A compound polysaccharide of Tremella fuciformis, Agaricus bisporus and Boletus edulis and its preparation method and application

Abstract

The present invention discloses a compound polysaccharide of tremella, Agaricus bisporus and boletus and its preparation method and application, belonging to the field of natural medicine development technology. The compound polysaccharide provided by the present invention is prepared from tremella, Agaricus bisporus and boletus as raw materials through water extraction and alcohol precipitation, and the weight ratio of the tremella, Agaricus bisporus and boletus is (1-5): (1-3): (1-3). It has been verified by experiments that the compound polysaccharide prepared by the present invention can slow down the formation of atherosclerotic plaques by reducing fat accumulation, reducing the content of triglycerides, total cholesterol and low-density lipoprotein cholesterol in serum, improving intestinal flora disorders, alleviating lipid metabolism disorders or reducing the level of atherosclerosis-related serum metabolites, and the effect is better than that of a single polysaccharide. It can be seen that the present invention provides a new idea for the clinical treatment of atherosclerosis.

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicine development, in particular to a compound polysaccharide of Tremella fuciformis, Agaricus bisporus and Boletus, and a preparation method and application thereof. Background Art

[0002] Atherosclerosis is a chronic inflammatory vascular disease caused by fibrous tissue calcification and lipid accumulation, and is an important pathological basis for various cardiovascular diseases. Atherosclerosis can lead to a series of diseases, such as ischemic stroke, heart disease, and peripheral vascular disease. In 2020, the World Health Organization reported that cardiovascular disease (ischemic heart disease, stroke) is one of the three leading causes of death worldwide. Over the past 20 years, atherosclerosis has remained the leading cause of cardiovascular morbidity and mortality worldwide, placing a huge economic burden on patients and their families.

[0003] Rosuvastatin or atorvastatin are commonly used drugs for the clinical treatment of atherosclerosis. While these drugs can effectively treat patients to a certain extent, up to one-third of patients still lack effective treatment for atherosclerosis. Furthermore, statin use can also cause adverse reactions such as liver damage, indigestion, and muscle pain. Currently, there is no effective and safe treatment for atherosclerosis.

[0004] Tremella fuciformis is a traditional medicinal fungus with extensive pharmacological activity. Existing Tremella fuciformis-related inventions are not targeted for the treatment of atherosclerosis, have minimal therapeutic effects, and their mechanisms of action are unclear. There are no prior reports on the use of Agaricus bisporus or Boletus as raw materials or excipients for improving atherosclerosis. Based on this, the present invention proposes a composite polysaccharide of Tremella fuciformis, Agaricus bisporus, and Boletus edulis for clinical application in the treatment of atherosclerosis. Summary of the Invention

[0005] The purpose of the present invention is to provide a compound polysaccharide of Tremella fuciformis, Agaricus bisporus and Boletus edulis, and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention uses Tremella fuciformis, Agaricus bisporus and Boletus edulis as raw materials and adopts a "water extraction and alcohol precipitation" method to obtain the compound polysaccharide, which has significant effects and conversion potential in preventing and treating atherosclerosis, and provides a new idea for the clinical treatment of atherosclerosis.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides a compound polysaccharide. The compound polysaccharide is prepared by taking tremella, Agaricus bisporus and boletus as raw materials through water extraction and alcohol precipitation. The weight ratio of the tremella, Agaricus bisporus and boletus is (1-5):(1-3):(1-3).

[0008] Furthermore, the weight ratio of the Tremella fuciformis, Agaricus bisporus and Boletus edulis is 3:2:2.

[0009] The present invention also provides a method for preparing the compound polysaccharide, comprising the following steps:

[0010] Washing, drying and crushing Tremella fuciformis, Agaricus bisporus and Boletus edulis respectively to obtain Tremella fuciformis powder, Agaricus bisporus powder and Boletus edulis powder;

[0011] Mixing Tremella powder, Agaricus bisporus powder and Boletus edulis powder according to a specified weight ratio;

[0012] Take the mixed powder, add deionized water, extract twice, combine the extracts, and concentrate under reduced pressure to obtain a concentrate;

[0013] The concentrated solution is dialyzed, ethanol is added after the dialysis, and the precipitate is collected by centrifugation. The precipitate is the compound polysaccharide.

