Allium macrostemon polysaccharide as well as preparation method and application thereof

Abstract

The invention provides allium macrostemon polysaccharide as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The method comprises the following steps: extracting and purifying small root garlic to obtain crude polysaccharides, namely AMBP 40, AMBP 60 and AMBP 80; separating to prepare a polysaccharide component AMBP60-1, and then separating to prepare the uniform polysaccharide AMBP-1a of the allium macrostemon. The AMBP-1a is mainly composed of galactose, glucose and arabinose in a molar ratio of 0.836: 0.158: 0.006, and the AMBP-1a is a neutral heteropolysaccharide. The structural characteristics of polysaccharide are related to activity, AMBP-1a can effectively improve the blood fat level of ApoE- / -atherosclerosis model mice, pharmacological activity of atherosclerosis plaques and the like, and atherosclerosis is intervened by regulating intestinal flora and short-chain fatty acid content.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to Allium spp. polysaccharide and a preparation method and application thereof. Background Art

[0002] Cardiovascular disease is the disease with the highest mortality rate among non-communicable diseases. According to the 2021 WHO statistics, the global mortality rate of cardiovascular disease has increased by 25% since 2000. In 2019, the number of deaths reached 17.9 million, accounting for 32% of all deaths, and has become the "number one killer" that seriously threatens human health. Among them, atherosclerosis (AS) is the main cause of the high incidence and high mortality of cardiovascular disease. Clinical treatment of AS often uses drug therapy and surgical treatment. Although it can relieve symptoms, it cannot be reversed. Long-term use will inevitably produce a variety of adverse reactions such as decreased appetite, abdominal pain, liver damage, rhabdomyolysis and nervous system damage. Surgical intervention can only temporarily improve blood flow conditions and is prone to complications such as restenosis and thrombosis. Chinese herbal polysaccharides have good biocompatibility and rich pharmacological activities, including antioxidant, anti-tumor, immunomodulatory, anti-inflammatory and other 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 for the prevention and improvement of AS is of great significance for the treatment of cardiovascular diseases related to AS in clinical practice.

[0003] Xiebai is the dried bulb of Allium macrostemon Bunge or Alliumchinense G.Don, a plant of the genus Allium in the Liliaceae family. Xiebai tastes pungent and bitter, and is warm in nature. It has the effects of promoting yang and dispersing nodules, stretching the qi, and unblocking qi stagnation. It has long been a good medicine used in traditional Chinese medicine to treat symptoms such as chest pain, heart pain, abdominal pain, bloating, and heaviness after diarrhea.

[0004] At present, the composition of garlic polysaccharides is complex. Some uniform polysaccharides have been reported, but most of them are mainly glucose or fructose, and it is unclear whether these uniform polysaccharides can play a pharmacological activity in improving AS in vivo. If uniform polysaccharides of garlic can be found to prevent or intervene in AS, it will provide a scientific basis for the development and application of garlic, which will be of great significance to the development of garlic in the biomedicine or health care industry. Summary of the invention

[0005] The technical problem to be solved by the present invention is to address the deficiencies of the above-mentioned prior art and to provide a Allium spp. polysaccharide, a preparation method and an application thereof.

[0006] In order to achieve the purpose of the present invention, the following technical scheme is adopted:

[0007] A method for preparing Allium sphaerocephalum polysaccharide comprises the following steps:

[0008] S1. Extracting and purifying garlic root to obtain crude polysaccharides, namely AMBP 40, AMBP 60 and AMBP80;

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

[0010] S3. Isolation and preparation of uniform polysaccharide AMBP-1a from Allium arvense.

