Application of atractylodes macrocephala polysaccharide in preparation of medicine for treating diabetes mellitus complicated with steatohepatitis

Abstract

The invention provides application of rhizoma atractylodis macrocephalae polysaccharide in preparation of a medicine for treating diabetes mellitus complicated with steatohepatitis, and belongs to the field of traditional Chinese medicines. The atractylodes macrocephala polysaccharide (APMs) composed of glucose, arabinose, galactose, galacturonic acid and glucuronic acid is prepared through a water extraction and alcohol precipitation method, can be used for treating diabetes mellitus complicated with steatohepatitis, and has a remarkable treatment effect on diabetes mellitus complicated with steatohepatitis; insulin resistance, lipid metabolism disorder, liver inflammation, liver injury and liver fibrosis caused by diabetes mellitus complicated with steatohepatitis can be improved.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicines and relates to a kind of Atractylodes macrocephala polysaccharide, and specifically relates to the application of the Atractylodes macrocephala polysaccharide in the preparation of a medicine for treating diabetes combined with fatty hepatitis. Background Art

[0002] Diabetes mellitus (DM) is mainly type 2 diabetes mellitus (T2DM), and metabolic associated fatty liver disease (MAFLD) is a common complication, including hepatic steatosis, nonalcoholic steatohepatitis (NASH), fibrosis and cirrhosis, which may lead to end-stage liver failure or hepatocellular carcinoma. A meta-analysis of 80 studies in 20 countries found that the global prevalence of MAFLD in 49,419 T2DM patients was 55.5%, which is twice that of the general population without T2DM. The relationship between MAFLD and T2DM may be bidirectional. T2DM increases the risk of liver-related complications such as cirrhosis, liver failure and liver cancer, and is the strongest predictor of NASH and liver fibrosis. In addition, the accumulation of lipids in the liver of MAFLD patients aggravates insulin resistance, thereby increasing blood sugar levels.

[0003] Many NASH candidate drugs have been rejected for marketing due to poor efficacy or many adverse reactions. In March 2024, the U.S. Food and Drug Administration (FDA) approved Rezdiffra, the world's first drug for the treatment of NASH. Its extensive effectiveness and safety still need to be studied in the future. Due to its multi-target and multi-pathway mode of action, Chinese herbal compound has shown good efficacy and safety for the systemic metabolic disease NASH and has broad application prospects. Chinese herbal polysaccharides can regulate glucose and lipid metabolism disorders, lower blood sugar and blood lipids, and play a therapeutic role in diabetes and fatty hepatitis. Polysaccharides of atractylodes macrocephala koidz (AMPs) are the main active ingredients of Atractylodes macrocephala. Studies have found that AMPs effectively prevent and treat MAFLD, reduce fatty degeneration and vacuolar degeneration of hepatocytes, and may reduce blood sugar by increasing the sensitivity of peripheral target organs to insulin, reducing plasma insulin levels, and improving insulin resistance. However, it is still unclear whether AMPs can treat type 2 diabetes combined with fatty liver hepatitis. The efficacy study of Atractylodes macrocephala polysaccharide in treating type 2 diabetes combined with fatty liver hepatitis will promote the development of new drugs for type 2 diabetes combined with fatty liver hepatitis.

[0004] T2DM and MAFLD share a common pathophysiological basis, with insulin resistance being their main feature. When the body first develops insulin resistance, it can compensate by increasing insulin secretion to maintain blood sugar at normal levels. However, long-term insulin resistance can cause irreversible damage to the physiological effects of insulin, and eventually the body will be unable to compensate for the increase in blood sugar through self-regulation. The high blood sugar in T2DM patients in the fasting state is caused by increased hepatic gluconeogenesis and endogenous glucose production caused by hepatic insulin resistance, among which the decrease in hepatic acetyl-CoA derived from adipose tissue decomposition serves as the main regulator of insulin inhibition of HGP. Continuous high blood sugar in the body can affect lipid metabolism and aggravate intracellular lipid accumulation. In MAFLD, the typical characteristics of dyslipidemia are increased levels of very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) and decreased levels of high-density lipoprotein cholesterol (HDL-C). The increase in VLDL may also further increase the risk of T2DM combined with MAFLD; and the increase in liver VLDL1-triglyceride and VLDL1-palmitic acid content in patients with relapse of T2DM after remission is greater than that in individuals without relapse.

[0005] Insulin is cleared by the liver and other tissues, and undergoes first metabolism in the liver (or liver extracts insulin), accounting for 50-80% of insulin clearance. Therefore, the liver can affect circulating insulin levels. Reduced insulin clearance in MAFLD patients is associated with insulin resistance, and changes in free fatty acids (FFA) may be an important regulatory signal. At the same time, a small increase in liver fat in MAFLD patients is associated with liver and skeletal muscle insulin resistance. Lipid mediators and genotypes also play an important role in insulin resistance. Increased liver lipid utilization and insufficient adaptation of mitochondrial function may induce the liver to produce DAG and ceramide, thereby affecting the progression of liver insulin resistance and MAFLD, and specific lipid species and their localization in the plasma membrane play a key role in insulin resistance. Compared with patients with PNPLA3I148M or PNPLA3M148M genotypes, MAFLD patients with PNPLA3I148I genotype have higher levels of insulin resistance.

[0006] There is no T2DM combined with NASH disease name in traditional Chinese medicine. T2DM belongs to the category of "diabetes" in traditional Chinese medicine, and NASH is mostly classified as "flank pain", "phlegm syndrome", "fat gas disease", "accumulation" and other categories. T2DM combined with NASH is mainly caused by factors such as insufficient endowment, irregular diet, emotional disorder, and improper work and rest, which lead to liver depression and spleen deficiency, phlegm and blood stasis, or internal dampness and heat, stagnating in the liver. Among them, spleen deficiency is the root of the pathogenesis, liver depression is the key link in the onset of the disease, and phlegm and blood stasis are the main pathological factors throughout the disease. Some doctors have carried out dialectical classification of T2DM combined with MAFLD based on clinical data. The main classifications include internal obstruction of phlegm and dampness, internal accumulation of dampness and heat, liver depression and spleen deficiency, phlegm and blood stasis, liver and kidney yin deficiency, etc. Based on the classification, the prescriptions and medicines for symptomatic treatment are selected, which often achieve good results. Wu Xinyan added Qingrequshi Decoction to conventional treatment to treat patients with type 2 diabetes combined with fatty liver hepatitis due to damp-heat stasis, which can effectively improve blood sugar control, reduce blood lipid levels, improve liver function and relieve clinical symptoms, with definite efficacy and high safety. Cui Yuhong found that Erchen Decoction plus other ingredients in the treatment of type 2 diabetes combined with fatty liver phlegm-dampness internal obstruction syndrome has definite clinical efficacy and few adverse reactions.

