DEAE Sepharose FastFlow column (3.0 × 60 cm). Gradient elution was performed sequentially using sodium chloride solutions of different concentrations (0, 0.1 M, 0.3 M, 0.5 M) at a flow rate of 1.5 mL / min. The eluents from each gradient were collected using an automatic fraction collector. The sugar content of the eluent in each tube was determined at 490 nm using the phenol-sulfuric acid method. The polysaccharide elution curve was plotted with the number of collection tubes as the x-axis and the absorbance value as the y-axis. Fractions with the same polysaccharide were combined, concentrated, and freeze-dried to obtain a major polysaccharide component, AMBP60-1. Further purification was performed using a Sephadex G-50 column (20.0×1200cm), eluted with ultrapure water at a flow rate of 0.5mL / min, and elution curves were plotted using the phenol-sulfuric acid method. The same polysaccharide fractions were combined, concentrated, and freeze-dried to obtain homogeneous polysaccharide from Allium tuberosum, which was named AMBP-1a.

[0039] Extraction and separation results: The yields of AMBP 40, AMBP 60, and AMBP 80 were 11.3%, 6.4%, and 16.2%, respectively. After purification by a DEAE Sepharose Fast Flow column, AMBP 60 was collected as a major component, AMBP60-1. Figure 1 A), with a yield of 2.9%. AMBP60-1 was subsequently purified using a Sephadex G-50 column to obtain a main peak (A). Figure 1 B), with a yield of 1.8%. Furthermore, such as... Figure 1 As shown in C, no characteristic absorption was observed in the ultraviolet spectrum of AMBP-1a at 260 nm and 280 nm, indicating that AMBP-1a does not contain protein or nucleic acid impurities.

[0040] Example 2: Characterization of polysaccharide structure in garlic.

[0041] Structural characterization methods:

[0042] (1) Purity and molecular weight determination

[0043] The absolute molecular weight of polysaccharide samples was determined using high performance size exclusion chromatography-multi-angle laser detector-differential detector (HPSEC-MALLS-RID). A Shi imadzu LC-10A high performance liquid chromatograph was used; the differential detector was a Shi imadzu RI-10A, the dynamic laser light scattering detector was a Wyatt DynaPro NanoStar, the laser wavelength was 658.1 nm, the specific refractive index increment dn / dc value was 0.1380 mg / L, and the light scattering model was the Zimm model; the chromatographic column was a BRT105-104-102 (8×300 mm) gel column used in series; the mobile phase was 0.05 M NaCl solution; the flow rate was 1.0 mL / min; the column temperature was 25 °C; and the injection volume was 20 μL. Weigh the polysaccharide sample and prepare a 5 mg / mL polysaccharide solution using the mobile phase. After centrifugation at 12000 rpm for 10 min, filter the supernatant through a 0.22 μm microporous membrane. Inject the filtrate into HPSEC-MALLS-RID for analysis and process the data using Astra8 software.

[0044] (2) Infrared analysis

[0045] Accurately weigh 1.0 mg of homogeneous polysaccharide, and thoroughly grind and mix it with 100.0 mg of dry potassium bromide powder under an infrared lamp. Press the mixture into thin sheets and heat at 4000-500 cm⁻¹. -1 Infrared spectral scanning was performed within the wavelength range.

[0046] (3) Methylation analysis

[0047] Preparation of polysaccharide hydrolysate: Weigh 5 mg of polysaccharide sample into an ampoule, add 2 mL of 3M TFA, and hydrolyze at 110 °C for 3 h. After drying the hydrolysate under nitrogen, add 5 mL of pure water, vortex to mix, take 50 μL and dilute to 1 mL, centrifuge at 12000 rpm for 5 min, and take the supernatant for HPAEC analysis. Preparation and calculation of standard solutions: Prepare a standard stock solution by taking 16 monosaccharide standards (glucose, ribose, mannose, fucose, galactose, rhamnose, fructose, arabinose, xylose, galacturonic acid, mannuronic acid, glucuronic acid, guluronic acid, glucosamine hydrochloride, galactosamine hydrochloride, and N-acetyl-D-glucosamine), and accurately prepare a mixed standard solution containing each standard. Ion chromatography conditions: Instrument: Dionex ICS-5000+ ion chromatograph; Column: Dionex Carbopac™ PA20 (3×150mm); Mobile phase: A: H2O; B: 15mM NaOH; C: 15mM NaOH, 100mM NaAC; Flow rate: 0.3mL / min; Injection volume: 5μL; Column temperature: 30℃; Detector: Electrochemical detector.

