Application of Atractylodes macrocephala polysaccharide in the preparation of drugs for treating diabetes-related diseases

Abstract

This invention provides the application of Atractylodes macrocephala polysaccharide in the preparation of drugs for treating diabetes-related diseases, belonging to the field of traditional Chinese medicine technology. The diabetes-related diseases include diabetic nephropathy and / or diabetic intestinal diseases. The Atractylodes macrocephala polysaccharide is prepared by water extraction and alcohol precipitation, and the obtained polysaccharide consists of glucose, arabinose, galactose, galacturonic acid, and glucuronic acid. This invention uses Atractylodes macrocephala polysaccharide to treat diabetic nephropathy and diabetic intestinal diseases, protecting kidney function, kidney structure, and colon structure, alleviating inflammation and kidney fibrosis caused by diabetic nephropathy, and repairing intestinal diseases caused by diabetes. It can restore colonic mucosal barrier function, restore the expression of intestinal epithelial marker proteins, regulate the expression of tight junction proteins Zo-1, E-cadherin, Ocln, Cldn2, and Cldn4, and can be used to regulate the gut microbiota in diabetic intestinal diseases.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, and relates to a polysaccharide of Atractylodes macrocephala, specifically the application of Atractylodes macrocephala polysaccharide in the preparation of drugs for treating diabetes-related diseases. Background Technology

[0002] Diabetic kidney disease (DKD) is a chronic kidney disease (CKD) caused by diabetes mellitus (DM). It is one of the major microvascular complications of diabetes and the most common cause of end-stage renal disease (ESRD) worldwide. DKD gradually progresses from low-grade kidney inflammation to kidney fibrosis and sclerosis, eventually leading to ESRD, as hyperglycemia develops. Epidemiological studies show that diabetes is one of the fastest-growing global health emergencies of the 21st century. With the prevalence of diabetes, the incidence of DKD is rapidly increasing. Approximately 30%-40% of diabetic patients develop DKD, and one-third of these patients further develop ESRD. DKD poses a significant challenge to my country's healthcare system, creating a huge social and economic burden, but currently, there are no effective drugs to slow or reverse the progression of DKD.

[0003] Pathologically, the kidneys of patients with DKD exhibit a variety of changes. Common intraglomerular features include capillary dilation, basement membrane thickening, extracellular matrix expansion, podocyte damage, and fibrosis; while in the tubulointerstitium, vacuolar degeneration, loose arrangement, and fibrosis are observed. Patients are typically diagnosed with glomerular hyperfiltration, microalbuminuria, and macroalbuminuria, with the glomerular filtration rate (GFR) decreasing as DKD progresses. In clinical practice, urinary protein (UP) levels, including the urinary albumin excretion rate (UAER) in patients with microalbuminuria or the 24-hour UP in patients with macroalbuminuria, are generally considered the primary indicator for detecting DKD. To date, the treatment and management strategies for DKD primarily focus on reducing weight, blood glucose, and blood pressure. Renin-angiotensin system inhibitors (RASIs), including angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs), are commonly used first-line treatments. However, satisfactory results are not always achieved when using these traditional methods to treat diabetic kidney disease (DKD). For example, it has been reported that ARBs are insufficient to prevent the progression of albuminuria in diabetic patients with normal blood pressure. Traditional Chinese medicine (TCM) has a long history as a commonly used treatment for DKD and related complications, demonstrating good efficacy in clinical practice. Several bioactive compounds in TCM that are beneficial to patients with diabetic nephropathy have been identified and purified, and their mechanisms of action have been extensively studied. Existing research indicates that TCM treatment can improve renal function and alleviate symptoms such as proteinuria and glomerulosclerosis. Therefore, exploring TCM with potential hypoglycemic effects may offer possibilities for the prevention and treatment of DKD and related complications.

[0004] Traditional Chinese medicine (TCM) polysaccharides are a class of natural macromolecules widely found in Chinese herbal medicines, possessing important pharmacological activities and abundant bioactive components. In recent decades, with the continuous development of biotechnology and modern chemical technology, the research on TCM polysaccharides has received increasing attention. TCM polysaccharides exhibit various biological activities, such as immunomodulation, antioxidation, antitumor, antiviral, hepatoprotection, and lipid-lowering effects. Currently, polysaccharides from traditional Chinese medicine (TCM) have been extensively studied in the field of glucose and lipid metabolism disorders. Lycium barbarum polysaccharides and Astragalus membranaceus polysaccharides, for example, have been shown to lower blood sugar. The mechanisms of action of these polysaccharides may be related to promoting insulin secretion, promoting glucose transport, and inhibiting glycogenase. Donkey-hide gelatin polysaccharides and Ganoderma lucidum polysaccharides have been shown to lower blood lipids by inhibiting fat synthesis, promoting fat breakdown, and regulating cholesterol metabolism. Diabetic patients often experience oxidative stress; Poria cocos polysaccharides and Cinnamomum cassia polysaccharides have significant antioxidant effects, and their mechanisms of action may be related to inhibiting free radical production and increasing antioxidant enzyme activity. In terms of immune regulation, Ganoderma lucidum polysaccharides and Lycium barbarum polysaccharides have been shown to exert their immunomodulatory effects by enhancing the body's immunity and regulating immune cell function. In conclusion, TCM polysaccharides, as a class of natural medicinal resources, have broad pharmacological activities and application prospects. With continued research, it is believed that more biological activities and pharmacological effects of TCM polysaccharides will be discovered and applied, providing possibilities for the prevention and treatment of diabetes mellitus (DM)-related complications.