[0007] Polysaccharides are a class of carbohydrate substances with complex molecular structures formed by the polymerization of more than 10 monosaccharide molecules through glycosidic bonds. They include animal polysaccharides, plant polysaccharides and microbial polysaccharides (including fungal polysaccharides and bacteria. In recent years, great progress has been made in the research on the hypoglycemic and lipid-lowering effects of polysaccharides, which has developed from basic pharmacological activity research to mechanism research. Astragalus polysaccharide is an important bioactive component of the traditional Chinese medicine Astragalus. Studies have found that astragalus polysaccharide increases the proliferation of pre-adipocytes in a dose-dependent manner, upregulates glucose transporter 4, increases glucose uptake, and improves insulin sensitivity, which may be related to the AMPK-related signaling pathway. Activation. Gynostemma pentaphyllum polysaccharide can inhibit the activity of α-glucosidase, affect glucose transporter 2, and thus lower blood sugar. Feeding hyperlipidemic rats with kumquat polysaccharide significantly increased the content of serum lipase, and increased the activity of total antioxidant superoxide dismutase, glutathione peroxidase and glutathione S-transferase, and reduced the content of malondialdehyde. This shows that kumquat polysaccharide may regulate lipid metabolism by enhancing the activity of antioxidant enzymes. Polysaccharides can also regulate lipid metabolism through the gut-liver axis. Studies have found that an active polysaccharide in Chlorella can improve lipid metabolism, accelerate the metabolism of cecal total bile acid (Bile acids, Bas) and short-chain fatty acids, and change the composition of intestinal flora, indicating that it can improve the composition of intestinal flora to improve lipid metabolism in hyperlipidemic rats. Astragalus polysaccharide may regulate lipid metabolism disorders by regulating the expression of intestinal sweet taste receptors and promoting intestinal hormone secretion.

[0008] The invention patent with the publication number CN1398630A discloses a white atractylodes sugar complex with hypoglycemic activity, a production method and a use thereof, wherein the white atractylodes water extract is obtained by soaking, an organic solvent is precipitated, the precipitate is dissolved in water, the supernatant is subjected to dialysis or membrane separation and concentrated and dried to obtain a crude product, and the crude product is separated by column chromatography to obtain a refined white atractylodes sugar complex. The white atractylodes sugar element analysis: C = 35%-45%, H = 5.5%-7.0%, N = 1.0%-3.0%; polysaccharide content: 40%-60%; uronic acid content: 15%-45%; protein content: 10%-25%; monosaccharide composition: neutral sugars include glucose, galactose, mannose, arabinose and rhamnose, and also contain glucuronic acid and galacturonic acid. The polysaccharide has no toxic side effects and has obvious hypoglycemic function.

[0009] Since the extraction and purification methods have a significant impact on the modification and biological activity of polysaccharides, strategies need to be improved in order to seek effective, safe and low-toxic new therapeutic drugs for the treatment of diabetic steatohepatitis. Summary of the invention

[0010] The present invention aims to solve the problems existing in the prior art and provides an application of Atractylodes macrocephala polysaccharide in the preparation of a drug for treating diabetes combined with fatty hepatitis. The Atractylodes macrocephala polysaccharide prepared by the present invention can improve insulin resistance, lipid metabolism disorder, liver inflammation and liver fibrosis caused by type 2 diabetes combined with fatty hepatitis.

[0011] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0012] In a first aspect, the present invention provides a use of Atractylodes macrocephala polysaccharide in the preparation of a medicament for treating diabetes combined with fatty hepatitis.

[0013] The diabetes combined with fatty liver hepatitis includes one or more of insulin resistance, lipid metabolism disorder, liver inflammation, liver damage and liver fibrosis.

[0014] The Atractylodes macrocephala polysaccharide is prepared by a water extraction and alcohol precipitation method, which is as follows: at 85-95° C., Atractylodes macrocephala and ultrapure water are extracted in a ratio of 1 g:15-25 mL, the extraction is performed 1-3 times, each extraction is 1-3 hours, the extraction is preferably performed 2 times, the extracts are combined and concentrated to obtain a concentrated solution; 2-4 times the volume of 90-100% ethanol solution is added to the concentrated solution, and alcohol precipitation is performed to obtain a precipitate, the precipitate is preferably washed with ethanol, and dried to obtain Atractylodes macrocephala polysaccharide.

[0015] In one embodiment, the water extraction and alcohol precipitation method is specifically:

[0016] Step 1): extract Atractylodes macrocephala and ultrapure water at a ratio of 1 g: 20 mL at 90°C for 2 h, centrifuge at 4000 rpm for 20 min, and collect the supernatant and residue;

[0017] Step 2): extract and centrifuge the residue for the second time according to the method of step 1), combine the supernatants of the two extractions and concentrate at 60° C. to obtain a concentrate;

[0018] Step 3): Add 3 times the volume of 95% ethanol solution to the concentrate, precipitate overnight at 4°C, collect the precipitate by centrifugation, wash the precipitate with ethanol and freeze-dry it to obtain Atractylodes macrocephala polysaccharide.

[0019] The atractylodes macrocephala polysaccharide is composed of glucose, arabinose, galactose, galacturonic acid and glucuronic acid.

[0020] Preferably, the Atractylodes macrocephala polysaccharide is composed of 82-85% glucose, 6-8% arabinose, 5-6% galactose, 2-4% galacturonic acid and 0.1-0.8% glucuronic acid.

[0021] In one embodiment, the Atractylodes macrocephala polysaccharide is composed of 83.92% glucose, 7.19% arabinose, 5.47% galactose, 3% galacturonic acid and 0.41% glucuronic acid.