[0048] (4) Nuclear magnetic resonance analysis

[0049] Weigh 50 mg of homogeneous polysaccharide, dissolve it in 0.5 mL of D2O, and place the NMR tube in a 600 MHz NMR spectrometer for NMR spectrum analysis.

[0050] Structural characterization results:

[0051] (1) Molecular weight distribution determination and monosaccharide composition analysis

[0052] The purity and absolute molecular weight of AMBP-1a were determined using the HPSEC-MALLS-RID method. The HPSEC spectrum of AMBP-1a showed a symmetrical single peak. Figure 1 D) indicates that AMBP-1a has good homogeneity. Its weight-average molecular weight (Mw) and number-average molecular weight (Mn) are 22.89 kDa and 18.47 kDa, respectively, and the dispersion factor (calculated as Mw / Mn) is 1.24, further indicating that its molecular weight distribution has high homogeneity.

[0053] Analysis of monosaccharide composition is fundamental and crucial for studying the structure and biological activity of polysaccharides. For example... Figure 1 As shown in E, AMBP-1a is mainly composed of galactose, glucose and arabinose, with a molar ratio of 0.836:0.158:0.006, indicating that AMBP-1a is a neutral heteropolysaccharide.

[0054] (2) Infrared spectroscopy analysis

[0055] The infrared spectrum of AMBP-1a is as follows: Figure 1 As shown in Figure F, it exhibits the characteristic absorption peak of polysaccharides. 3404 cm⁻¹ -1 The strong and broad peak is caused by the stretching vibration of OH, at 2937 cm⁻¹. -1 and 1423cm -1 The nearby absorption peaks are attributed to the stretching and bending vibrations of CH, respectively, at 1130 cm⁻¹. -1 1061cm -1 and 1029cm -1 There are three absorption peaks at 814 cm⁻¹, which are attributed to the stretching vibrations of CO, COC, and COH on the pyrose ring, respectively. Additionally, at 814 cm⁻¹... -1 and 880cm -1 These are characteristic absorption peaks for α- and β-type glycosidic bonds, respectively.

[0056] (3) Methylation analysis

[0057] Infrared spectra of AMBP-1a methylated products, such as Figure 2 As shown in A, 3400cm -1 The complete disappearance of the nearby OH absorption peak indicates that AMBP-1a is fully methylated. The GC-MS TIC spectrum is shown below. Figure 2 As shown in Figure B, based on the mass spectrometry of fragment ions of partially methylated aldosterone acetates (PMAAs), we determined the glycosidic bond types of methylated AMBP-1a. Based on retention time and characteristic ion fragments, six glycosidic bond linkages were identified in AMBP-1a: 1,5-Araf (3.55%), T-Galp (8.33%), 1,3-Glcp (5.90%), 1,4-Galp (72.57%), 1,2,4-Galp (2.45%), and 1,4,6-Glcp (7.02%). Among the various sugar residues, 1,4-Galp had the largest proportion, indicating that it is likely the main backbone of AMBP-1a. Furthermore, AMBP-1a contains 3.55% arabinose, 83.35% galacturonic acid, and 12.92% glucose, ratios close to those of monosaccharides.

[0058] (4) NMR analysis

[0059] The structure of AMBP-1a was further investigated using NMR. 1 H-NMR, 13 C-NMR, 1 H- 1 H COSY, HSQC, HMBC, and NOESY Figure 3 AF). 1 H and 13The C-NMR signals are concentrated in the 3.4–5.4 ppm and 60–105 ppm regions, which are typical polysaccharide signals. The terminal proton signals and terminal carbon signals are located in the δ 4.3–5.5 ppm and δ 96–110 ppm ranges, respectively. Figure 3 (AB) indicates that the AMBP-1a structure contains both α- and β-configuration glycosidic bonds.