[0005] The gastrointestinal tract of healthy individuals is home to a vast array of microorganisms. These microorganisms, known as the gut microbiota, constitute a large and complex ecosystem. Research indicates that the gut microbiota is a crucial component of the gut micro-ecosystem, playing a vital role in host physiological functions and processes, such as nutrient absorption, growth and development, biological barrier function, immune regulation, lipid metabolism, and anti-tumor activity. Under normal circumstances, the human gut microbiota forms a dynamic equilibrium system, mutually regulating each other to maintain the body's micro-ecological balance and participating in functions such as digestion, metabolism, immune regulation, energy conversion, and maintaining intestinal mucosal defense. Once influenced by changes in the host itself and the external environment, the balance between the host and the microbiota is disrupted, leading to an imbalance in the gut micro-ecosystem, which in turn results in impaired bodily functions and disease.

[0006] Diabetic patients often develop intestinal diseases, resulting in intestinal mucosal damage, intestinal microbiota homeostasis imbalance, barrier damage, and decreased immune function. In severe cases, it can induce complications such as stroke, coronary artery occlusion, retinopathy, and decline and failure of organ function such as liver and kidney.

[0007] In recent years, the interaction between the bioactive components of Traditional Chinese Medicine (TCM) and the gut microbiota has become a focus of much research. When TCM enters the digestive tract, some bioactive components cannot be well absorbed by the intestines, resulting in low bioavailability. TCM components undergo metabolism or biotransformation through the gut microbiota, generating new bioactive molecules and promoting drug absorption into circulation. Simultaneously, TCM components influence the composition and structure of the gut microbiota, thereby affecting the remote function of diseased organs / tissues through the systemic effects of the gut microbiota. Studies have shown that TCM polysaccharides can increase the number of beneficial gut bacteria such as Bifidobacteria and Lactobacillus, while inhibiting the growth of harmful bacteria such as Escherichia coli and Enterococcus. These beneficial bacteria can produce beneficial metabolites such as short-chain fatty acids, which help maintain the integrity of the intestinal mucosal barrier, suppress inflammatory responses, and promote nutrient absorption and metabolism. In addition, some Chinese herbal polysaccharides also have the effect of regulating intestinal metabolism. For example, astragalus polysaccharides can lower blood sugar, blood lipids and weight by regulating intestinal flora and metabolism; aloe polysaccharides can improve intestinal barrier function and prevent diseases such as enteritis by promoting the growth and metabolism of beneficial flora.

[0008] The gut microbiota is a microbial community closely related to the host, playing a crucial role in maintaining intestinal homeostasis and participating in the occurrence and development of metabolic diseases such as obesity, diabetes, and chronic kidney disease (CKD). Gut microbiota dysbiosis includes structural alterations and functional abnormalities. Structural changes in the gut microbiota often manifest as a significant reduction in probiotics and the proliferation of opportunistic pathogens, accompanied by functional changes that affect host physiological processes such as metabolism and immune responses, increasing the risk of disease and creating a vicious cycle. Disruption of the intestinal barrier is a significant cause of metabolic inflammation. Unhealthy dietary habits, such as high-sugar and high-fat diets, lead to changes in the composition of the gut microbiota, promoting pathogen growth and inhibiting the growth of symbiotic bacteria. With the increase of invasive pathogens, these bacteria can degrade intestinal mucus and reach the epithelial barrier. Subsequently, bacterial components such as LPS bind to TLRs on the surface of intestinal epithelial cells, recruiting immune cells in the gut, releasing pro-inflammatory factors such as IFN-γ and IL-1β, inhibiting the expression of intestinal tight junction proteins, disrupting the intestinal epithelial barrier, promoting the passage of intestinal bacteria through the intestinal plate, and further damaging the intestinal vascular barrier. Recent studies have shown that the gut microbiota is involved in the progression of DKD. Bacterial metabolites have been shown to influence the occurrence and progression of chronic kidney disease, and progression to kidney failure leads to a worsening of gut microbiota dysbiosis.

[0009] CKD patients often exhibit gut microbiota dysbiosis, accumulation of bacterial metabolites, impaired intestinal barrier function, and chronic inflammation. Most CKD patients have gut bacterial overgrowth, but bacterial diversity is reduced. Furthermore, *Eggerthellalenta*, *Flavonifractor* spp., *Alistipes* spp., *Ruminococcus* spp., and *Fusobacterium* spp. are significantly enriched in ESRD patients, while the abundance of bacteria producing short-chain fatty acids (SCFAs), particularly butyrate-producing bacteria, gradually decreases with the malignant progression of ESRD. It has been reported that remodeling the gut microbiota by inhibiting oxidative stress and inflammation can partially improve diabetes-related kidney damage. The enrichment of bacteria producing SCFAs (i.e., acetate, butyrate, and propionate) suppresses disease progression by modulating gut inflammation and host immunity. Therefore, the gut microbiota and its derived metabolites may serve as novel therapeutic targets for DKD.

[0010] Previous studies have shown that traditional Chinese medicine and its natural extracts may have specific protective effects on kidney function. These effects may slow the progression of kidney disease, but the exact mechanisms remain unclear. It is speculated that plant polysaccharides may exert their pharmacological effects by regulating the gut microbiota, restoring the integrity of the intestinal barrier, and inhibiting inflammation. Treatment strategies targeting the gut microbiota hold great potential for the future, opening up a new perspective and direction for the treatment of DKD.