[0022] Compared with the prior art, the present invention has the following beneficial effects: the Atractylodes macrocephala polysaccharide (APMs) prepared by the present invention has a significant therapeutic effect on diabetes combined with fatty liver hepatitis, and can improve liver damage, inflammation, lipid metabolism and fibrosis caused by diabetes combined with fatty liver hepatitis. The present invention found that AMPs can regulate sugar metabolism through blood sugar and OGTT detection, and found that AMPs have the effect of improving liver function and lowering blood lipids through serum biochemical detection; AMPs have the effect of improving liver lipid deposition through H&E and Oil Red O staining; AMPs can reduce liver inflammation through CD68 immunohistochemistry; AMPs can reduce liver collagen deposition through Sirius red staining; AMPs can improve liver lipid metabolism, inflammation and fibrosis at the gene and protein levels by detecting mouse liver-related gene and protein expression through qPCR and WB. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the ion chromatogram of the monosaccharide composition of AMPs;

[0024] Figure 2 is the Fourier-infrared spectrum of AMPs;

[0025] Figure 3 is the scanning electron microscope image of AMPs;

[0026] Figure 4 This is a flowchart of the mouse experiment;

[0027] Figure 5 Figure 2 is a graph showing lipid metabolism in mice, where A is a graph showing the levels of TG in mouse liver, TC in serum, and LDL-C, and B is a graph showing the mRNA levels of Ppar-γ and Cd36, genes related to lipid metabolism in mouse liver ( ### P<0.001VS.CON group, * P<0.05VS.DM group, ** P<0.01VS.DM group, *** P < 0.001 vs. DM group);

[0028] Figure 6 The mRNA levels of inflammation-related genes Ccl2, Il6, and Tnf-α in mice ( ### P<0.001VS.CON group, * P<0.05VS.DM group, ** P<0.01VS.DM group, *** P < 0.001 vs. DM group);

[0029] Figure 7 Figure 2 is the mRNA level of liver fibrosis-related genes Tgf-b1, Col1a1, and Timp1 in mice (###P<0.001VS.CON group, *P<0.05VS.DM group, **P<0.01VS.DM group, ***P<0.001VS.DM group);

[0030] Figure 8 Figure 1 is a graph of mouse body weight, liver index, blood glucose, and glucose tolerance. Figure A is a graph of mouse body weight and liver index, and Figure B is a graph of mouse blood glucose and the area under the OGTT curve (AUC of OGTT), and mouse fasting blood glucose (Blood glucose). ### P<0.001VS.CON group, * P<0.05VS.DM group, ** P<0.01VS.DM group, *** P < 0.001 vs. DM group);

[0031] Fig. 9 Figure 1 is a diagram of mouse lipid metabolism, where A is the appearance of mouse liver, H&E staining of liver tissue, and Oil Red O staining of liver tissue; and B is a diagram of the levels of Pparγ and ATGL, proteins related to mouse lipid metabolism;

[0032] Fig.10Figure 1 is a graph showing liver inflammation in mice, where A is a graph showing CD68 immunohistochemical staining of mouse liver tissue and statistical graph of the positive area, B is a graph showing the activity of ALT and AST in mouse serum, and C is a graph showing the levels of inflammation-related proteins NLRP3 and MCP1 in mice;

[0033] Fig.11 The graphs are for mouse liver fibrosis, where A is a statistical graph of mouse Sirius red staining and positive area, and B is a graph of the levels of Collagen-1 and α-SMA, proteins related to mouse liver fibrosis. DETAILED DESCRIPTION

[0034] It is worth noting that, unless otherwise specified, the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0035] The following sources of raw materials are provided for illustrative purposes:

[0036] The main reagents are shown in Table 1:

[0037] Table 1 Main reagents

[0038]

[0039]

[0040] The main instruments and equipment are shown in Table 2.

[0041] Table 2 Main instruments and equipment

[0042]

[0043]

[0044] Data analysis was performed using statistical methods:

[0045] Measurement data were expressed as mean ± standard deviation (Mean ± SD), count data were expressed as frequency and percentage (f, %), and efficacy evaluation indicators were expressed as frequency or percentage (f, %) after conversion. For comparison of means between two groups, homogeneity test was performed first. If the variances were equal, t test was used. If the variances were unequal, non-parametric t' test was used. Comparison between multiple groups was performed by one-way analysis of variance (ANOVA). Paired t / t' test was used for comparison of measurement data of each group before and after treatment in vivo studies. χ test was used for comparison of count data of four-cell tables between two groups. 2 The rank sum test was used for comparison of rank data. P < 0.05 was considered statistically significant. SPSS 26.0 and Graphpad prism 9 were used for statistical analysis.

[0046] Example 1

[0047] Preparation and analysis of Atractylodes macrocephala polysaccharide

[0048] 1. Preparation of Atractylodes macrocephala polysaccharides (AMPs):

[0049] After the Atractylodes macrocephala was powdered, 10.0 g of Atractylodes macrocephala powder was accurately weighed and the first extraction was performed: at 90°C, the Atractylodes macrocephala powder and ultrapure water were stirred and extracted for 2 hours at a solid-liquid ratio of 1:20 (g / mL) to obtain an extract, and the extract was centrifuged at 4000 rpm for 20 minutes in a high-speed refrigerated centrifuge to collect the supernatant and residue of the first extraction. The residue was extracted for the second time according to the method of the first extraction to obtain the supernatant of the second extraction. The supernatants of the two extractions were combined and concentrated to 1 / 3 of the original volume by vacuum rotary evaporation at 60°C to obtain a concentrated solution. The iodine-potassium iodide reagent was used for detection, and the results showed that there was no starch in the concentrated solution. Three times the amount of 95% ethanol solution was added to the concentrated solution, stirred and mixed, and placed at 4°C for alcohol precipitation overnight. The mixed solution after alcohol precipitation was centrifuged at 4000×r for 10 minutes, the supernatant was removed, and the precipitate was collected. The precipitate was then washed twice with ethanol of equal volume fraction to make the polysaccharide components more uniform. Finally, the precipitate was evenly dissolved in ultrapure water and vacuum freeze-dried for 48 hours to obtain Atractylodes macrocephala polysaccharides (AMPs).

[0050] Determine the total polysaccharide content of AMPs. The phenol-sulfuric acid method was used for determination, specifically: accurately weigh 2-5 mg of sample, add 1 mL of sterile water, ultrasonically extract for 10 min, and centrifuge at 12000 rpm for 10 min. Pipette 0.4 mL of supernatant into a new test tube and dilute to a certain volume with dd water. Then, add the diluted sample solution to the test tube and mix it with phenol-concentrated sulfuric acid. After the reaction, measure the absorbance at 490 nm. Draw a standard curve with glucose to calculate the total polysaccharide content. The extraction rate of Atractylodes macrocephala polysaccharide and its total sugar content are shown in Table 3.

[0051] Table 3 Extraction rate and total polysaccharide content of Atractylodes macrocephala polysaccharides (AMPs)

[0052]

[0053] The results in Table 3 show that the extraction rate of AMPs was 20.07% and the total sugar content was 87.06%.