[0014] Furthermore, the volume ratio of the mixed powder to the deionized water is 1:30.

[0015] Furthermore, the extraction is carried out at 80°C.

[0016] Furthermore, the dialysis is performed by placing the precipitate under 3500Da conditions and dialyzing it at 4°C for 72 hours.

[0017] Furthermore, the volume concentration of the ethanol is 95%; the volume ratio of the concentrated liquid to the ethanol is 1:5.3.

[0018] The present invention also provides the use of the compound polysaccharide in preparing a drug for preventing or treating atherosclerosis.

[0019] Furthermore, the compound polysaccharide plays a role in preventing and / or treating atherosclerosis by reducing fat accumulation, lowering the levels of triglycerides, total cholesterol and low-density lipoprotein cholesterol in serum, alleviating intestinal flora disorders, alleviating lipid metabolism disorders or reducing the levels of serum metabolites related to atherosclerosis.

[0020] The present invention also provides a medicine for preventing and treating atherosclerosis, comprising the compound polysaccharide.

[0021] The present invention discloses the following technical effects:

[0022] The present invention uses three kinds of edible fungi, and through careful processing and preparation, the compound polysaccharide can exert the best therapeutic effect and provide targeted treatment for atherosclerosis. The experimental verification shows that the compound polysaccharide prepared by the present invention can improve ApoE by reducing the levels of TC, TG and LDL-C in serum. - / - Lipid metabolism function in atherosclerotic mice; alleviating ApoE - / -Organ damage in atherosclerotic mice; reduced lipid droplet accumulation and vacuolization in adipose tissue; improved lipid metabolism by reducing the proportion of white fat and increasing the proportion of brown fat; improved ApoE - / - The intestinal flora of mice with atherosclerosis was disrupted and the levels of serum metabolites associated with atherosclerosis were reduced. The effect was superior to that of a single polysaccharide. This suggests that the present invention provides new insights into the clinical treatment of atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] # indicates p < 0.05 compared with the normal diet blank group, ## indicates p < 0.01 compared with the normal diet blank group, ### indicates p < 0.001 compared with the normal diet blank group; * indicates p < 0.05 compared with the high-fat diet model group, ** indicates p < 0.01 compared with the high-fat diet model group, *** indicates p < 0.001 compared with the high-fat diet model group; ns indicates no statistically significant difference.

[0025] Figure 1 Figure 2 is the weight change of mice in each group;

[0026] Figure 2 The Oil Red O staining images of the aorta of mice in each group; NCD: normal diet blank group, HFD: high-fat diet model group, HFD+TF: Tremella fuciformis alone administration group, HFD+AB: Agaricus bisporus alone administration group, HFD+BE: Boletus edulis alone administration group, HFD+CCP (3:2:2): compound polysaccharide administration group;

[0027] Figure 3 H&E staining images of adipose tissues of mice in each group; iWAT: inguinal white adipose tissue, eWAT: epididymal white adipose tissue, pWAT: perirenal white adipose tissue, BAT: brown adipose tissue;

[0028] Figure 4 Statistical graphs of adipose tissue index in each group of mice; A: inguinal white adipose tissue index; B: epididymal white adipose tissue index; C: perirenal white adipose tissue index; D: brown adipose tissue index;

[0029] Figure 5 Statistical graph of triglyceride (A), total cholesterol (B) and low-density lipoprotein cholesterol (C) levels in the serum of mice in each group;

[0030] Figure 6 Statistical graphs of liver (A), heart (B), pancreas (C), spleen (D), thymus (E) and kidney (F) indexes of mice in each group;

[0031] Figure 7 Statistical graph of the number of intestinal flora species in each group of mice;

[0032] Figure 8 The number of intestinal flora species in each group of mice;

[0033] Figure 9 Statistical diagram of differential metabolites in each group of mice;

[0034] Figure 10 Statistical graph of metabolite changes in each group of mice;

[0035] Figure 11 Pathway enrichment statistics for each group of mice. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Example 1

[0042] 1. Preparation of compound polysaccharides and single polysaccharides

[0043] Preparation of compound polysaccharide:

[0044] Cleaning and physical crushing: Wash and dry the three edible fungi (tremella, Agaricus bisporus, and porcini) separately. Cut the Tremella, Agaricus bisporus, and porcini with a diameter exceeding 1 cm into pieces or slices approximately 1 cm in diameter. Crush the three edible fungi separately and pass through a 100-mesh sieve.