[0011] Traditional Chinese medicine (TCM) polysaccharides, as biological macromolecules, are typically composed of 10 or more types of monosaccharides and have become a research focus in recent years due to their diverse biological activities. These polysaccharides are long-chain polycarbohydrates formed by linking multiple monosaccharide units through glycosidic bonds, which can produce monosaccharides or oligosaccharides upon hydrolysis. Their general formula is Cx(H₂O)y, where the x value is usually between 200 and 2500. Currently, there are various extraction methods for TCM polysaccharides, which significantly affect the types, physicochemical properties, and biological activities of the final extract. Hot water extraction and ethanol precipitation are the most common extraction methods, with advantages such as simple operation, no need for specialized equipment, and low cost. Although some new extraction techniques have been developed to extract polysaccharides with different properties, the purity of the extracted polysaccharides is usually low due to technical limitations. Therefore, the purification and structural identification of TCM polysaccharides are particularly crucial. The purification process is complex, and obtaining high-purity homogeneous polysaccharide components is difficult. Increasing research shows that extraction and purification methods have a significant impact on the modification and biological activity of polysaccharides. Therefore, the extraction and purification of TCM polysaccharides are an important foundation for analyzing their biological activity and chemical modification.

[0012] Studies have shown that polysaccharides are closely related to the regulation of immune function, cell division and differentiation, and the occurrence and development of diseases. Atractylodes macrocephala is a plant with spleen-tonifying and qi-boosting properties, and polysaccharides are its active ingredients. Atractylodes macrocephala polysaccharides have strong activity in regulating intestinal microecological balance and intestinal mucosal immune function. Furthermore, Atractylodes macrocephala polysaccharides can reduce inflammatory damage and oxidative stress in mice and play a protective role in the early stages of LPS invasion of the liver. Atractylodes macrocephala polysaccharides (AMPs) are important active components of Atractylodes macrocephala and cannot be directly absorbed by the human body.

[0013] The invention patent with publication number CN116726040A discloses the application of Atractylodes macrocephala polysaccharide in the preparation of drugs for the prevention and treatment of psoriasis. The polysaccharide is obtained by water extraction and alcohol precipitation, specifically including the following steps: Atractylodes macrocephala is dried and pulverized, then water is added, and extraction is carried out at 60-110℃ for 0.5-8 hours. This extraction is repeated several times. The extracts are combined and filtered, concentrated, and precipitated with a solution of 70% or higher ethanol. The precipitate is then allowed to stand at 4-30℃ for 6-96 hours. The precipitate is collected by filtration, yielding a solution containing Atractylodes macrocephala polysaccharide, which is then freeze-dried to obtain the polysaccharide. The polysaccharide is composed of mannose, rhamnose, galacturonic acid, glucose, galactose, and arabinose in a molar mass ratio of 2.48:6.17:3.58:66.82:9.10:11.68, primarily in the β-configuration.

[0014] Chinese patent application CN114073710A discloses a novel use of Atractylodes macrocephala polysaccharide: its application in preparing health products or functional foods that reduce the relative abundance ratio of Firmicutes (F) and Baceroidetes (B) in the gut microbiota of individuals with spleen deficiency, regulate the F / B balance, and prevent or treat obesity caused by gut microbiota imbalance due to spleen deficiency. The Atractylodes macrocephala polysaccharide is a heteropolysaccharide composed of five monosaccharides: rhamnose (Rha), galacturonic acid (GalA), glucose (Glu), galactose (Gal), and arabinose (Ara), and possesses specific fingerprint characteristics.

[0015] Chinese patent application CN1398630A discloses a hypoglycemic Atractylodes macrocephala glycosuria complex, its production method, and its uses. The method involves soaking to obtain an aqueous extract of Atractylodes macrocephala, precipitating it with an organic solvent, dissolving the precipitate in water, and then dialysis or membrane separation followed by concentration and drying to obtain a crude product. The crude product is then separated by column chromatography to obtain a refined Atractylodes macrocephala glycosuria complex. The Atractylodes macrocephala glycosuria complex has the following elemental 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 it also contains glucuronic acid and galacturonic acid. This polysaccharide has no toxic side effects and exhibits a significant hypoglycemic effect.

[0016] Extraction and purification methods have a significant impact on the modification and bioactivity of polysaccharides. In order to find effective, safe and low-toxicity new treatments for diabetic nephropathy, strategies need to be improved. Summary of the Invention

[0017] This invention addresses the problems existing in the prior art by providing the application of Atractylodes macrocephala in the preparation of drugs for treating diabetes-related diseases. The Atractylodes macrocephala polysaccharide prepared by this invention using water extraction and alcohol precipitation can improve renal function damage, inflammation, and renal fibrosis in diabetic nephropathy, repair the colonic mucosal barrier function caused by diabetes, restore the expression of intestinal epithelial marker proteins, and regulate the expression of tight junction proteins Zo-1, E-cadherin, Ocln, Cldn2, and Cldn4. It can be used in drugs to regulate intestinal flora dysbiosis caused by diabetic intestinal diseases.

[0018] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0019] In a first aspect, the present invention provides the application of Atractylodes macrocephala polysaccharide in the preparation of a medicament for treating diabetes-related diseases, including diabetic nephropathy and / or diabetic intestinal diseases.

[0020] Preferably, the Atractylodes macrocephala polysaccharide is used to prepare a drug for treating diabetic nephropathy and diabetic intestinal disease mixed disease, or diabetic nephropathy, wherein diabetic nephropathy includes one or more of renal function impairment, inflammation, and renal fibrosis.