[0054] 2. Preparation of Atractylodes macrocephala polysaccharide (AMPs-CM):

[0055] After the Atractylodes macrocephala was powdered, 10.0 g of Atractylodes macrocephala powder was accurately weighed and the first extraction was performed: at 80°C, the Atractylodes macrocephala powder and ultrapure water were stirred and extracted for 5 hours at a solid-liquid ratio of 1:15 (g / mL) to obtain an extract, and the extract was centrifuged at 4000 rpm for 20 minutes in a high-speed refrigerated centrifuge to collect the supernatant and residue of the first extraction. The residue was extracted for the second and third times according to the method of the first extraction, and the supernatants of the three extractions were combined and concentrated to 1 / 3 of the original volume by vacuum rotary evaporation at 60°C to obtain a concentrate. The iodine-potassium iodide reagent was used for detection, and the results showed that there was no starch in the concentrate. Three times the amount of 85% ethanol solution was added to the concentrate, stirred and mixed, and placed at 4°C for alcohol precipitation overnight. The mixed solution after alcohol precipitation was centrifuged at 4000×r for 10 minutes, the supernatant was removed, and the precipitate was collected. The precipitate was then washed twice with ethanol of equal volume fraction to make the polysaccharide components more uniform. Finally, the precipitate was evenly dissolved in ultrapure water and vacuum freeze-dried for 48 hours to obtain Atractylodes macrocephala polysaccharide (AMPs-CM).

[0056] 3. Monosaccharide composition analysis

[0057] The monosaccharide composition of AMPs and AMPs-CM was determined by electrochemical detection using a Thermo ICS5000 ion chromatography system. 6.0 mg of AMPs and AMPs-CM were hydrolyzed with trifluoroacetic acid (2.0 mol / L) at 121 ° C for 2 h, connected to nitrogen, and blown dry. Then add 99.99% methanol to wash, blow dry, and repeat the washing 3 times. Add sterile water to dissolve and transfer to a chromatographic bottle as the sample to be tested. Each sample was repeated 3 times, and the 3 repeats of the AMPs sample were named AMPs-1, AMPs-2, and AMPs-3. The mixed standard (MD) is prepared in a series of concentration gradients by proportions of fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), fructose (Fru), ribose (Rib), galacturonic acid (Gal-UA), glucuronic acid (Glc-UA), mannuronic acid (Man-UA) and guluronic acid (Gul-UA). TM CarboPac TM PA20 (150*3.0mm, 10μm) liquid chromatography column.

[0058] The monosaccharide compositions of AMPs and AMPs-CM are shown in Table 4 .

[0059] Table 4 Monosaccharide composition percentage of AMPs and AMPs-CM

[0060]

[0061] The results in Table 4 show that both AMPs and AMPs-CM are composed of monosaccharides glucose, arabinose, galactose, galacturonic acid and glucuronic acid. The ion chromatogram of the monosaccharide composition of AMPs is shown in Figure 1 As shown in the figure, AMPs are mainly composed of glucose (83.92%). In addition, they also contain a small amount of arabinose (7.19%), galactose (5.47%), galacturonic acid (3.00%) and glucuronic acid (0.41%). The structure of AMPs is relatively pure, and the main type of sugar in AMPs is dextran.

[0062] 4. Infrared spectroscopy analysis

[0063] The infrared spectra of AMPs were analyzed using a Fourier transform infrared spectrometer to identify the functional group characteristics contained. First, a small amount of AMPs was weighed and mixed with 200 mg of potassium bromide, then pressed into a sheet with a thickness of 1 mm and then tested on the machine. A Fourier transform infrared spectrometer (Nicolet iZ-10) was used in the frequency range (4000-400 cm -1 ) to perform scanning analysis and record the infrared spectrum. Figure 2 As shown, Figure 2 This is the Fourier-infrared spectrum of AMPs.

[0064] Depend on Figure 2 As shown, at 3200cm -1 To 3600cm -1 The vibration region shown between 3000-2800cm -1 Within the range (2930cm -1 ) is the CH absorption peak, which includes the stretching vibrations of CH, CH2 and CH3. -1 The vibration range that appeared was caused by the vibration of the ester sugar group (CO-C) and the pyranose ring (COH), which proved that AMPs contain pyranose. Specifically, 905-876cm -1 The vibration range that appears in the range is caused by the β-configuration of glucan. 855-833cm -1 The vibration range that appears in the range is caused by the α-configuration of glucan.

[0065] 5. Scanning electron microscope (SEM) analysis

[0066] A high-resolution field emission scanning electron microscope (Zeiss Merlin Compact) was used to perform electron microscopic scanning analysis of Atractylodes macrocephala polysaccharides. The polysaccharide sample was passed through a 100-mesh sieve, and a small amount was taken on a conductive carbon tape. After gold spraying, it was scanned and photographed using an electron microscope with a magnification of 100-4000 times. Representative polysaccharide structures were observed at different magnifications (5k, 10k, and 20k). The results are shown in Figure 2. Figure 3 As shown, Figure 3 This is a scanning electron microscope image of AMPs.

[0067] Figure 3 The results showed that the surface of AMPs was loose and rough, and distributed in irregular multi-layer flakes or multi-layer rose petals.

[0068] In summary, the present invention uses hot water extraction and ethanol precipitation to extract AMPs, with an extraction rate of 20.07% and a polysaccharide retention rate of 87.06%. The obtained AMPs are composed of glucose, arabinose, galactose, galacturonic acid, and galacturonic acid, and contain functional groups such as ester sugar groups and pyranose rings. SEM results show that the surface of AMPs is loose and rough, and is distributed in irregular multi-layer flakes or multi-layer rose petals.

[0069] Example 2

[0070] Study on the efficacy of Atractylodes macrocephala polysaccharide in treating diabetic mice with fatty hepatitis

[0071] 1. Experimental methods

[0072] 1.1 Experimental drugs and animals

[0073] Experimental animal treatment Figure 4 As shown, Figure 4 The flowchart of the mouse experiment was as follows. Male C57BL / 6 mice of 8 weeks old and weighing 20-25g were selected. They were fed with standard laboratory animal food and had free access to water. They were placed in a restricted access room with controlled temperature and light / dark (12h / 12h) cycle. A mouse model of type 2 diabetes combined with fatty hepatitis was established by high-fat diet plus intraperitoneal injection of streptozotocin (STZ): 60 mice were adaptively fed for 1 week; in the second week, the control group (CON, 10 mice) was given a normal diet (Control diet), and the modeling group (50 mice) was given a high-fat diet (High-fat diet); in the fourth week, the modeling group was intraperitoneally injected with STZ (ipSTZ (biw)); at the end of the fifth week, the blood glucose of the modeling group mice was >11.1mmol / L, indicating that the modeling was successful.