[0045] Compound formula: Mix the three edible fungi powders according to the weight ratio of 24 parts of Tremella fuciformis, 16 parts of Agaricus bisporus, and 16 parts of Boletus edulis (3:2:2);

[0046] Polysaccharide extraction: Take 200g of the mixed powder, add 6000mL of deionized water, extract at 80℃ for 4h, twice, combine the extracts to obtain the aqueous extract, and concentrate the extract under reduced pressure to about 150mL.

[0047] Dialysis: The concentrated solution was placed in a 3500Da dialysis bag and dialyzed in a chromatography cabinet at 4°C for 72 h.

[0048] Alcohol precipitation: add 95% ethanol in a volume ratio of 1:5.3, let it stand at 4°C overnight, then centrifuge at 5000r / min for 10min, collect the precipitate, and the precipitate is the compound polysaccharide.

[0049] Preparation of individual polysaccharides:

[0050] The preparation of individual polysaccharides of Tremella fuciformis, Agaricus bisporus and Boletus edulis was the same as the above operation, except that the compound combination was not performed. After washing and physical crushing, the three edible fungi were subjected to subsequent operations such as polysaccharide extraction.

[0051] 2. Modeling and grouping

[0052] 8-week-old male ApoE - / - Thirty-six mice were randomly divided into six groups: a normal diet blank group (NCD), a high-fat diet model group (HFD), a Tremella fuciformis (HFD+TF) group, an Agaricus bisporus (HFD+AB) group, a Boletus edulis (HFD+BE) group, and a compound polysaccharide group (HFD+CCP (3:2:2)). Modeling was performed for 70 days (weeks 1-10) and drug administration was continued for 28 days (weeks 11-14).

[0053] ApoE - / -During the high-fat diet modeling period (weeks 1-10), high-fat and standard feeds were checked daily at 9:00 AM. Insufficient feed was replenished or oxidized feed was replaced promptly. During the treatment period (weeks 11-14), 5 mL / kg of normal saline was administered orally at 9:00 AM daily for the NCD and HFD groups. The HFD+TF, HFD+AB, HFD+BE, and HFD+CCP (3:2:2) groups were given 50 mg / kg of the corresponding polysaccharide by oral gavage. Dosing continued for 28 days. Mice were weighed and weighted daily.

[0054] 3. Compound polysaccharide improves ApoE - / - Body weight changes in atherosclerotic mice

[0055] Obesity and weight gain are important characteristics of atherosclerosis caused by a long-term high-fat diet. Figure 1 As shown. From the beginning of the experiment (week 6) to the end of modeling (week 10), the weight of mice on HFD was significantly higher than that of mice on NCD. After the end of administration (week 14), the weight of atherosclerotic mice on HFD+TF, HFD+AB, HFD+BE and HFD+CCP (3:2:2) decreased significantly. This shows that compound polysaccharides can improve ApoE - / - Body weight changes in atherosclerotic mice.

[0056] 4. Compound polysaccharide alleviates ApoE - / - Atherosclerotic lesions in the aorta of atherosclerotic mice

[0057] Aortic lipid deposition is the main cause of atherosclerosis. After the end of drug administration, the aorta of the mice was dissected and placed in 4% paraformaldehyde tissue fixative. OCT embedding, frozen sectioning, staining and preparation of tissue sections were performed. Histological analysis by Oil Red O staining was performed. The results are as follows Figure 2 As shown in the results, the relative staining area of Oil Red O in HFD mice was significantly higher than that in NCD mice. Compared with HFD, the relative staining area of HFD+TF, HFD+AB, HFD+BE and HFD+CCP (3:2:2) was significantly reduced, and the relative staining area of Oil Red O in HFD+CCP (3:2:2) group mice was less than that in HFD+TF, HFD+AB and HFD+BE. This shows that compound polysaccharide can more effectively reduce lipid accumulation in the aorta and alleviate ApoE - / - Atherosclerotic lesions in the mouse aorta.