[0021] Preferably, the Atractylodes macrocephala polysaccharide is used to prepare a drug for treating a mixed disease of diabetic nephropathy and diabetic intestinal disease, or diabetic intestinal disease, wherein the diabetic intestinal disease is intestinal mucosal damage, and the intestinal mucosal damage is characterized by: suppressed expression of tight junction proteins of the intestinal mucosa or intestinal flora dysbiosis, wherein the tight junction proteins include one or more of Zo-1, E-cadherin, Ocln, Cldn2 and Cldn4.

[0022] The Atractylodes macrocephala polysaccharide was prepared by water extraction and alcohol precipitation. The water extraction and alcohol precipitation method is as follows:

[0023] Atractylodes macrocephala and ultrapure water are extracted at a ratio of 1g:15-25mL at an extraction temperature of 85-95℃, preferably 90℃, for 1-3 extractions, preferably 2 extractions, each extraction lasting 1-3 hours. The extracts are combined and concentrated to obtain a concentrated solution. A 90-100% (v / v) ethanol-water solution is added to the concentrated solution, with a volume ratio of the concentrated solution to the ethanol-water solution of 1:2-4, and the solution is precipitated by alcohol precipitation. The precipitate is then dried to obtain Atractylodes macrocephala polysaccharide. Preferably, the precipitate is further washed with ethanol.

[0024] In one embodiment, the water extraction and alcohol precipitation method specifically comprises:

[0025] Step 1): Extract Atractylodes macrocephala and ultrapure water at a ratio of 1g:20mL at 90℃ for 2h, centrifuge at 4000×r for 20min, and collect the supernatant and residue.

[0026] Step 2): Following the method in Step 1), the residue is extracted and centrifuged a second time. The supernatants from the two extractions are combined and concentrated to 1 / 3 of the original volume at 60°C to obtain a concentrated solution.

[0027] Step 3): Add three volumes of 95% ethanol aqueous solution to the concentrate, precipitate overnight at 4°C, centrifuge at 4000 rpm for 10 min, collect the precipitate, wash the precipitate twice with 95% ethanol aqueous solution, and freeze-dry to obtain Atractylodes macrocephala polysaccharide. The Atractylodes macrocephala polysaccharide consists of 83.93% glucose, 7.19% arabinose, 5.47% galactose, 3.00% galacturonic acid, and 0.41% glucuronic acid.

[0028] The surface of the Atractylodes macrocephala polysaccharide is loose and rough, and it is irregularly multi-layered or multi-layered rose petal-like.

[0029] This invention provides the application of Atractylodes macrocephala polysaccharide in the preparation of a drug for treating diabetes-related diseases, including diabetic nephropathy and / or diabetic intestinal diseases. Compared with the prior art, this invention has the following beneficial effects:

[0030] This invention utilizes the prepared Atractylodes macrocephala polysaccharide in a drug for treating diabetic nephropathy. This can protect kidney function, kidney structure, and colon structure, alleviate inflammation and kidney fibrosis caused by diabetic nephropathy, and reduce the expression of CD68, a macrophage marker in the kidney.

[0031] This invention utilizes the prepared Atractylodes macrocephala polysaccharide in the treatment of diabetic intestinal diseases. It can repair the colonic mucosal barrier function caused by diabetic intestinal diseases, restore the expression of intestinal epithelial marker proteins, and regulate the expression of tight junction proteins Zo-1, E-cadherin, Ocln, Cldn2 and Cldn4. It can improve diabetic intestinal diseases by regulating the intestinal flora. Attached Figure Description

[0032] Figure 1 The monosaccharide composition ion chromatogram of AMPs;

[0033] Figure 2 Fourier-infrared spectra of AMPs;

[0034] Figure 3 Scanning electron microscope images of AMPs;

[0035] Figure 4The plot shows the mRNA levels of inflammatory factors Ccl2, Il-1β, and Nlrp3 (# indicates a significant difference from the CON group, #p<0.05, ##p<0.01, ###p<0.001, * indicates a significant difference from the DKD group, *p<0.05, **p<0.01, ***p<0.001, ns indicates no significant difference from the DKD group);

[0036] Figure 5 The plot shows the mRNA levels of fibrosis-related markers fibronectin (Fn1), collagen I (Col1a1), and transforming growth factor β1 (Tgfb1) (# indicates a significant difference from the CON group, #p<0.05, ##p<0.01, ###p<0.001, * indicates a significant difference from the DKD group, *p<0.05, **p<0.01, ***p<0.001, ns indicates no significant difference from the DKD group);

[0037] Figure 6 mRNA levels of colonic tight junction molecules Zo-1, E-cadherin, Ocln, Cldn2, and Cldn4 (# indicates significant difference from the CON-FMT group, #p<0.05, ##p<0.01, ###p<0.001, * indicates significant difference from the DKD-FMT group, *p<0.05, **p<0.01, ***p<0.001, ns indicates no significant difference from the DKD group);

[0038] Figure 7 The graph shows the effects of AMPs on biochemical indicators in DKD mice. In the graph, A represents the urinary protein level of each group of mice, B represents the urinary protein / creatinine ratio of each group of mice, and C represents the serum creatinine content of each group of mice.

[0039] Figure 8 The graph shows the effects of AMPs on body weight and blood glucose in DKD mice. (A represents the change in body weight of mice over 8 weeks; B represents the fasting blood glucose of mice at week 8; # indicates a significant difference from the CON group, #p<0.05, ##p<0.01, ###p<0.001, * indicates a significant difference from the DKD group, *p<0.05, **p<0.01, ***p<0.001);

[0040] Figure 9The effect of AMPs on glucose tolerance in DKD mice is shown in the figure (A is a line graph of oral glucose tolerance test in each group of mice; B is the area under the curve (AUC) of the OGTT line graph; # indicates a significant difference with the CON group, #p<0.05, ##p<0.01, ###p<0.001, * indicates a significant difference with the DKD group, *p<0.05, **p<0.01, ***p<0.001).