[0074] After successful modeling, the experimental mice in the modeling group were divided into model group (DM), low-dose Atractylodes macrocephala polysaccharide group (AMPs-L), high-dose group (AMPs-H), positive control metformin group (MET) and Atractylodes macrocephala polysaccharide group (AMPs-CS), 10 mice in each group, and the drugs were administered for 8 weeks. The AMPs-L and AMPs-H groups were given low-dose (100 mg / kg) and high-dose (300 mg / kg) Atractylodes macrocephala polysaccharide suspension (normal saline solution) by gavage, the AMPs-CS group was given AMPs-CS (300 mg / kg) suspension (normal saline solution) by gavage, the MET group was given metformin (200 mg / kg) suspension (normal saline solution) by gavage, and the CON group was given normal saline by gavage once a day.

[0075] 1.2 Sample collection and preparation

[0076] After the drug intervention cycle, the mice were anesthetized and killed, and the eye canthal venous blood was collected. After standing, the blood was centrifuged and the supernatant was collected. At the same time, fresh liver tissue was collected. The tissue for pathological sections was placed in an embedding box, and the tissue for mRNA detection was added with RNA protective agent and stored in a -20℃ refrigerator. The liver tissue for Western blot was quickly frozen in liquid nitrogen and then moved to a -80℃ refrigerator for storage.

[0077] 1.3 Detection

[0078] (1) Serum biochemical test: Biochemical kits were used to detect serum TC and LDL-C levels and liver tissue TG (triglyceride) levels. All operations were performed according to the instructions.

[0079] (2) Real-time fluorescence quantitative PCR detection:

[0080] ① Extraction of total RNA from tissues using Trizol method:

[0081] Cut the tissue into pieces, weigh 25 mg and put it into an EP tube. Add 0.5 mL Trizol and two steel balls to each EP tube. After standing for 5 minutes, homogenize with a tissue homogenizer at 60 Hz for 2 minutes, twice; add 200 μl of chloroform, shake vigorously for 15 seconds, and stand for 10 minutes. Centrifuge at 4°C, 12000 rpm for 15 minutes. At this time, the sample is divided into three layers: the bottom layer is a red phenol-chloroform phase, the middle layer is protein, and the upper layer is an aqueous phase. RNA is dissolved in the upper aqueous phase. Transfer about 0.2 ml of the upper aqueous phase into a new EP tube. Add 200 μL of isopropanol to precipitate RNA, invert 30 times to mix, and stand at room temperature for 10 minutes. Centrifuge at 4°C, 12000 rpm for 10 minutes, discard the supernatant, add 1 ml of DEPC-treated 75% ethanol, and vortex for a moment to wash the precipitate once. Centrifuge at 4°C, 7500 rpm for 5 min, discard the supernatant, and dry the RNA in a fume hood for 5-10 min until the RNA is semi-dry and pale white. Add 20 μL of DEPC water and place in a 65°C oven for 10 min to dissolve the RNA.

[0082] ②Reverse transcription

[0083] 1) Remove DNA:

[0084] The system is shown in Table 5.

[0085] Table 5 RNA removal system

[0086]

[0087] SYBR Green method, 1 μg RNA in 10 μL system, 42°C room temperature for 5 minutes.

[0088] 2) Reverse transcription reaction:

[0089] The system is shown in Table 6.

[0090] Table 6 Reverse transcription reaction system

[0091]

[0092]

[0093] A total of 20 μL

[0094] Reaction conditions: as shown in Table 7.

[0095] Table 7 Reverse transcription reaction conditions

[0096]

[0097] ③Fluorescence quantitative PCR:

[0098] The fluorescence quantitative PCR system is shown in Table 8.

[0099] Table 8 Real-time fluorescence quantitative PCR system

[0100]

[0101] (3) WB test:

[0102] ① Extraction of liver tissue protein

[0103] Weigh about 20 mg of liver tissue and add RIPA lysis buffer to the protease inhibitor at a ratio of 100:1 for homogenization. After homogenization, lyse on ice for 15 minutes, transfer to a centrifuge, and centrifuge at 4°C, 12,000 rpm for 15 minutes. Carefully aspirate the supernatant.

[0104] ② BCA quantitative method

[0105] According to the instructions of the BCA protein quantification kit, the standard was diluted in a gradient manner, the reagent was added to detect the protein absorbance, and the standard curve was drawn.

[0106] Take the protein supernatant prepared in (1), dilute it 5 times with PBS, detect the protein concentration with BCA protein quantification kit, substitute it into the standard curve to calculate the actual protein concentration, multiply it by the dilution factor to get the protein stock concentration, quantify each sample to the same concentration at 30 μg / 20 μL, add protein loading buffer at a ratio of 4:1, boil it in a metal bath at 100°C for 10 min to denature, then load the sample or put it in a -20°C refrigerator for later use.

[0107] ③ Electrophoresis, membrane transfer, antibody incubation

[0108] According to the molecular weight of the protein, 8%, 10%, 12%, and 15% electrophoresis gels were prepared, and 8 μL of denatured protein sample was added to each well. The specific operation steps are shown in Table 9.

[0109] Table 9 Specific steps of Western Blot experiment

[0110]

[0111] Mix developer A and developer B in a ratio of 1:1, soak the PVDF membrane in the developer for 1.5 min, and then put it into the developer for development.

[0112] ④Results analysis

[0113] Image J software was used for statistical analysis of the grayscale values ​​of the bands.

[0114] 2. Experimental results

[0115] 2.1 Effects of Atractylodes macrocephala polysaccharide on systemic and hepatic lipid metabolism in diabetic mice with steatohepatitis

[0116] The results are as follows Figure 5 As shown, Figure 5 The above diagrams are for the lipid metabolism of mice, where A is the graph of mouse liver TG, serum TC and LDL-C levels, and B is the graph of the mRNA levels of Ppar-γ and Cd36 genes related to lipid metabolism in mouse liver.

[0117] like Figure 5 Figure A in the middle shows that compared with the CON group, the liver TG (triglyceride) of mice in the DM group has an upward trend, but due to the large dispersion, there is no statistical significance. The serum TC (total cholesterol) and LDL-C (low-density lipoprotein) of mice increased significantly. Compared with the DM group, the liver TG of mice in the AMPs-L group decreased significantly, and the liver TG of the AMPs-H group, AMPs-CM group and MET group showed a downward trend, but the difference was not significant; the serum TG and LDL-C of mice in the MET group and AMPs-H group decreased significantly, while the serum TG and LDL-C of mice in the AMPs-L group and AMPs-CM group were not much different from those in the DM group.

[0118] Figure 5 Figure B in the middle shows that compared with the CON group, the mRNA expression of lipid synthesis and transport-related genes Ppar-γ and Cd36 in the liver of the DM group mice increased significantly. Compared with the DM group, the mRNA expression of Ppar-γ and Cd36 in the liver of the AMPs-L and AMPs-H groups decreased significantly, while the mRNA expression of Ppar-γ and Cd36 in the liver of the MET group mice was not much different from that of the DM group; the mRNA expression of Ppar-γ in the liver of the AMPs-CM group mice was significantly reduced, and the mRNA expression of Cd36 was not much different.