[0058] 5. Compound polysaccharide alleviates ApoE - / - Lipid droplet accumulation and fat vacuolization in adipose tissue of atherosclerotic mice

[0059] Long-term high-fat diet can lead to the appearance of a large number of fat vacuoles in the adipose tissue of mice, a significant increase in the number of lipid droplets, and a high degree of fat infiltration, which can lead to lipid metabolism disorders. Figure 3 As shown in the results, compared with HFD, HFD+TF, HFD+AB, HFD+BE, and HFD+CCP (3:2:2) showed fewer fat vacuoles, fewer lipid droplets, and smaller lipid droplets. The compound polysaccharide group also showed better results, indicating that the combination can better reduce lipid accumulation in adipose tissue and reduce lipid infiltration. This further proves that the compound polysaccharide has a better lipid-lowering effect.

[0060] 6. Compound polysaccharide improves ApoE - / - Lipid metabolism in atherosclerotic mice

[0061] After administration, the mice were dissected, and their inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), perirenal white adipose tissue (pWAT), and brown adipose tissue (BAT) were collected. The remaining blood on the surface was blotted with absorbent paper and then weighed immediately. The data were recorded and the fat index was calculated.

[0062] Fat index (%) = fat weight (g) / mouse weight before sacrifice (g) × 100

[0063] A high-fat diet can cause ApoE - / - The WAT index of mice increased and the BAT index decreased, which led to the slowdown of normal lipid metabolism and fat accumulation in mice. Figure 4 As shown in the results, compared with HFD, the WAT index of HFD+TF, HFD+AB, HFD+BE and HFD+CCP (3:2:2) was downregulated and the BAT index was upregulated, and the eWAT index of HFD+CCP (3:2:2) was downregulated and the BAT index was upregulated, indicating that the compound polysaccharide can effectively alleviate lipid metabolism disorders.

[0064] 7. Compound polysaccharide improves ApoE - / - Serum lipid metabolism function in atherosclerotic mice

[0065] After administration, blood was collected from the tail tip of the mice and centrifuged at 2500 rpm for 15 minutes. The supernatant was serum. Dyslipidemia is a major cause of atherosclerosis. Serum levels of triglycerides (TG), TC, and LDL-C can, to a certain extent, reflect the body's lipid metabolism and serve as important indicators for monitoring changes in blood lipids. Studies have shown that hyperlipidemia can lead to elevated levels of TC, TG, and LDL-C.

[0066] Triglyceride levels were measured according to the instructions for the A110-1-1 triglyceride test kit from the Nanjing Jiancheng Bioengineering Institute. The principle is a glycerol phosphate oxidase-peroxidase activator coupled method. Sample preparation: Serum was diluted tenfold with normal saline. The corresponding samples or standards were added to a 96-well plate according to Table 1. Serum triglyceride content (mmol / L) was determined as follows.

[0067]

[0068] Table 1 TG level measurement - 96-well plate sample loading reference table

[0069]

[0070] TC levels were determined according to the instructions for the A111-1 total cholesterol test kit from the Nanjing Jiancheng Bioengineering Institute. The principle is cholesterol oxidase-peroxidase coupled method. Sample preparation: Serum was diluted tenfold with physiological saline; refer to

[0071] The corresponding samples or standards in Table 2 were added to a 96-well plate; the serum TC content (mmol / L) was determined as follows.

[0072]

[0073] Table 2TC level measurement - 96-well plate sample loading reference table

[0074]

[0075] LDL-C levels were measured according to the instructions for the A113-1-1 total cholesterol test kit from the Nanjing Jiancheng Bioengineering Institute. This method utilizes a microplate assay. Sample preparation: Serum was diluted tenfold with normal saline. The corresponding samples or standards were added to a 96-well plate according to Table 3. Serum LDL-C levels (mmol / L) were determined as follows.

[0076]

[0077] Table 3 LDL-C level measurement - 96-well plate sample loading reference table

[0078]

[0079] ApoE in each group - / - The results of the effects on TC, TG and LDL-C levels in mouse serum are as follows Figure 5As shown in the figure. Compared with NCD, the serum LDL-C, TC, and TG levels of HFD mice were significantly increased (p < 0.001). Compared with the HFD results, the serum TC, TG, and LDL-C concentrations of HFD+TF, HFD+AB, HFD+BE, and HFD+CCP (3:2:2) were all reduced, and the HFD+CCP (3:2:2) had a better reduction effect, indicating that the compound polysaccharide is more effective.