[0060] 1 In the H-NMR spectrum, δ5.28, 4.46, 4.66, 4.65, 4.98, and 4.45 ppm represent terminal matrix proton signals. Figure 3 A) The chemical shift at δ 3.4–4.2 ppm is a characteristic signal of the sugar ring. According to 1 H- 1 H COSY( Figure 3 C) Determine the H1 / H2 ratio for each residue. The signals at δ 5.28 / 3.29, 4.46 / 3.54, 4.66 / 3.70, 4.65 / 3.68, 4.98 / 4.11, and 4.45 / 3.51 ppm represent the H1 / H2 ratios of residues A through F, respectively. Similarly, the remaining proton signals H-3, H-4, H-5, and H-6 on the sugar ring of residue AF can be determined according to... 1 H- 1 The H COSY spectrum was assigned values. 13 In the C-NMR spectrum, the terminal carbon signals of AMBP-1a were located at δ 107.43, 103.56, 104.22, 104.34, 97.67, and 103.64 ppm, respectively. The corresponding terminal proton signals, determined by HSQC spectroscopy, were located at δ 5.28, 4.46, 4.66, 4.65, 4.98, and 4.45 ppm, respectively. Figure 3 (B and 3D). Six related peaks appeared at 107.43 / 5.28, 103.56 / 4.46, 104.22 / 4.66, 104.34 / 4.65, 97.67 / 4.98 and 103.64 / 4.45 ppm, respectively, and were attributed to →5)-α-L-Araf-(1→(A), β-D-Galp-(1→(B), →3)-β-D-Glcp-(1→(C), →4)-β-D -Galp-(1→(D), →2,4)-α-D-Galp-(1→(E) and →4,6)-β-D-Glcp-(1→(F). Furthermore, the chemical shifts of sugar residue AF from H-2 / C-2 to H-6 / C-6 are similarly assigned. Combining the results of NMR spectroscopy, monosaccharide composition, and methylation analysis, and based on the chemical position values ​​of similar sugar residues in relevant literature, the main types of sugar residues in ABMP60-1a are... 1 H and 13 The C chemical shift signal was assigned values, as shown in Table 1.

[0061] Table 1

[0062]

[0063] The linkage mechanism of sugar residues in AMBP-1a was further elucidated using HMBC and NOESY spectra. Figure 3 (EF). In the HMBC spectrum, the correlation peak at 4.98 / 65.46 ppm (HH1 / A C5) indicates that the C-5 of residue A is linked to the H-1 of residue E. Similarly, the correlation peaks at 4.65 / 77.68ppm (DH1 / E C4), 4.65 / 77.63ppm (DH1 / C4), 4.11 / 103.56ppm (E H2 / B C1), 4.18 / 103.64ppm (DH4 / F C1), 4.18 / 104.34ppm (DH4 / C1), and 3.64 / 104.22ppm (F H6 / C C1) indicate that the C-4 of residue E is linked to the H-1 of residue D, the C-4 of residue D is linked to the H-1 of residue D, the C-1 of residue B is linked to the H-2 of residue E, the C-1 of residue F is linked to the H-4 of residue D, the C-1 of residue D is linked to the H-4 of residue D, and the C-1 of residue C is linked to the H-6 of residue F. The NOESY spectrum further confirmed the connection between these sugar residues, with related peaks appearing at 4.65 / 3.75ppm (DH1 / F H5), 3.95 / 3.71ppm (CH5 / B H5), and 3.93 / 4.11ppm (DH5 / E H2), indicating the presence of links between →4)-β-D-Galp-(1→ and →4,6)-β-D-Glcp-(1→, β-D-Galp-(1→ and →3)-β-D-Glcp-(1→, →4)-β-D-Galp-(1→ and →2,4)-α-D-Galp-(1→).

[0064] In summary, AMBP-1a is composed of repeating units, with branched structures of β-D-Galp-(1→3)-β-D-Glcp-(1→) and β-D-Galp-(1→), linked to C-6 of →4,6)-β-D-Glcp-(1→) and C-2 of →2,4)-α-D-Galp-(1→). Based on monosaccharide composition, methylation, and NMR analysis data, the possible structure of AMBP-1a is as follows: Figure 4 As shown.