[0119] Figure 5 The results showed that AMPs could significantly alleviate the increase of liver TG, serum TC and LDL-C, and the mRNA expression of Ppar-γ and Cd36, genes related to liver lipid synthesis and transport, in mice with diabetes and steatohepatitis. The therapeutic effect of AMPs-CM on mice with diabetes and steatohepatitis was lower than that of AMPs.

[0120] 2.2 Effect of Atractylodes macrocephala polysaccharide on liver inflammation in diabetic mice with fatty liver disease

[0121] The results are as follows Figure 6 As shown, Figure 6Figure 2 is a graph of the mRNA levels of inflammation-related genes Ccl2, Il6, and Tnf-α in mice. Compared with the CON group, the mRNA levels of inflammation-related genes Ccl2, Il6, and Tnf-α in the liver of mice in the DM group were significantly increased. Compared with the mRNA expression of inflammation-related genes Ccl2 and Il6 in the liver of mice in the DM group, the mRNA expression levels of the mRNA in the MET group, the AMPs-L group, and the AMPs-H group were significantly reduced, and there was little difference in the AMPs-CM group. Compared with the mRNA expression level of Tnf-α in the liver of mice in the DM group, the mRNA expression level of the inflammation-related gene Tnf-α in the liver of mice in the AMPs-L group was significantly reduced, and there was little difference in the MET group, the AMPs-H group, and the AMPs-CM group. The results showed that AMPs can significantly improve the mRNA expression of inflammation-related genes Ccl2, Il6, and Tnf-α in the liver of mice and relieve inflammation. AMPs-CM can improve the mRNA expression of inflammation-related genes Ccl2, Il6, and Tnf-α in the liver of mice, but its therapeutic effect is significantly lower than that of AMPs.

[0122] 2.3 Effect of Atractylodes macrocephala polysaccharide on liver fibrosis in diabetic mice with fatty liver hepatitis

[0123] The results are as follows Figure 7 As shown, Figure 7 This is a graph of the mRNA levels of liver fibrosis-related genes Tgf-b1, Col1a1, and Timp1 in mice. Compared with the CON group, the mRNA levels of liver fibrosis-related genes in the DM group were significantly increased.

[0124] Compared with the mRNA expression levels of Tgf-b1, a gene related to liver fibrosis in mice in the DM group, those in the MET group, AMPs-L group, AMPs-H group and AMPs-CM group were significantly decreased, indicating that MET, AMPs and AMPs-CM can significantly reduce the mRNA expression of Tgf-b1, a gene related to liver fibrosis in mice and improve liver fibrosis in mice.

[0125] Compared with the mRNA expression of Col1a1, a gene related to liver fibrosis in mice in the DM group, the mRNA expression of Col1a1 in the MET group, AMPs-L group, AMPs-H group and AMPs-CM group was significantly reduced, and the AMPs-CM group was significantly higher than the MET group, AMPs-L group and AMPs-H group. This indicates that MET, AMPs and AMPs-CM can significantly reduce the mRNA expression of Tgf-b1, a gene related to liver fibrosis in mice, improve liver fibrosis in mice, and the therapeutic effect of MET and AMPs on liver fibrosis is better than AMPs-CM.

[0126] Compared with the expression of Timp1 mRNA, a gene related to liver fibrosis, in the MET group and AMPs-H group, it was significantly reduced; the expression of Timp1 mRNA in the AMPs-L group and AMPs-CM group was reduced, but there was no significant difference. The results showed that MET (200 mg / kg) and high-dose AMPs (300 mg / kg) could significantly improve the expression of Timp1 mRNA, a gene related to fibrosis, with significant therapeutic effects, while low-dose AMPs (100 mg / kg) and AMPs-CM (300 mg / kg) had no significant therapeutic effects.

[0127] In summary, the therapeutic effect of AMPs on mice with type 2 diabetes and fatty liver disease is better than that of MET and AMPs-CM. Furthermore, the therapeutic effect of AMPs on mice with type 2 diabetes and fatty liver disease was comprehensively determined.

[0128] Example 3

[0129] 1. Experimental methods

[0130] 1.1 Experimental drugs and animals

[0131] The mouse model was established by the method of Example 2 (1.1 Experimental drugs and animals). After the model was established successfully, the experimental mice in the model group were divided into a model group (DM), a low-dose group of Atractylodes macrocephala polysaccharide (AMPs-L), a high-dose group (AMPs-H) and a positive control metformin group (MET), with 10 mice in each group, and the drug was administered for 8 weeks. The AMPs-L and AMPs-H groups were given low-dose (100 mg / kg) and high-dose (300 mg / kg) suspensions (physiological saline solution) by gavage, respectively, the MET group was given metformin (200 mg / kg) suspension (physiological saline solution) by gavage, and the CON group was given physiological saline solution by gavage, once a day. The body weight of the mice was recorded weekly, and the fasting blood glucose of the mice was recorded every 2 weeks. The glucose tolerance (OGTT) of the mice was tested at the end of the 7th week of administration: 1 g / kg of glucose was gavaged, and the blood glucose level of the mice was tested at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min after gavage.

[0132] 1.2 Sample collection and preparation

[0133] After the drug intervention cycle, the mice were anesthetized and killed, and the eye vein blood was collected. After standing, the blood was centrifuged and the supernatant was collected. At the same time, fresh liver tissue was collected and the tissue for pathological sections was placed in an embedding box. When the liver tissue material was taken, the mouse body weight and liver weight were weighed and the liver index was calculated. The tissue for mRNA detection was added with RNA protective agent and stored in a -20℃ refrigerator. The liver tissue for Western blot was quickly frozen in liquid nitrogen and then moved to a -80℃ refrigerator for storage.

[0134] 1.3 Detection

[0135] (1) Serum biochemical test: Serum ALT and AST levels were detected using a biochemical kit, and all operations were performed according to the instructions.

[0136] (2) Tissue fixation, dehydration, embedding, and sectioning:

[0137] Fresh liver tissue was placed in an embedding box and immersed in tissue fixative. Paraffin sections were dehydrated using gradient ethanol dehydration, then immersed in wax, embedded in paraffin, and placed on a paraffin slicer for slicing. Frozen sections were dehydrated using sucrose dehydration, embedded in OTC embedding agent, and placed on a cryostat for slicing.