[0011] Preferably, in step S1, 200 g of dried garlic root is weighed accurately, 95% ethanol is added at a solid-liquid ratio of 1:20 (w / v), heated to reflux at 100°C for 1h, repeated 3 times to remove low-polar impurities, and the residue is collected by gauze filtration, distilled water is added at a solid-liquid ratio of 1:20, and boiling water extraction is repeated 3 times. After centrifugation at 4500 rpm for 10 minutes, the supernatant is combined, concentrated under reduced pressure to 1 / 4 of the original volume, and 95% ethanol is slowly added to the concentrate while stirring to a final concentration of 40%, 60% and 80%, respectively, and left to stand overnight. The precipitates from different alcohol precipitation parts are freeze-dried to obtain crude polysaccharides, which are named AMBP 40, AMBP 60 and AMBP80, respectively.

[0012] Preferably, in step S2, 400 mg of AMBP 60 is dissolved in 10 mL of distilled water and centrifuged to obtain the supernatant, which is purified by a DEAE Sepharose Fast Flow column (3.0×60 cm), and different concentrations of sodium chloride solutions (0, 0.1 M, 0.3 M, 0.5 M) are used in sequence for gradient elution at a flow rate of 1.5 mL / min. The eluate of each gradient is collected by an automatic fraction collector, and the sugar content of the eluate in each tube is determined by the phenol-sulfuric acid method at a wavelength of 490 nm. The elution curve of the polysaccharide is drawn with the number of collected tubes as the horizontal axis and the absorbance value as the vertical axis. The same polysaccharide fractions are collected and combined, concentrated and freeze-dried to obtain a main polysaccharide component AMBP60-1.

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

[0014] Preferably, the 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] Further preferably, the main skeleton 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→), and the branch structures are β-D-Galp-(1→3)-β-D-Glcp-(1→ and β-D-Galp-(1→, which are 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: use of Allium giganteum polysaccharide in the preparation of a drug for preventing and treating atherosclerosis or improving blood lipid levels, wherein the Allium giganteum polysaccharide is AMBP-1a.

[0017] Preferably, the Allium spp. polysaccharide is used in the preparation of a drug for preventing and treating atherosclerosis or improving blood lipid levels, and the AMBP-1a regulates intestinal flora and short-chain fatty acids to improve atherosclerosis.

[0018] Further preferably, the Allium spp. polysaccharide is used in the preparation of a drug for preventing and treating atherosclerosis or improving blood lipid levels, and the drug is in the form of any one of capsules, tablets, pills, powders or solutions.

[0019] Still further preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0020] Beneficial effects:

[0021] Compared with the structural characteristics of uniform polysaccharides reported from garlic, the AMBP-1a separated by this method has a more novel structure. The monosaccharide composition is mostly galactose, with a small amount of glucose and arabinose; and its glycosidic bond connection mode is mainly →4)-Galp-(1→, accounting for as high as 72.57%, while the uniform polysaccharide reported previously from Allium macrostemon (garlic, leek) does not have this structural feature. The structural characteristics of polysaccharides are related to activity. AMBP-1a can effectively improve the ApoE - / - The pharmacological activities include blood lipid levels and atherosclerotic plaques in atherosclerosis model mice, and intervention in atherosclerosis by regulating the composition of intestinal flora and short-chain fatty acids. However, the uniform polysaccharides of Allium tuberosum reported so far have not been found to have such activity. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0024] Figure 2 This is the infrared spectrum and methylation result diagram of AMBP-1a.

[0025] Figure 3 This is the nuclear magnetic resonance result of AMBP-1a.

[0026] Figure 4 This is the structural result diagram of AMBP-1a.

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

[0028] Figure 6 AMBP-1a effectively improves ApoE - / - Figure 2. The composition of intestinal flora (α diversity) in mice with atherosclerosis model.

[0029] Figure 7 AMBP-1a effectively improves ApoE - / - Figure 2. The composition of intestinal flora (phylum and genus levels) in mice with atherosclerosis model.

[0030] Figure 8 AMBP-1a effectively regulates ApoE - / - Short-chain fatty acid results in atherosclerosis model mice. DETAILED DESCRIPTION

[0031] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments; in the following description, specific details such as specific configurations are provided only to help fully understand the embodiments of the present invention. Therefore, it should be clear to those skilled in the art 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, in the absence of conflict, the embodiments of the present invention and the features of the embodiments may be combined with each other.