[0065] Example 3: Evaluation of in vivo anti-atherosclerotic activity

[0066] (1) Animal model construction and drug administration

[0067] After one week of acclimatization, the mice were randomly divided into 6 groups (n=8 per group). Eight C57BL / 6J mice served as the normal control group, and 32 ApoE mice were used as the control group. - / - Mice were randomly divided into five groups: a model group (Mode 1), a positive control group (Simvastatin SV), a low-dose group (AMBP-1a-L), a medium-dose group (AMBP-1a-M), and a high-dose group (AMBP-1a-H). Except for the control group, which was fed a normal diet for 8 weeks, the other groups were fed a high-fat diet for 8 weeks. Furthermore, mice in the SV, AMBP-1a-L, AMBP-1a-M, and AMBP-1a-H groups were administered 5.2 mg / kg simvastatin solution, 50 mg / kg AMBP-1a solution, 100 mg / kg AMBP-1a solution, and 200 mg / kg AMBP-1a solution daily by gavage, respectively, while the control and model groups were administered an equal volume of physiological saline daily by gavage. Mouse weight was recorded weekly. After the last administration, mice were fasted for 12 hours but allowed free access to water, and blood samples, heart, aortic tissue, and cecal contents were collected for further analysis.

[0068] (2) Histopathological observation

[0069] After the entire aorta was removed, it was placed in physiological saline to dissect peripheral tissues. It was then fixed with 4% paraformaldehyde, stained with Oil Red O, and photographed. The removed aortic root was rinsed with physiological saline to remove residual blood, then fixed with 4% paraformaldehyde for 48 hours. After OCT embedding, 10 μm thick frozen sections were prepared transversely. Intact frozen sections were selected for Oil Red O staining to observe plaque area and lipid deposition. Pathological examination was performed under an optical microscope, and photographs were taken. The plaque area was then statistically analyzed using ImageJ software. The relative severity of atherosclerosis was calculated as the percentage of plaque area to the total aortic vessel area and the percentage of plaque area to the transverse section of the aortic root.

[0070] (3) Blood lipid measurement

[0071] Blood samples were collected and left at room temperature for 2 hours, then centrifuged at 6000 rpm for 15 minutes at 4°C. The supernatant was collected as serum. Serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using a Roche fully automated biochemical analyzer.

[0072] To evaluate the protective effect of AMBP-1a against atherosclerosis, we fed ApoE patients a high-fat diet. - / - Mouse animal model, experimental design diagram see Figure 5A. The body weight of the model group mice was significantly different from that of the control group, and higher than that of other drug-treated groups ( Figure 5 B). Oil Red O staining analysis of the entire aorta and aortic root to determine the effect of AMBP-1a on ApoE. - / - Effects on atherosclerotic plaque formation in mice. For example... Figure 5 As shown in Figure C, compared with the control group, the overall aortic plaque area in the model group was significantly increased (p<0.05), while AMBP-1a significantly reduced the aortic plaque area compared with the model group (p<0.05). Furthermore, Figure 5 Oil Red O staining results of the transverse section of the aortic root in group D also showed that, compared with the model group, the plaque area in the AMBP-1a treatment group was significantly reduced (p<0.05). These results indicate that AMBP-1a can effectively alleviate ApoE induced by a high-fat diet. - / - The development of atherosclerotic lesions in mice.

[0073] Dyslipidemia is a significant risk factor for atherosclerosis, typically referring to elevated levels of total cholesterol (TC) and triglycerides (TG) in the blood, and also including elevated levels of low-density lipoprotein cholesterol (LDL-C) and decreased levels of high-density lipoprotein cholesterol (HDL-C). Figure 5 As shown in Figure E, compared with the control group, the serum TG, TC, and LDL-C levels in the model group were significantly increased, while the HDL-C level was significantly decreased (p<0.05). Compared with the model group, high-dose AMBP-1a administration significantly reduced TG, TC, and LDL-C levels and significantly increased HDL-C levels (p<0.05). These results indicate that AMBP-1a can significantly improve ApoE in patients fed a high-fat diet. - / - Abnormal blood lipids in mice.