[0041] Figure 10 The images show the morphology of mouse kidneys (A is the appearance of representative mouse kidneys from each group; B is the kidney weight / body weight ratio; C is the H&E staining of the kidneys; # indicates a significant difference from the CON group, #p<0.05, ##p<0.01, ###p<0.001, * indicates a significant difference from the DKD group, *p<0.05, **p<0.01, ***p<0.001);

[0042] Figure 11 The effect of AMPs on renal inflammation in DKD mice is shown in the figure (A is the CD68 immunohistochemical staining of mouse kidneys, scale bar = 20 μm; B is the statistical area of ​​CD68 positive regions; # indicates a significant difference with the CON group, #p < 0.05, ##p < 0.01, ###p < 0.001, * indicates a significant difference with the DKD group, *p < 0.05, **p < 0.01, ***p < 0.001);

[0043] Figure 12 The image shows the expression of MCP-1 and NLRP3 in the kidneys of DKD mice detected by Western blotting. GAPDH was used as an internal standard for statistical analysis (# indicates a significant difference from the CON group, #p<0.05, ##p<0.01, ###p<0.001, * indicates a significant difference from the DKD group, *p<0.05, **p<0.01, ***p<0.001).

[0044] Figure 13 The effect of AMPs on renal fibrosis in DKD mice is shown in the figure (A is Masson staining of mouse kidneys, scale bar = 20 μm; B is the statistical area of ​​positive areas stained by Masson staining; # indicates significant difference with the CON group, #p < 0.05, ##p < 0.01, ###p < 0.001, * indicates significant difference with the DKD group, *p < 0.05, **p < 0.01, ***p < 0.001).

[0045] Figure 14The effect of AMPs on renal tubulointerstitial fibrosis in DKD mice is shown in the figure (A is CD68 immunohistochemical staining of mouse kidneys, scale bar = 20 μm; B is the statistical area of ​​FN positive regions; # indicates significant difference with the CON group, #p < 0.05, ##p < 0.01, ###p < 0.001, * indicates significant difference with the DKD group, *p < 0.05, **p < 0.01, ***p < 0.001).

[0046] Figure 15 Figure 1 shows the expression of FN, Collage-1, and TGF-β1 in the kidneys of DKD mice detected by Western blotting; GAPDH was used as an internal standard for statistical analysis (# indicates significant difference from the CON group, #p<0.05, ##p<0.01, ###p<0.001, * indicates significant difference from the DKD group, *p<0.05, **p<0.01, ***p<0.001).

[0047] Figure 16 The effect of AMPs on the intestinal barrier of DKD mice is shown in the figure (A is H&E staining of colonic tissue, scale bar = 50 μm; B is ZO-1 (red) immunofluorescence staining of colon, scale bar = 20 μm; C is the statistical analysis of the area of ​​ZO-1 positive region; # indicates significant difference with the CON-FMT group, #p < 0.05, ##p < 0.01, ###p < 0.001, * indicates significant difference with the DKD-FMT group, *p < 0.05, **p < 0.01, ***p < 0.001);

[0048] Figure 17 The expression levels of colonic tight junction molecules (ZO-1 and Occludin) are plotted; GAPDH was used as an internal standard for statistical analysis (# indicates a significant difference from the CON-FMT group, #p<0.05, ##p<0.01, ###p<0.001, * indicates a significant difference from the DKD-FMT group, *p<0.05, **p<0.01, ***p<0.001). Detailed Implementation

[0049] It is worth noting that, unless otherwise specified, the raw materials used in this invention are all commercially available products, and their sources are not specifically limited.

[0050] Example 1

[0051] 1. Main experimental reagents are listed in Table 1.

[0052] Table 1 Main experimental reagents

[0053]

[0054]

[0055] 2. Major instruments and equipment are listed in Table 2.

[0056] Table 2 Main Instruments and Equipment

[0057]

[0058]

[0059] 3. Preparation of Atractylodes macrocephala polysaccharides (AMPs):

[0060] Step 1): After Atractylodes macrocephala is ground into powder, accurately weigh 10.0 g of the powder and extract it at 90℃ with stirring for 2 h using a ratio of Atractylodes macrocephala powder to ultrapure water of 1:20 (g / mL). Centrifuge the extract at 4000×r for 20 min in a high-speed refrigerated centrifuge and collect the supernatant and precipitate.

[0061] Step 2): Following the method in Step 1), the precipitate was extracted and centrifuged a second time. The supernatants from 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 concentrated solution was tested using an iodine-potassium iodide reagent, and the results showed that there was no starch.

[0062] Step 3): Add three times the volume of 95% (v / v) ethanol aqueous solution to the concentrate, stir and mix well, and let it precipitate overnight at 4°C. Centrifuge the mixture after precipitation at 4000 rpm for 10 min, remove the supernatant, and collect the precipitate. Wash the precipitate twice with 95% (v / v) ethanol aqueous solution to make the polysaccharide components more homogeneous. Finally, dissolve the precipitate evenly in ultrapure water and freeze-dry under vacuum for 48 h to obtain Atractylodes macrocephala polysaccharides (AMPs).

[0063] The extraction rate and total sugar content of AMPs are shown in Table 3.

[0064] Table 3 Extraction rate and total polysaccharide content of Atractylodes macrocephala polysaccharides

[0065]

[0066] The results showed that the extraction rate of AMPs was 20.07%, and the total sugar content was 87.06%.