[0033] Unless otherwise specified, the materials, practices and experimental equipment involved in the implementation cases of the present invention are all in line with commercially available products in the relevant chemical and biotechnology fields.

[0034] Embodiment 1, allium arvense polysaccharide extraction and separation method:

[0035] (1) Extraction and purification:

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

[0037] (2) Separation and preparation:

[0038] 400 mg of AMBP 60 was dissolved in 10 mL of distilled water and centrifuged. The supernatant was taken and purified by a DEAE Sepharose FastFlow column (3.0×60 cm). Different concentrations of sodium chloride solution (0, 0.1 M, 0.3 M, 0.5 M) were used for gradient elution at a flow rate of 1.5 mL / min. The eluate of each gradient was collected by an automatic fraction collector. The sugar content of the eluate in each tube was determined by the phenol-sulfuric acid method at a wavelength of 490 nm. The elution curve of the polysaccharide was plotted with the number of collected tubes as the horizontal axis and the absorbance value as the vertical axis. The same polysaccharide fractions were collected and combined, concentrated and freeze-dried to obtain a major polysaccharide component AMBP60-1. It was further purified by Sephadex G-50 column (20.0×1200cm), eluted with ultrapure water at a flow rate of 0.5mL / min, and the elution curve was drawn by the phenol-sulfuric acid method. The same polysaccharide fractions were combined, concentrated and freeze-dried to obtain a uniform polysaccharide of Allium spp., 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. AMBP 60 was purified by DEAE Sepharose Fast Flow column and a major component AMBP60-1 ( Figure 1 A), with a yield of 2.9%. AMBP60-1 was then purified by Sephadex G-50 column to obtain a main peak ( Figure 1 B), the yield is 1.8%. Figure 1 As shown in C, no characteristic absorption was observed in the UV spectrum of AMBP-1a at 260 nm and 280 nm, indicating that AMBP-1a does not contain protein and nucleic acid impurities.

[0040] Example 2: Characterization of the structure of Allium spp. polysaccharide

[0041] Structural Characterization Methods:

[0042] (1) Purity and molecular weight determination

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

[0044] (2) Infrared analysis

[0045] Accurately weigh 1.0 mg of homogeneous polysaccharide, grind and mix with 100.0 mg of dry potassium bromide powder under infrared light, press into thin slices, and heat at 4000-500 cm -1 Infrared spectrum scanning was performed in the wavelength range.

[0046] (3) Methylation analysis

[0047] Preparation of polysaccharide hydrolyzate: weigh 5 mg of polysaccharide sample into an ampoule, add 2 mL of 3M TFA, and hydrolyze at 110 ° C for 3 h. After the hydrolyzate is blown dry with nitrogen, add 5 mL of pure water, vortex to mix, draw 50 μL from it and dilute to 1 mL, centrifuge at 12000 rpm for 5 min, and take the supernatant for HPAEC analysis. Preparation and calculation method of standard solution: take 16 monosaccharide standards (glucose, ribose, mannose, fucose, galactose, rhamnose, fructose, arabinose, xylose, galacturonic acid, mannuronic acid, glucuronic acid, guluronic acid, glucosamine hydrochloride, galactosamine hydrochloride, N-acetyl-D-glucosamine) to prepare standard stock solution, and accurately prepare mixed standard solution containing each standard. Ion chromatography conditions: instrument: Dionex ICS-5000+ ion chromatograph; chromatographic column: Dionex CarbopacTM 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] 50 mg of homogeneous polysaccharide was weighed and dissolved in 0.5 mL of D2O. The NMR tube was placed in a 600 MHz NMR instrument 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 by HPSEC-MALLS-RID method. The HPSEC spectrum of AMBP-1a showed a symmetrical single peak ( Figure 1 D), indicating that AMBP-1a has good uniformity. Its weight average molecular weight (Mw) and number average molecular weight (Mn) are 22.89 kDa and 18.47 kDa respectively, and the dispersion coefficient (calculated as Mw / Mn) is 1.24, further indicating that its molecular weight distribution has high uniformity.