[0080] 8. Compound polysaccharide alleviates ApoE - / - Damage to major organs in atherosclerotic mice

[0081] The organ index is an important indicator of the health of the mouse's major organs. After dosing, the heart, liver, spleen, kidney, thymus, and pancreas were dissected and weighed. Any remaining blood on the surface was blotted with absorbent paper, and the weight was immediately weighed. The data was recorded and the organ index was calculated.

[0082] Organ index (%) = organ weight (g) / mouse weight before sacrifice (g) × 100

[0083] like Figure 6 As shown, high-fat diet caused an increase in the heart index (Heart Index), spleen index (Spleen Index), pancreas index (pancreas Index), kidney index (Kidney Index), liver index (LiverIndex) and a decrease in the thymus index (Thymus Index) of mice on HFD. Compared with the results of HFD, the pancreas index of mice on HFD+BE was not decreased, and the thymus index of mice on HFD+AB was not increased. Compared with the results of HFD, the heart index, spleen index, kidney index and liver index of mice on HFD+TF, HFD+AB, HFD+BE and HFD+CCP (3:2:2) were decreased. Compared with the results of HFD, the thymus index of mice on HFD+TF and HFD+CCP (3:2:2) was increased and the pancreas index was decreased. This shows that compound polysaccharides can better improve ApoE - / - Damage to major organs in atherosclerotic mice.

[0084] 9. Effect of compound polysaccharide on ApoE - / - Effects of intestinal flora on atherosclerosis in mice

[0085] The intestinal microbiome is the largest and most complex microbial community in the human body and plays a vital role in the occurrence and development of atherosclerosis. - / - The intestinal flora of mice with atherosclerosis is disturbed and the number of flora decreases. By comparing the relative abundance distribution of different microorganisms and genus levels in each group of mice, we can understand the effect of compound polysaccharides on the intestinal flora of mice.

[0086] Table 4 Primer sequences for V3-V4 variable regions

[0087]

[0088] Table 5 PCR reaction program

[0089]

[0090] After administration, cecal contents of HFD+CCP (3:2:2) mice were dissected and genomic DNA was extracted using the OMEGA Mag-bind soil DNA kit. The DNA purity and concentration were determined. PCR amplification of the selected V3-V4 variable regions was performed using barcoded primers and a high-fidelity DNA polymerase, based on the selected sequencing regions. Genomic DNA from intestinal microbiota served as the PCR template, and 30 cycles of PCR were performed using barcoded primers (see Table 4) based on the sequencing regions. The PCR reaction system consisted of 15 μL of Phusion Master Mix (2x), 3 μL of Primer (2 μM), 10 μL of gDNA (1 ng / μL) (5-10 ng), and 2 μL of H₂O. The PCR cycle was 98°C for 10 sec; 50°C for 30 sec; 72°C for 30 sec, followed by pre-denaturation at 98°C for 1 min (see Table 5). The PCR products were detected by 2% agarose gel electrophoresis, and the target fragments were excised and recovered by gel excision, wherein the Quant-iT PicoGreen dsDNA Assay Kit was used for gel recovery. Referring to the preliminary quantitative results of electrophoresis, the PCR amplification and recovery products were detected and quantified using the Microplate reader (dereplication) BioTek, FLx800 fluorescence quantitative system, wherein the corresponding proportions were mixed according to the sequencing amount requirements of each sample. The library was constructed using Illumina's TruSeq Nano DNA LT Library Prep Kit. The constructed library was quality-checked by Agilent Bioanalyzer2100 and Promega QuantiFluor, and the library was sequenced after passing the quality inspection. After obtaining the original sequencing data, the sequencing data quality was assessed, and ASV / OUT was clustered and annotated. According to the results of ASVs cluster analysis and research needs, the Venn diagram of NCD, HFD, and CCP (3:2:2) was drawn; according to the species annotation results, the top 30 species with the largest abundance at the genus level of NCD, HFD, and HFD+CCP (3:2:2) were selected, and the species relative abundance column cumulative graph was drawn using the Ouyi Cloud platform.