[0067] 4. Preparation of Atractylodes macrocephala polysaccharides (AMPs-CM):

[0068] After being ground into powder, 10.0 g of Atractylodes macrocephala powder was accurately weighed for the first extraction: At 80℃, the powder and ultrapure water were stirred and extracted for 5 hours at a ratio of 1:15 (g / mL) to obtain the extract. The extract was then centrifuged at 4000 rpm for 20 minutes in a high-speed refrigerated centrifuge, and the supernatant and residue from the first extraction were collected. The residue was subjected to a second and third extraction using the same method. The supernatants from the three extractions were combined and concentrated to 1 / 3 of their original volume under vacuum rotary evaporation at 60℃ to obtain the concentrated solution. Iodine-potassium iodide reagent analysis showed that the concentrated solution contained no starch. Three times the volume of 85% (v / v) ethanol aqueous solution was added to the concentrated solution, stirred thoroughly, and allowed to precipitate overnight at 4℃. The mixture after ethanol precipitation was then centrifuged at 4000 × r for 10 minutes, the supernatant was removed, and the precipitate was collected. The precipitate was then washed twice with an equal volume fraction of ethanol-water solution to make the polysaccharide components more homogeneous. Finally, the precipitate was uniformly dissolved in ultrapure water and freeze-dried under vacuum for 48 hours to obtain Atractylodes macrocephala polysaccharide (AMPs-CM).

[0069] 5. Monosaccharide composition analysis

[0070] The monosaccharide composition of AMPs and AMPs-CM was determined using a Thermo ICS5000 ion chromatography system with an electrochemical detector. 6.0 mg of AMPs and AMPs-CM were hydrolyzed with trifluoroacetic acid (2.0 mol / L) at 121 °C for 2 h, followed by nitrogen purging and drying. The samples were then washed with 99.99% methanol, dried, and the washing process was repeated 2-3 times. The solutions were then dissolved in sterile water and transferred to chromatographic vials as the test samples. Each setup was repeated in triplicate. AMPs samples were AMPs-1, AMPs-2, and AMPs-3. A mixed standard (MD) was prepared as a series of concentration gradients from fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid (Gal-UA), glucuronic acid (Glc-UA), mannuronic acid (Man-UA), and guluronic acid (Gul-UA). Dionex was used. TM CarboPac TM The detection was performed using a PA20 (150*3.0mm, 10μm) liquid chromatography column.

[0071] The monosaccharide composition of AMPs and AMPs-CM is shown in Table 4.

[0072] Table 4. Monosaccharide composition percentage of AMPs and AMPs-CM

[0073]

[0074]

[0075] The results showed that both AMPs and AMPs-CM were composed of the monosaccharides glucose, arabinose, galactose, galacturonic acid, and glucuronic acid. The ion chromatograms of the monosaccharide composition of AMPs are shown below. Figure 1 As shown, AMPs are mainly composed of glucose (83.93%), and also contain small amounts of arabinose (7.19%), galactose (5.47%), galacturonic acid (3.00%) and glucuronic acid (0.41%).

[0076] 6. Infrared spectroscopy and scanning electron microscopy analysis of Atractylodes macrocephala polysaccharides (AMPs)

[0077] The infrared spectra of AMPs were analyzed using Fourier transform infrared spectroscopy to identify the functional group characteristics. A small amount of AMPs was weighed and mixed with 200 mg of potassium bromide, then pressed into a sheet structure with a thickness of 1 mm for analysis. Three replicates were set up, with AMPs samples AMPs-1, AMPs-2, and AMPs-3, and the samples were scanned and analyzed using a Fourier transform infrared spectroscopy (Nicolet iZ-10).

[0078] Fourier transform infrared spectrometer was used in the frequency range (4000-400cm). -1 Record infrared spectra. Figure 2 The Fourier-infrared spectra of AMPs are provided by Figure 2 It can be seen that at 3200cm -1 up to 3600cm -1 The vibrational region shown between 3000-2800 cm⁻¹ is the characteristic band of the hydroxyl group. -1 Within the range (2930cm) -1 The fluctuations observed are CH absorption peaks, which include stretching vibrations of CH, CH2, and CH3. These peaks occur in the 1000-1200 cm⁻¹ range. -1 The observed range of vibrations is caused by the vibrations of the ester sugar group (CO-C) and the pyranose ring (COH), proving that AMPs contain pyranose. Specifically, 905-876 cm⁻¹ -1 The vibrational range observed is caused by the β-configuration of the dextran. 855-833 cm⁻¹ -1 The range of vibrations that occur is caused by the α-configuration of dextran.

[0079] High-resolution field emission scanning electron microscopy (Zeiss Merlin Compact) was used to analyze the polysaccharides (AMPs) of Atractylodes macrocephala. The polysaccharide samples were passed through a 100-mesh sieve, and a small amount was taken onto conductive carbon tape, sputtered with gold, and then scanned and photographed using an electron microscope at magnifications of 100-4000x.

[0080] The results are as Figure 3 shown, Figure 3 which are the scanning electron microscope images of AMPs. At different magnifications (5k, 10k, and 20k), polysaccharide structures were observed. It was found that the surface of AMPs was loose and rough, showing an irregular multi-layered flaky or multi-layered rose petal-like distribution.