[0053] The analysis of monosaccharide composition is the basis and key to studying the structure and biological activity of polysaccharides. 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 F, it has the characteristic absorption peak of polysaccharide. 3404cm -1 The strong and broad peak at 2937 cm is caused by the stretching vibration of OH. -1 and 1423cm -1 The absorption peaks near 1130 cm-1 are attributed to the stretching vibration and bending vibration of CH. -1 、1061cm -1 and 1029cm -1 There are three absorption peaks at 814cm, which are attributed to the stretching vibrations of CO, COC and COH on the pyridine ring. -1 and 880cm -1 They are the characteristic absorption peaks of α- and β-type glycosidic bonds respectively.

[0056] (3) Methylation analysis

[0057] The infrared spectrum of the methylation product of AMBP-1a is as follows Figure 2 As shown in A, 3400cm -1 The OH absorption peak near the methylation peak completely disappeared, indicating that AMBP-1a was completely methylated. Figure 2 As shown in Figure B, based on the mass spectra of the fragment ions of partially methylated acetic acid alditol (PMAAs), we determined the glycosidic bond type of methylated AMBP-1a. According to the retention time and characteristic ion fragments, six glycosidic bond connection modes were identified in AMBP-1a, namely 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 accounts for the largest proportion, indicating that it may be the main skeleton of AMBP-1a. In addition, AMBP-1a contains 3.55% arabinose, 83.35% galacturonic acid, and 12.92% glucose, and the ratios are close to the monosaccharide composition ratio.

[0058] (4) NMR analysis

[0059] The structure of AMBP-1a was further investigated by 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 3.4-5.4ppm and 60-105ppm regions, which are typical polysaccharide signals. The terminal proton signal and terminal carbon signal are located in the δ4.3-5.5ppm range and δ96-110ppm range, respectively ( Figure 3 AB), indicating that the AMBP-1a structure has both α-configuration 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 are terminal proton signals ( Figure 3 A), the chemical shift at δ3.4-4.2ppm is the characteristic signal of sugar ring. 1 H- 1 H COSY( Figure 3 C), determine the H1 / H2 of each residue, and the signals at δ5.28 / 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 of residues A to F, respectively. Similarly, the remaining proton signals H-3, H-4, H-5 and H-6 on the sugar ring of residues AF can be calculated according to 1 H- 1 The H COSY spectrum was assigned. 13 In the C-NMR spectrum, the terminal carbon signals of AMBP-1a are located at δ107.43, 103.56, 104.22, 104.34, 97.67 and 103.64 ppm, respectively. The corresponding terminal proton signals are determined by HSQC spectrum to be located at δ5.28, 4.46, 4.66, 4.65, 4.98 and 4.45 ppm, respectively. Figure 3 B and 3D). Six relevant 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, which were respectively 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). In addition, the chemical shift assignments of sugar residues AF from H-2 / C-2 to H-6 / C-6 were also similar. Combining the results of NMR spectrum, monosaccharide composition and methylation analysis, the main types of sugar residues in ABMP60-1a were identified based on the chemical position values ​​of similar sugar residues in the relevant literature. 1 H and 13 The C chemical shift signals were assigned values, as shown in Table 1.

[0061] Table 1

[0062]