[0074] Example 4: AMBP-1a regulates gut microbiota and improves atherosclerosis

[0075] (1) 16S rRNA gene sequencing

[0076] Total DNA of the microbial community was extracted from the contents of the mouse cecum using a kit from Omega Medical Materials (USA). DNA quality was assessed using agarose gel electrophoresis and a micro-nucleic acid and protein analyzer. Then, PCR amplification of the V3-V4 region of the 16S rRNA gene was performed using forward primers (5'-ACTCCTACGGGAGGCAGCA-3') and reverse primers (5'-GGACTACHVGGGTWTCTAAT-3'). PCR products from the same sample were mixed and purified using 2% agarose gel electrophoresis, followed by quantitative analysis. Finally, after library construction, sequencing analysis was performed using the Miseq PE300 platform.

[0077] (2) Determination of short-chain fatty acid (SCFA) content

[0078] 20 mg of cecal contents were placed in a 2 mL grinding tube, and grinding beads and 1000 μL of 0.5% phosphoric acid aqueous solution were added. The sample was cryogenically ground for 1 min (50 Hz), sonicated for 10 min, and then centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was collected. Then, 200 μL of the supernatant was transferred to a 1.5 mL tube, and 500 μL of MTBE containing the internal standard was added for extraction. The solution was then vortexed for 3 min, sonicated at low temperature for 5 min, and centrifuged at 12000 rpm for 10 min at 4 °C. Finally, the supernatant was collected for analysis, and the short-chain fatty acid content was determined using an 8890B-7000D gas chromatograph.

[0079] 16S rRNA sequencing was used to analyze the effect of AMBP-1a on gut microbiota abundance. α-diversity analysis showed that the cecal microbiota abundance (Chao1 index and ACE index) and Shannon index were significantly reduced in the model group, while AMBP-1a administration significantly reversed these changes in α-diversity indices. Figure 6 AC). Regarding β diversity, PCA, PCoA, and NMDS analyses revealed that AMBP-1a administration significantly altered ApoE. - / - Microbial community structure of mice Figure 6 AMBP-1a (DF) enhanced the richness and diversity of the microbial community. Phylogenetic analysis revealed an increased Firmicutes / Bacteroidetes ratio in the model group, which was reversed by AMBP-1a administration. Figure 7 A). The Firmicutes / Bacteroidetes ratio is generally considered an indicator of gut dysbiosis and also a biomarker for coronary heart disease. At the genus level, the top 10 microorganisms with the largest abundance changes among the three groups were analyzed. Compared with the model group, administration of AMBP-1a increased the abundance of Faecalibaculum and Akkermansia, and decreased the abundance of Blautia and Bifidobacterium. Figure 7 B). LEfSe was used to further analyze the differentially expressed bacterial communities among the three groups. Significant changes were observed in Faecalibaculum, Akkermansia, and Blautia after AMBP-1a administration, which may be potential biomarkers. Figure 7 (CG). Furthermore, Akkermansia acts as a barrier against exogenous pathogens, and its abundance is negatively correlated with atherosclerosis. It has been reported that Akkermansia muciniphila treatment reduces macrophage infiltration, chemokines, and pro-inflammatory cytokines, protects the integrity of the intestinal barrier, and slows ApoE.- / - Progression of atherosclerotic lesions in mice. Under a high-fat diet with ApoE... - / - In mice, AMBP-1a can improve atherosclerosis by regulating the gut microbiota, especially the abundance of Faecalibaculum and Akkermansia.

[0080] SCFAs, acting as a link between the microbiota and host homeostasis, play a crucial role in regulating atherosclerosis and intestinal barrier function. In the gut, acetate, propionate, and butyrate are common SCFAs produced by specific microbial communities. Acetate and propionate are primarily produced by Bacteroidetes, while Firmicutes are the main contributors to butyrate production. The changes in the seven SCFAs in each group are shown below. Figure 8 As shown in the AG, the levels of acetic acid, butyric acid, valeric acid, hexanoic acid, and isobutyric acid in the model group were significantly lower than those in the control group. Compared with the model group, the levels of SCFAs (acetic acid, butyric acid, hexanoic acid, and isobutyric acid) in the AMBP-1a treatment group were significantly increased, while the levels of propionic acid and isovaleric acid did not change significantly in any group. These results indicate that AMBP-1a alters the ApoE levels of a high-fat diet. - / - Metabolite content in mice.