[0091] like Figure 7 As shown in Figure 3, the number of microbiota in the CCP (3:2:2) group mice was increased compared with the HFD group. Figure 8As shown, the populations of g_Dubosiella and f_Atopobiaceae|g_uncultured decreased in mice fed a HFD, while those in the CCP (3:2:2) group increased compared to the HFD group. The populations of f_Lachnospiraceae|g_uncultured decreased in mice fed a HFD, while those in the CCP (3:2:2) group increased compared to the HFD group. This suggests that the compound polysaccharide alleviated intestinal flora disturbances in mice with atherosclerosis.

[0092] 10. Effect of compound polysaccharide on ApoE - / - Effects of atherosclerosis on body metabolism in mice

[0093] After the administration, the serum of mice was collected (the method was the same as that of “7. Compound polysaccharide improves ApoE - / - Serum lipid metabolism function in atherosclerotic mice"). The main steps for determining body metabolites include: sample preparation, QC preparation, sample LC-MS / MS mass spectrometry analysis, and data analysis.

[0094] Metabolite extraction: After HFD+CCP (3:2:2) mouse serum samples were slowly thawed at 4°C, an appropriate amount of sample was added to a pre-chilled methanol / acetonitrile / water solution (2:2:1, v / v), vortexed, sonicated at low temperature for 30 min, allowed to stand at -20°C for 10 min, and centrifuged at 14,000 g for 20 min at 4°C. The supernatant was vacuum dried and, for mass spectrometry analysis, reconstituted with 100 μL of acetonitrile-water solution (acetonitrile:water = 1:1, v / v), vortexed, and centrifuged at 14,000 g for 15 min at 4°C. The supernatant was then sampled for analysis. Analysis was then performed by chromatography-mass spectrometry.

[0095] Chromatographic conditions: Samples were separated using an Agilent 1290 Infinity LC ultra-high performance liquid chromatography (UHPLC) system with a HILIC column; column temperature was 25°C; flow rate was 0.5 mL / min; injection volume was 2 μL; mobile phase composition was A: water + 25 mM ammonium acetate + 25 mM ammonia, B: acetonitrile; gradient elution program was as follows: 95% B from 0 to 0.5 min; linear B from 95% to 65% from 0.5 to 7 min; linear B from 65% to 40% from 7 to 8 min; B maintained at 40% from 8 to 9 min; B linear from 40% to 95% from 9 to 9.1 min; B maintained at 95% from 9.1 to 12 min. Samples were maintained in an autosampler at 4°C throughout the analysis. To minimize the influence of instrument signal fluctuations, samples were analyzed sequentially in random order. QC samples were inserted into the sample queue to monitor and evaluate system stability and the reliability of the experimental data.

[0096] Mass spectrometry conditions: Primary and secondary spectra of the samples were acquired using an AB Triple TOF 6600 mass spectrometer. Samples were separated using an Agilent 1290 Infinity LC ultra-high performance liquid chromatography (UHPLC) system and analyzed by mass spectrometry using a Triple TOF 6600 mass spectrometer (AB SCIEX), employing electrospray ionization (ESI) in positive and negative ion modes, respectively. The ESI source setting parameters are as follows: nebulizer gas auxiliary heating gas 1 (Gas1): 60, auxiliary heating gas 2 (Gas2): 60, curtain gas (CUR): 30 psi, ion source temperature: 600℃, spray voltage (ISVF) ±5500 V (positive and negative modes); primary mass-to-charge ratio detection range: 60-1000 Da, secondary product ion mass-to-charge ratio detection range: 25-1000 Da, primary mass spectrometry scan accumulation time: 0.20 s / spectra, secondary mass spectrometry scan accumulation time 0.05 s / spectra; secondary mass spectra were acquired using data-dependent acquisition mode (IDA), and peak intensity value screening mode was adopted, declustering voltage (DP): ±60 V (positive and negative modes), collision energy: 35±15 eV, IDA settings are as follows: dynamic exclusion of isotope ions range: 4 Da, 10 fragment spectra were collected for each scan.

[0097] Data Analysis: Raw data were converted to .mzXML format using ProteoWizard, and then peak alignment, retention time correction, and peak area extraction were performed using XCMS software. The XCMS-extracted data were first subjected to metabolite structure identification and data preprocessing (null value filtering: removing ion peaks with >50% missing values; null value filling: KNN fill; data filtering: filtering features with RSD >50%), followed by data quality assessment and final data analysis.