[0063] The connection mode of sugar residues in AMBP-1a was further elucidated by HMBC and NOESY spectra. Figure 3 In the HMBC spectrum, the correlation peak at 4.98 / 65.46 ppm (HH1 / AC5) indicated that C-5 of residue A was connected to H-1 of residue E. Similarly, the correlation peaks at 4.65 / 77.68 ppm (D H1 / E C4), 4.65 / 77.63 ppm (D H1 / C4), 4.11 / 103.56 ppm (E H2 / B C1), 4.18 / 103.64 ppm (D H4 / F C1), 4.18 / 104.34 ppm (D H4 / C1), and 3.64 / 104.22 ppm (F H6 / C C1) indicate that C-4 of residue E is connected to H-1 of residue D, C-4 of residue D is connected to H-1 of residue D, C-1 of residue B is connected to H-2 of residue E, C-1 of residue F is connected to H-4 of residue D, C-1 of residue D is connected to H-4 of residue D, and C-1 of residue C is connected to H-6 of residue F. The NOESY spectra further confirmed the connection between these sugar residues, with correlation peaks appearing at 4.65 / 3.75 ppm (DH1 / F H5), 3.95 / 3.71 ppm (C H5 / B H5), and 3.93 / 4.11 ppm (D H5 / E H2), respectively, indicating the existence of →4)-β-D-Galp-(1→ and →4,6)-β-D-Glcp-(1→, β-D-Galp-(1→ as well as →3)-β-D-Glcp-(1→, →4)-β-D-Galp-(1→ and →2,4)-α-D-Galp-(1→).

[0064] In summary, the repeating unit 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→) is composed of two branch structures, β-D-Galp-(1→3)-β-D-Glcp-(1→) and β-D-Galp-(1→, which are connected to C-6 of →4,6)-β-D-Glcp-(1→) and C-2 of →2,4)-α-D-Galp-(1→. Based on the data of monosaccharide composition, methylation and NMR analysis, the possible structure of AMBP-1a is as follows Figure 4 shown.

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

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

[0067] After one week of adaptive feeding, the mice were randomly divided into 6 groups (8 mice in each group). 8 C57BL / 6J mice served as the normal control group (Controll) and 32 ApoE - / - Mice were randomly divided into 5 groups: model group (Mode l), positive drug simvastatin group (SV), low-dose group (AMBP-1a-L), medium-dose group (AMBP-1a-M, and high-dose group (AMBP-1a-H). Except for the control group, which was fed with a normal diet for 8 weeks, the other groups were fed with a high-fat diet for 8 weeks. In addition, mice in the SV, AMBP-1a-L, AMBP-1a-M, and AMBP-1a-H groups were gavaged with 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, respectively, daily, while the control group and model group were gavaged with an equal volume of saline daily. At the same time, the body weight of mice was recorded weekly. After the last administration, the mice were fasted but not watered for 12 hours, and blood samples, heart, aortic tissues, and cecal contents were collected for further analysis.

[0068] (2) Histopathological observation

[0069] After the aorta was removed as a whole, it was placed in saline to strip the peripheral tissue, then fixed with 4% paraformaldehyde, stained with Oil Red O and photographed. The removed aortic root was rinsed with saline to remove residual blood, then fixed with 4% paraformaldehyde for 48 hours, embedded in OCT, and cross-sectioned with 10 μm thick frozen sections. The frozen sections with complete morphology were selected, and the plaque area and lipid deposition were observed by Oil Red O staining. The pathological conditions were observed under an optical microscope and photographed, and the plaque area was counted using Image J software. The relative severity of atherosclerosis was calculated as the percentage of plaque area to the total area of ​​the aorta, and the percentage of plaque area to the cross-section of the aortic root.

[0070] (3) Blood lipid measurement

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

[0072] To evaluate the protective effect of AMBP-1a on atherosclerosis, we used ApoE - / - Mouse animal model, experimental design schematic diagram see Figure 5A. The body weight of mice in the model group was significantly different from that in the control group and was higher than that in the other drug-treated groups ( Figure 5 B). Oil red O staining analysis of the aorta as a whole and the aortic root to determine the effect of AMBP-1a on ApoE - / - Effects of atherosclerotic plaque formation in mice. Figure 5 As shown in C, compared with the control group, the overall aortic plaque area in the model group increased significantly (p<0.05), and compared with the model group, AMBP-1a could significantly reduce the aortic plaque area (p<0.05). Figure 5 The results of Oil Red O staining of the aortic root cross section in D also showed that the plaque area in the AMBP-1a treatment group was significantly reduced compared with the model group (p<0.05). The above results show that AMBP-1a can effectively reduce ApoE induced by a high-fat diet. - / - Development of atherosclerotic lesions in mice.