[0138] (3) H&E staining:

[0139] Paraffin sections were placed in a 65°C oven for 1 hour, dewaxed in xylene three times, 10 minutes each time, and washed with various levels of ethanol: 100% ethanol twice for 5 minutes each time, 95% ethanol twice for 5 minutes each time, 80% ethanol twice for 5 minutes each time, and washed with water twice for 5 minutes. Stained with hematoxylin for 5 minutes, washed with distilled water for 5 minutes. Differentiated with 1% hydrochloric acid alcohol solution for 5 seconds, rinsed with running water to return to blue for 15 minutes. After staining in 0.5% eosin aqueous solution for 1 minute, rehydrated with various levels of ethanol: 80% ethanol for 2 minutes, 95% ethanol for 5 minutes each time, and 100% ethanol for 5 minutes each time. After being transparent in xylene twice for 5 minutes each time, the slides were sealed with neutral gum.

[0140] (4) Oil Red O staining:

[0141] The frozen sections were placed at room temperature for 30 minutes before staining. They were first fixed in 4% paraformaldehyde for 5 minutes, soaked in pure water for 5 minutes, stained with Oil Red O working solution for 10 minutes, rinsed once with pure water, placed in 60% isopropanol, differentiated until the stroma was transparent, differentiated with pure water, counterstained with hematoxylin for 3 minutes, rinsed with pure water, differentiated with 0.5% hydrochloric acid ethanol for 1 second, rinsed with pure water, soaked in PBS for 5 minutes for anti-blueing, and sealed with glue.

[0142] (5) Sirius red staining:

[0143] The steps of drying and dewaxing are the same as those of H&E staining. After soaking in pure water for 5 minutes, place the slices in a wet box and stain with Sirius red stain for 30 minutes. Keep the wet box moist, rinse with pure water twice, rehydrate to make it transparent, follow the steps of HE staining, and seal the slices with neutral gum.

[0144] (6) Immunohistochemical staining (CD68):

[0145] The steps of drying and dewaxing are the same as H&E staining, and the slides are rinsed with tap water for 5 minutes. Prepare sodium citrate buffer, add water to the pressure cooker, boil until boiling, pour sodium citrate solution into the beaker, put in the tissue slices, seal with tin foil, cover the pot and boil until steam is generated, and count for 15 minutes. After venting, take out the beaker and cool naturally to room temperature. Soak in distilled water for 2 minutes, let stand at room temperature for 10 minutes with 3% hydrogen peroxide, and wash with PBS buffer (PH = 7.2-7.4) for 5 minutes × 3 times. Incubate with primary antibody: first use filter paper to absorb water, block with 1% BSA serum for 1 hour, and place at room temperature. During this period, prepare the primary antibody working solution (FN / CD68: 1% BSA = 1: 500). Shake off the blocking solution, and wash with PBS for 5 minutes × 3 times. Add the primary antibody diluent to completely cover the tissue surface, and incubate overnight at 4°C. The next day, rewarm for 30 minutes and wash with PBS 3 times / 5 minutes. Incubate with secondary antibody: Prepare secondary antibody diluent with 1% BSA in advance and incubate at room temperature for 1 hour. After shaking off the secondary antibody working solution on the slide, wash 3 times / 5min with PBS buffer (PH=7.2-7.4). Transfer to a dark place and develop with DAB colorimetric agent. Pay attention to control the color development time to avoid excessive color development and false positive area. Stain with hematoxylin for 30s and run water for 5 minutes. Differentiate with 1% hydrochloric acid ethanol differentiation solution and wash with running water for 15 minutes. Rehydrate and make it transparent, the steps are the same as H&E staining, and seal with neutral gum.

[0146] (7) Perform real-time fluorescence quantitative PCR and WB detection according to the method of Example 2.

[0147] 2. Experimental results

[0148] Effects of AMPs on body weight, liver index, blood glucose, and glucose tolerance in diabetic mice with steatohepatitis

[0149] The body weight of mice was recorded once a week, and the blood glucose of mice was recorded once every 2 weeks. The glucose tolerance of mice was tested at the end of the 7th week of drug administration. The body weight and liver weight of mice were weighed when samples were collected 8 weeks after drug administration, and the liver index was calculated. Figure 8 As shown in A and B, Figure 8 Figure 1 is a graph of mouse body weight, liver index, blood sugar, and glucose tolerance, wherein A is a graph of mouse body weight (Body weight) and liver index (Liverindex), and Figure B is a graph of mouse blood sugar and the area under the OGTT curve (AUC of OGTT), and mouse fasting blood sugar (Bloodglucose).

[0150] Figure 8As shown in Figure A, compared with the CON group, the body weight of mice in the DM group decreased significantly, and the liver index increased significantly; compared with the DM group, the body weight of mice in the AMPs-L, AMPs-H, and MET groups did not change much, but the liver index decreased significantly, and the high dose was better than the low dose group. AMPs can reduce the relative liver weight of mice. Figure 8 As shown in B, compared with the CON group, the blood glucose of mice in the DM group showed a significant upward trend, and the area under the OGTT curve increased significantly; compared with the DM group, the blood glucose of mice in the AMPs-L, AMPs-H and MET groups showed a significant downward trend; the area under the OGTT curve of mice in the MET and AMPs-H groups decreased significantly. The results show that AMPs can reduce blood glucose in mice, improve glucose tolerance in mice, and regulate glucose metabolism in mice.

[0151] Effects of AMPs on systemic and hepatic lipid metabolism in diabetic mice with steatohepatitis

[0152] The results are as follows Fig. 9 As shown, Fig. 9 The above diagram is a diagram of mouse lipid metabolism, where A is the appearance of mouse liver, H&E staining of liver tissue, and Oil Red O staining of liver tissue; and B is a diagram of the levels of mouse lipid metabolism-related proteins Pparγ and ATGL.

[0153] When obtaining liver tissue materials, observe the appearance of the mouse liver and take pictures of the ex vivo liver. Perform H&E staining and Oil red O staining on the liver sections and observe them. Fig. 9 As shown in A, compared with the CON group, the liver volume of mice in the DM group was significantly increased, HE staining showed obvious hepatocyte necrosis, lipid vacuoles and inflammatory cell infiltration in the DM group, and Oil Red O staining showed that there were more lipid droplets in the DM group; compared with the DM group, the liver volume of mice in the AMPs-L, AMPs-H and MET groups was significantly reduced, HE staining showed that hepatocyte necrosis, lipid vacuoles and inflammatory cell infiltration in the AMPs-L, AMPs-H and MET groups were alleviated, and Oil Red O staining showed that the lipid droplets in the AMPs-L, AMPs-H and MET groups were significantly reduced.