[0098] The overlap of significantly differential metabolites screened for each comparison group was displayed in the form of a Venn diagram, which helps to screen core metabolite modules related to biological processes. Correlation analysis of differential metabolites can measure significant differences (VIP>1, p<0.05). KEGG pathway enrichment analysis uses KEGG pathways as units and the metabolic pathways involved in the species or closely related species as the background. The significance level of metabolic pathway enrichment is analyzed and calculated using Fisher's Exact Test.

[0099] like Figure 9 As shown in Figure 3, there were 134 differential metabolites between the NCD group and the HFD group, 20 differential metabolites between the HFD group and HFD+CCP (3:2:2), and 13 common differential metabolites between the two groups. Figure 10 As shown in the results, after a high-fat diet, the metabolites of Aspartic acid, L-Asparagine, L-Aspartate, L-Malic acid, L-Phenylalanine, L-Valine, Phenylalanine, D-glutamine, Glutamic acid, and Histidine increased in the mice in the HFD group. However, CCP (3:2:2) administration alleviated the increase of Aspartic acid, L-Asparagine, L-Aspartate, L-Malic acid, L-Phenylalanine, L-Valine, Phenylalanine, D-Glutamine, Glutamic acid, and Histidine. Figure 11 As can be seen in the results, the central carton metabolism in cancer pathway is enriched, which can lead to the activation of the HIF-1α-NF-κB inflammatory pathway and aggravate atherosclerosis. The results show that after the compound polysaccharide is administered, the atherosclerotic inflammation of mice is alleviated.

[0100] In summary, the present invention uses three kinds of edible fungi and, through careful processing and blending, brings out the best therapeutic effect of compound polysaccharide to treat atherosclerosis in a targeted manner. Compound polysaccharide can reduce the levels of TC, TG and LDL-C in serum; alleviate ApoE - / - The study also showed that the main organ damage in atherosclerotic mice was reduced; lipid droplet accumulation and vacuolization in adipose tissue were reduced, and lipid metabolism was improved; the proportion of white fat was reduced and the proportion of brown fat was increased; ApoE was reduced. - / -It also alleviated intestinal flora disturbances and reduced the levels of atherosclerosis-related serum metabolites in mice with atherosclerosis, with effects superior to those of a single polysaccharide.

[0101] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A compound polysaccharide, characterized in that: The compound polysaccharide is prepared by using Tremella fuciformis, Agaricus bisporus and Boletus as raw materials through water extraction and alcohol precipitation, wherein the weight ratio of Tremella fuciformis, Agaricus bisporus and Boletus is 3:2:2; The preparation method of the compound polysaccharide comprises the following steps: The Tremella fuciformis, Agaricus bisporus and Boletus edulis are respectively washed, dried and crushed to obtain Tremella fuciformis powder, Agaricus bisporus powder and Boletus edulis powder; Mixing Tremella powder, Agaricus bisporus powder and Boletus edulis powder according to a specified weight ratio; Take the mixed powder, add deionized water, extract twice, combine the extracts, and concentrate under reduced pressure to obtain a concentrate; The concentrated solution is dialyzed, ethanol is added after dialysis, and the precipitate is collected by centrifugation, where the precipitate is the compound polysaccharide; The dialysis is performed by placing the concentrated solution in a 3500 Da dialysis bag and dialyzing at 4°C for 72 hours; The volume ratio of the mixed powder to the deionized water is 1:30; The extraction is carried out at 80°C; The volume concentration of the ethanol is 95%; the volume ratio of the concentrated liquid to the ethanol is 1:5.

3.

2. Use of the compound polysaccharide according to claim 1 in the preparation of a drug for preventing and / or treating atherosclerosis.

3. The use according to claim 2, characterized in that The compound polysaccharide plays a role in preventing and / or treating atherosclerosis by reducing fat accumulation, lowering the levels of triglycerides, total cholesterol and low-density lipoprotein cholesterol in serum, improving intestinal flora disorders, improving lipid metabolism or reducing the levels of serum metabolites related to atherosclerosis.

4. A drug for preventing and treating atherosclerosis, characterized in that: Contains the compound polysaccharide according to claim 1.

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