[0081] Example 2

[0082] Experimental drugs and experimental animals

[0083] Six-week-old male specific pathogen-free C57BL / 6 mice (16 - 18 g) were purchased from the Guangdong Provincial Medical Animal Center. Animal production license number: SCXK(Guangdong)2022 - 0002. All mice were housed in a SPF-class sterile environment in the Animal Experiment Center of Guangdong Pharmaceutical University. The mice were maintained in an environment (12 h light-dark cycle, 20 - 25 °C, relative humidity 60 ± 5%), with free access to food and water. All experimental mice were acclimated for 1 week before the experiment. All animal experiments were conducted in accordance with the relevant regulations of the Chinese Animal Welfare Law and were approved by the Laboratory Animal Ethics Committee of Guangdong Pharmaceutical University (No. gdpulacspf2022097).

[0084] Method for establishing a DKD (diabetic kidney disease) mouse model: The mice were first fed a high-fat diet (18% fat, 50% carbohydrates, 19% protein) for two weeks, and then STZ (streptozotocin, 40 mg / kg) was intraperitoneally injected daily for 5 consecutive days to induce diabetes. Seven days later, the FBG (fasting blood glucose) of the mice fasting for 6 h was measured. If the FBG exceeded 11.1 mmol / L, the mice were considered diabetic.

[0085] Mice with different administration treatments were set up: All diabetic mice were randomly divided into 5 groups (8 mice in each group), namely the DKD model group (DKD, Saline), the positive drug losartan group (LOS, 30 mg / kg / d), the positive drug metformin (MET, 200 mg / kg / d), the low-dose AMPs group (AMPs-L, 100 mg / kg / d), the high-dose AMPs group (AMPs-H, 300 mg / kg / d), and the AMPs-CM group (300 mg / kg / d). A control group was set up. The mice in the control group (CON, Saline) were fed a normal diet and injected with citrate buffer for 5 consecutive days (8 mice in each group).

[0086] Losartan, as an antihypertensive drug, can effectively lower blood pressure, but it has the following adverse reactions: First, effects on the nervous and mental system, which may manifest as headache, dizziness, and insomnia in some patients. These adverse reactions are relatively mild and transient, and generally do not require discontinuation of treatment. Second, hypotension, which is more common when the dosage is too high or the patient is sensitive to the antihypertensive drug. Third, allergic reactions, including dermatitis, rash, and allergic edema. Fourth, gastrointestinal adverse reactions, mainly including nausea, vomiting, and abnormal liver function.

[0087] Example 3

[0088] Effects of Atractylodes macrocephala polysaccharide on renal inflammation in DKD mice

[0089] Hyperglycemia-induced kidney damage is closely related to the release of various inflammatory factors. In a hyperglycemic state, kidney cells are damaged, leading to the initiation of an inflammatory response. This process involves the release of various inflammatory cytokines; excessive release of these inflammatory factors can trigger an inflammatory response, leading to pathological changes in kidney tissue, including glomerular sclerosis, interstitial fibrosis, and tubular damage. With the progression of kidney disease (DKD), kidney fibrosis often occurs, leading to irreversible loss of kidney function and accelerating the progression of kidney failure. Collagen fibers are widely distributed in the body and primarily reflect the degree of damage and fibrosis in the kidneys. The intestinal mucosa performs its crucial protective function; hyperglycemia-induced kidney damage may allow harmful bacteria and toxins to enter the bloodstream through the intestines, increasing the risk of enterogenic infections.

[0090] Mice with different drug treatments obtained in Example 2 were used in the experiment. After 8 weeks of drug administration, the mice were anesthetized and sacrificed, and samples were collected. The mRNA levels of inflammatory factors Ccl2, Il-1β, and Nlrp3, as well as fibrosis-related indicators fibronectin (Fn1), collagen I (Col1a1), and transforming growth factor β1 (Tgfb1) in the kidneys were detected by q-PCR.

[0091] Establishment of FMT mouse model: In the fecal microbiota transplantation experiment, healthy male C57BL / 6 mice were acclimatized and divided into a fecal microbiota transplantation normal control group (CON-FMT) and a model group. The DKD mouse model was established according to the same method as in Example 2 above. After successful modeling, the model group mice were randomly divided into the fecal microbiota transplantation model group (DKD-FMT) (n=8), the fecal microbiota transplantation positive control group (LOS-FMT, 30mg / kg / d) (n=8), the fecal microbiota transplantation Atractylodes macrocephala polysaccharide AMPs low-dose treatment group (AMPs-L-FMT, 100mg / kg / d) (n=8), the fecal microbiota transplantation Atractylodes macrocephala polysaccharide AMPs high-dose treatment group (AMPs-H-FMT, 300mg / kg / d) (n=8), and the fecal microbiota transplantation Atractylodes macrocephala polysaccharide AMPs-CM treatment model group (AMPs-CM-FMT, 300mg / kg / d) (n=8). Before the experiment, all mice were pretreated by gavage for one week with a mixture of antibiotics including ampicillin (10 g / L), metronidazole (8 g / L), neomycin sulfate (10 g / L), and vancomycin (5 g / L). Approximately 200 mg of fresh feces from donor mice was mixed with physiological saline and centrifuged for 3 minutes to separate insoluble substances. The supernatant was then administered orally once daily. After 8 weeks, urine was collected from the mice, and they were anesthetized and euthanized. Blood, kidney, and intestinal tissue samples were then collected and stored at -80°C. The mRNA expression of colonic tight junction molecules Zo-1, E-cadherin, Ocln, Cldn2, and Cldn4 was detected.

[0092] The specifics of quantitative real-time PCR (q-PCR) are as follows:

[0093] Trizol method for extracting total RNA from tissues:

[0094] ① Cut approximately 20 mg of thawed kidney or colon tissue into 1.5 mL EP tubes. Place two grinding steel balls into each EP tube and grind thoroughly. Then add 1 mL of Trizol and homogenize in a tissue homogenizer (60 Hz, 2 min × 3 times). The homogenizing module should be pre-cooled at -20°C before use. After homogenization, let the homogenate stand at room temperature for 10 min.