[0154] Fig. 9 As shown in B, compared with the CON group, the level of PPARγ, a protein related to lipid synthesis, in the liver of mice in the DM group showed an increasing trend, and the expression of ATGL, a protein related to lipid decomposition, showed a decreasing trend; compared with the DM group, the level of Pparγ protein in the AMPs-L, AMPs-H and MET groups showed a decreasing trend, and the level of ATGL protein showed an increasing trend.

[0155] The results showed that AMPs could regulate serum lipid metabolism and alleviate hepatic lipid deposition by reducing lipid synthesis and transport and increasing lipid degradation.

[0156] Effects of AMPs on liver inflammation in diabetic mice with steatohepatitis

[0157] The results are as follows Fig.10 As shown, Fig.10 These are graphs showing liver inflammation in mice, where A is a graph showing CD68 immunohistochemical staining of mouse liver tissue and statistical graph of the positive area, B is a graph showing the activity of mouse serum ALT and AST, and C is a graph showing the levels of mouse inflammation-related proteins NLRP3 and MCP1.

[0158] Immunohistochemical staining was performed on liver sections and observed. Fig.10 As shown in Figure A, compared with the CON group, the CD68-positive area in the liver of mice in the DM group increased significantly; compared with the DM group, the CD68-positive area in the liver of mice in the AMPs-L and AMPs-H groups decreased significantly. Fig.10 As shown in Figure B, compared with the CON group, the serum ALT and AST of mice in the DM group were significantly increased; compared with the DM group, the serum ALT and AST of mice in the AMPs-H and MET groups were significantly decreased. This indicates that the hepatocyte damage in diabetic mice is more serious, and the administration of AMPs can reduce the hepatocyte damage caused by inflammation, thereby improving liver function. Fig.10 As shown in C, compared with the CON group, the expressions of NLRP3 and MCP1, the inflammation-related proteins in the liver, in the DM group showed an upward trend; compared with the DM group, the expressions of AMPs-L, AMPs-H and MET groups showed a downward trend.

[0159] The results showed that the model mice were accompanied by significant hepatocellular damage and liver inflammation, while AMPs administration significantly improved liver damage and inflammation.

[0160] Effects of AMPs on liver fibrosis in diabetic mice with steatohepatitis

[0161] The results are as follows Fig.11 As shown, Fig.11 These are graphs showing liver fibrosis in mice, where A is a statistical graph showing Sirius red staining and positive area, and B is a graph showing the levels of Collagen-1 and α-SMA, proteins associated with liver fibrosis in mice.

[0162] Liver sections were stained with Sirius red and observed. Fig.11 As shown in Figure A, compared with the CON group, the Sirius red-positive area of ​​the liver of the DM group mice increased significantly; compared with the DM group, the Sirius red-positive area of ​​the liver of the MET group and the AMPs-L group mice tended to decrease, and the Sirius red-positive area of ​​the liver of the AMPs-H group mice decreased significantly. WB detection of liver fibrosis-related proteins in mice, such as Fig.11As shown in B, compared with the CON group, the expression of collagen-1 and α-SMA, the fibrosis-related proteins in the liver of mice in the DM group, increased; compared with the DM group, the expression of collagen-1 and α-SMA in the liver of mice in the MET group and AMPs-L and AMPs-H groups decreased. The results showed that AMPs can reduce collagen deposition in the liver of mice and improve liver fibrosis.

[0163] In summary, the present invention adopts a mouse model based on type 2 diabetes combined with fatty liver hepatitis to study the improvement effect of AMPs on liver damage, inflammation, lipid metabolism, and fibrosis. Through blood glucose and OGTT detection, it was found that AMPs can regulate the glucose metabolism of mice; through serum biochemical detection, it was found that AMPs have the effect of improving liver function and lowering blood lipids; through H&E and Oil Red O staining, it was found that AMPs have the effect of improving liver lipid deposition; through CD68 immunohistochemistry, it was found that AMPs can reduce mouse liver inflammation; through Sirius red staining, it was found that AMPs can reduce mouse liver collagen deposition. By qPCR and WB to detect the expression of mouse liver-related genes and proteins, it was found that AMPs can improve mouse liver lipid metabolism, inflammation, and fibrosis at the gene and protein levels. In summary, AMPs have a significant therapeutic effect on diabetes combined with fatty liver hepatitis.

[0164] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. An application of Atractylodes macrocephala polysaccharide in the preparation of a drug for treating diabetes combined with fatty liver disease.

2. The use according to claim 1, characterized in that: The diabetes combined with fatty liver hepatitis includes one or more of insulin resistance, lipid metabolism disorder, liver inflammation, liver damage and liver fibrosis.

3. The application according to claim 1, characterized in that: Atractylodes macrocephala polysaccharide was prepared by water extraction and alcohol precipitation.

4. The use according to claim 3, characterized in that: The water extraction and alcohol precipitation method comprises: extracting Atractylodes macrocephala and ultrapure water in a ratio of 1 g:15-25 mL at 85-95° C., extracting 1-3 times, each time for 1-3 hours, combining the extracts and concentrating to obtain a concentrated solution; adding 2-4 times the volume of 90-100% ethanol solution to the concentrated solution, precipitating with alcohol to obtain a precipitate, and drying to obtain Atractylodes macrocephala polysaccharide.

5. The application according to claim 4, characterized in that: The extraction was performed twice.

6. The use according to claim 4, characterized in that: The precipitate was washed with ethanol.

7. The use according to any one of claims 3 to 6, characterized in that: The water extraction and alcohol precipitation method is specifically as follows: Step 1): extract Atractylodes macrocephala and ultrapure water at a ratio of 1 g: 20 mL at 90°C for 2 h, centrifuge at 4000 rpm for 20 min, and collect the supernatant and residue; Step 2): extract and centrifuge the residue for the second time according to the method of step 1), combine the supernatants of the two extractions and concentrate at 60° C. to obtain a concentrate; Step 3): Add 3 times the volume of 95% ethanol solution to the concentrate, precipitate overnight at 4°C, collect the precipitate by centrifugation, wash the precipitate with ethanol and freeze-dry it to obtain Atractylodes macrocephala polysaccharide.

8. The use according to claim 7, characterized in that: Atractylodes macrocephala polysaccharide is composed of glucose, arabinose, galactose, galacturonic acid and glucuronic acid.

9. The use according to claim 8, characterized in that: The Atractylodes macrocephala polysaccharide is composed of 82-85% glucose, 6-8% arabinose, 5-6% galactose, 2-4% galacturonic acid and 0.1-0.8% glucuronic acid.

10. The use according to claim 9, characterized in that: The Atractylodes macrocephala polysaccharide is composed of 83.92% glucose, 7.19% arabinose, 5.47% galactose, 3% galacturonic acid and 0.41% glucuronic acid.

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