[0073] Dyslipidemia is an important risk factor for the formation of atherosclerosis, which usually refers to the increase of TC (cholesterol) and TG (triglyceride) levels in the blood, and also includes the increase of LDL-C (low-density lipoprotein cholesterol) and the decrease of HDL-C (high-density lipoprotein cholesterol). Figure 5 As shown in 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 administration of AMBP-1a can significantly reduce the levels of TG, TC and LDL-C, and significantly increase the level of HDL-C (p<0.05). The above results show that AMBP-1a can significantly improve the ApoE - / - Dyslipidemia in mice.

[0074] Example 4: AMBP-1a regulates intestinal flora to improve atherosclerosis

[0075] (1) 16S rRNA gene sequencing

[0076] In the experiment, the total DNA of the microbial community in the cecal contents of mice was extracted according to the kit of Omega Company in the United States, and the quality of DNA was tested by agarose gel electrophoresis and micro-nucleic acid protein analyzer. Then, the V3-V4 region of the 16S rRNA gene was PCR amplified using the forward primer (5'-ACTCCTACGGGAGGCAGCA-3') and the reverse primer (5'-GGACTACHVGGGTWTCTAAT-3'). After the PCR products of the same sample were mixed, the PCR products were recovered and purified by 2% agarose gel electrophoresis, and quantitative analysis was performed. Finally, after library construction, the Miseq PE300 platform was used for sequencing analysis.

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

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

[0079] 16SrRNA sequencing was used to analyze the effect of AMBP-1a on the abundance of intestinal flora. α diversity analysis showed that the richness of cecal flora (Chao1 index and ACE index) and Shannon index in the model group were significantly reduced, while the administration of AMBP-1a significantly reversed the changes in α diversity index ( Figure 6 AC). Regarding β diversity, PCA, PCoA and NMDS analysis found that administration of AMBP-1a significantly altered ApoE - / - Microbial community structure of mice ( Figure 6 DF), which increased the richness and diversity of the microbial community. At the phylum level, the Firmicutes / Bacteroidetes ratio was found to be enhanced in the model group, and the administration of AMBP-1a could reverse this enhancement ( Figure 7 A). The Firmicutes / Bacteroidetes ratio is generally considered an indicator of intestinal dysbiosis and is also considered a marker of coronary heart disease. At the genus level, the top 10 microorganisms with the highest abundance changes among the three groups were analyzed. Compared with the model group, the 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 differential microbiota among the three groups. After AMBP-1a administration, Faecalibaculum, Akkermansia, and Blautia changed significantly, which may be potential biomarkers ( Figure 7 CG). In addition, Akkermansia is a barrier to exogenous pathogens, and its abundance is negatively correlated with atherosclerosis. Akkermansia muciniphila treatment reportedly reduced macrophage infiltration, chemokines, and proinflammatory cytokines, protected the integrity of the intestinal barrier, and slowed down ApoE- / - Progression of atherosclerotic lesion formation in mice. - / - In mice, AMBP-1a can improve atherosclerosis by regulating intestinal flora, especially the abundance of Faecalibaculum and Akkermansia.

[0080] SCFAs, as a link between the microbiota and host homeostasis, play an important role in regulating atherosclerosis and intestinal barrier function. In the intestine, acetate, propionate, and butyrate are common SCFAs produced by specific microbiota. Acetate and propionate are mainly produced by Bacteroidetes, while Firmicutes is the main contributor of butyrate. The changes of the 7 SCFAs in each group are shown in Figure 2. Figure 8 As shown in AG, the content of acetate, butyrate, valerate, caproate and isobutyrate in the model group was significantly lower than that in the control group. Compared with the model group, the content of SCFAs in the AMBP-1a-treated group increased significantly (acetate, butyrate, caproate and isobutyrate), while the levels of propionate and isovaleric acid in each group did not change significantly. These results indicate that AMBP-1a changes the ApoE - / - Metabolite content in mice.