[0081] Those skilled in the art to which this application pertains may make various modifications or additions to the specific embodiments described, or adopt similar methods to replace them, without departing from the inventive concept of this application or exceeding the scope defined by the appended claims.

Claims

1. The application of a polysaccharide from *Allium tuberosum* in the preparation of a drug for preventing and treating atherosclerosis that improves atherosclerosis by regulating intestinal flora and short-chain fatty acids, characterized in that... The polysaccharide obtained from small-root garlic includes the following steps: S1. Crude polysaccharides were obtained from small garlic by extraction and purification, namely AMBP 40, AMBP 60 and AMBP 80; S2. Separation and preparation of polysaccharide component AMBP60-1; S3. Preparation of homogeneous polysaccharide AMBP-1a from small-rooted garlic; In step S1, 200 g of dried garlic root was weighed accurately. 95% ethanol was added at a material-to-liquid ratio of 1:20 w / v, and the mixture was heated to reflux at 100°C for 1 h. This process was repeated three times to remove low-polarity impurities. The residue was collected by gauze filtration. Distilled water was added at a material-to-liquid ratio of 1:20, and the mixture was extracted with boiling water for 2 h. This process was repeated three times. After centrifugation at 4500 rpm for 10 minutes, the supernatants were combined and concentrated under reduced pressure to 1 / 4 of the original volume. 95% ethanol was slowly added to the concentrate while stirring until the final concentrations were 40%, 60%, and 80%, respectively. The mixture was allowed to stand overnight. The precipitates from different alcohol-precipitated fractions were freeze-dried to obtain crude polysaccharides, which were named AMBP 40, AMBP 60, and AMBP 80, respectively. In step S2, 400 mg AMBP 60 was dissolved in 10 mL of distilled water, centrifuged, and the supernatant was collected and purified by DEAE Sepharose Fast Flow column. Gradient elution was performed sequentially with 0 M, 0.1 M, 0.3 M, and 0.5 M sodium chloride solutions at a flow rate of 1.5 mL / min. The eluents of each gradient were collected using an automatic fraction collector. The sugar content of the eluent in each tube was determined at a wavelength of 490 nm using the phenol-sulfuric acid method. The polysaccharide elution curve was plotted with the number of collection tubes as the x-axis and the absorbance value as the y-axis. The polysaccharide fractions with the same collection were combined, concentrated, and freeze-dried to obtain a polysaccharide fraction AMBP60-1 eluted with 0 M sodium chloride solution. The backbone of AMBP-1a consists of →4)- β -d-Glcp-(1→4)- β -d-Galp-(1→4)- β -d-Galp-(1→4)- β -d-Galp-(1→4)- α -d Galp-(1→5)- α -l-Araf-(1→ repeating units, and the branched structures are respectively β -d-Galp-(1→3)- β -d-Glcp-(1→ and β -d-Galp-(1→, and is linked to the C-6 of →4,6)- β -d-Glcp-(1→ and →2,4)- α -d-Galp (1→ is linked to the C-2; AMBP-1a is composed of galactose, glucose and arabinose in a molar ratio of 0.836:0.158:0.006, and is a neutral heteropolysaccharide. The AMBP-1a regulates the gut microbiota, specifically by adjusting... Faecalibaculum and Akkermansia Abundance can improve the effect of atherosclerosis.

2. The application according to claim 1, characterized in that, In step S3, the polysaccharide was purified using a Sephadex G-50 column, eluted with ultrapure water at a flow rate of 0.5 mL / min, and an elution curve was plotted using the phenol-sulfuric acid method. The same polysaccharide fractions were combined, concentrated, and freeze-dried to obtain homogeneous polysaccharide from small garlic roots, named AMBP-1a.

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