[0081] Those skilled in the art to which the present application belongs may make various modifications or additions to the specific embodiments described or replace them in a similar manner, but they will not deviate from the inventive concept of the present application or exceed the scope defined by the attached claims.

Claims

1. A method for preparing Allium spp. polysaccharide, characterized in that: The steps include: S1. extracting and purifying garlic root to obtain crude polysaccharides, namely AMBP 40, AMBP 60 and AMBP 80; S2. Isolation and preparation of polysaccharide component AMBP60-1; S3. Isolation and preparation of uniform polysaccharide AMBP-1a from Allium arvense.

2. The method for preparing Allium spp. polysaccharide according to claim 1, characterized in that: In step S1, 200 g of dried garlic root was accurately weighed, 95% ethanol was added at a solid-liquid ratio of 1:20 (w / v), heated to reflux at 100°C for 1 h, and repeated 3 times to remove low-polar impurities, and the residue was collected by gauze filtration, and distilled water was added at a solid-liquid ratio of 1:20, and boiling water extraction was performed for 2 h, which was repeated 3 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, and 95% ethanol was slowly added to the concentrated solution while stirring to final concentrations of 40%, 60% and 80%, respectively, and allowed to stand overnight; the precipitates from different alcohol precipitation parts were freeze-dried to obtain crude polysaccharides, which were named AMBP 40, AMBP 60 and AMBP 80, respectively.

3. The method for preparing Allium spp. polysaccharide according to claim 2, characterized in that: In the step S2, 400 mg of AMBP 60 is dissolved in 10 mL of distilled water and centrifuged. The supernatant is taken and purified by a DEAE Sepharose Fast Flow column. Different concentrations of sodium chloride solutions are used for gradient elution at a flow rate of 1.5 mL / min. The eluate of each gradient is collected by an automatic fraction collector. The sugar content of the eluate in each tube is determined at a wavelength of 490 nm by the phenol-sulfuric acid method. The elution curve of the polysaccharide is drawn with the number of collected tubes as the horizontal axis and the absorbance value as the vertical axis. The same polysaccharide fractions are collected and combined, concentrated and freeze-dried to obtain a main polysaccharide component AMBP60-1.

4. The method for preparing Allium spp. polysaccharide according to claim 3, characterized in that: In the step S3, the polysaccharide was purified by Sephadex G-50 column, eluted with ultrapure water at a flow rate of 0.5 mL / min, and the elution curve was drawn by the phenol-sulfuric acid method. The same polysaccharide fractions were combined, concentrated and freeze-dried to obtain a uniform polysaccharide of Allium spp., which was named AMBP-1a.

5. The method for preparing Allium spp. polysaccharide according to claim 1, characterized in that: The AMBP-1a is mainly composed of galactose, glucose and arabinose with a molar ratio of 0.836:0.158:0.006, and is a neutral heteropolysaccharide.

6. The method for preparing Allium spp. polysaccharide according to claim 1, characterized in that: The main skeleton of AMBP-1a is composed 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 branch structures are β -d-Galp-(1→3)- β -d-Glcp-(1→ and β -d-Galp-(1→, with→4,6)- β -d-Glcp-(1→C-6 connected and→2,4)- α -d-Galp-(1→) is connected to C-2.

7. The Allium spp. polysaccharide prepared by the preparation method according to any one of claims 1 to 6, characterized in that: The Allium spp. polysaccharide is AMBP-1a.

8. Use of the Allium spp. polysaccharide prepared by the preparation method of any one of claims 1 to 6 or the Allium spp. polysaccharide according to claim 7 in the preparation of a drug for preventing and treating atherosclerosis or improving blood lipid levels.

9. The use according to claim 8, characterized in that: The AMBP-1a regulates intestinal flora and short-chain fatty acids to improve atherosclerosis.

10. The use according to claim 8 or 9, characterized in that: The dosage form of the drug is any one of capsules, tablets, pills, powders or solutions, and also includes pharmaceutically acceptable excipients.

Images