[0095] ② Add 200 μL of chloroform to each EP tube, shake vigorously to mix the solution thoroughly, and place it on the fume hood table to stand for 10 minutes.

[0096] ③ Set the centrifuge to 4 degrees Celsius and 12,000 rpm. After the centrifuge has pre-cooled to 4 degrees Celsius, place the EP tube inside and set the timer for 15 minutes. Tighten the cap and start centrifuging. After the centrifuge finishes and the speed indicator shows 0, open the cap and carefully remove the EP tube. The upper layer of the EP tube will be a clear, colorless solution containing the total RNA required for the experiment. Use a pipette to pipette 400 μL of this clear, colorless solution. Take another set of RNase-free EP tubes, label the caps with the sample information, and use them to hold the solution you just pipetted. Be extremely careful in this step to avoid taking any solution from other impurity layers in the tube.

[0097] ④ Accurately add 400 μL of isopropanol to the EP tube using a pipette, gently invert it to mix thoroughly, and repeat the inversion 30 times. After inversion, place it on the lab bench to allow precipitation to occur, which takes approximately 10 minutes. Then, set the centrifuge to 12000 rpm at 4°C for 10 minutes. Once the centrifuge has finished centrifuging and the speed display shows 0, open the cap and carefully remove the EP tube.

[0098] ⑤ At this point, a white precipitate will be observed in the EP tube. This is the precipitated RNA. Discard the supernatant and add 1 mL of pre-chilled 75% ethanol (pre-cooled on ice) to the EP tube to thoroughly rinse the precipitate, repeating 3-5 times. After thorough washing, centrifuge at 4°C and 12000 rpm for 15 minutes. Once the centrifuge has finished centrifuging and the speed indicator shows 0, open the cap, carefully remove the EP tube, and carefully and slowly aspirate the supernatant using a pipette. Avoid aspirating the precipitate, as this will cause RNA loss.

[0099] ⑥ At this point, the tube contains only RNA precipitate moistened with 75% ethanol. To reduce ethanol impurities in the RNA, open the EP tube cap and place it in a fume hood to allow the ethanol to evaporate. When the RNA precipitate turns a translucent pale white, it indicates that the precipitate is semi-dry. Add 25 μL of nuclease-free water to each EP tube. This step is to dissolve the RNA. To ensure complete dissolution, place the EP tube in a 65°C oven and let it stand for 10 minutes. Once dissolution is complete, this is the RNA stock solution.

[0100] ⑦ To determine the quality of RNA, its concentration and purity were measured. If the stock solution concentration was too high, DEPC water was added to dilute the RNA solution to 500 ng / μL. The A260 / A280 ratio of the RNA solution was measured; a ratio between 1.8 and 2.2 indicated acceptable purity. RNA that did not meet the purity standard was not used in subsequent experiments.

[0101] Reverse transcription reaction: A portion of the sample was subjected to agarose gel electrophoresis to assess RNA integrity. Intact RNA bands included 28S, 18S, and 5S ribosomal RNA. 28S / 18S > 2 indicates high RNA integrity. A DNA-free reaction system was prepared (DNA Resizer Buffer: gDNA Eraser = 2:1), diluted to 1 μg per μL based on the total RNA concentration of each sample, with a total volume of 10 μL. The reaction was carried out at room temperature for 15 minutes. The next step was to prepare the reverse transcription RNA system, with the system ratios shown in Table 5.

[0102] Table 5. Reverse Transcription RNA System

[0103]

[0104] Mix well on ice, add to the DNA removal system, and mix thoroughly. Finally, perform amplification cycles (conditions: 37℃ for 15 min, 85℃ for 5 s) to obtain cDNA, and store at 4℃.

[0105] q-PCR and data processing: Prepare the PCR system according to Table 6:

[0106] Table 6 PCR System

[0107]

[0108] After thorough mixing, add the samples to a 96-well plate in the order specified. Perform analysis. After amplification cycles, analyze the data (using gapdh or β-actin as internal control genes for relative quantification).

[0109] The mRNA levels of inflammatory factors (pro-inflammatory cytokines Ccl2, Il-1β, and inflammasome Nlrp3) in each treatment group are as follows: Figure 4 As shown, Figure 4 The image shows the mRNA levels of inflammatory factors Ccl2, Il-1β, and Nlrp3 (Relative mRNA represents the fold change in expression of the corresponding related intestinal tight junction protein mRNA). The results indicate that Ccl2, Il-1β, and Nlrp3 were significantly upregulated in DKD mice and significantly reduced after AMPs administration. The therapeutic effect of AMPs-CM was lower than that of AMPs.

[0110] mRNA levels of fibrosis-related markers in each treatment group are as follows: Figure 5 As shown, Figure 5This image shows the mRNA levels of fibrosis-related markers fibrosis-related proteins fibronectin (Fn1), collagen I (Col1a1), and transforming growth factor β1 (Tgfb1). The results indicate that the mRNA expression levels of Fn1, Col1a1, and Tgfb1 (TGFB1) were significantly increased in the kidney tissue of DKD mice. Treatment with AMPs, AMPs-CM, and LOS significantly reduced the expression levels of Fn1, Colage-1, and TGFB1, with LOS showing the best efficacy, while AMPs were significantly more effective than AMPs-CM. Increased levels of Fn1, Colage-1, and TGFB1 are key markers of renal interstitial fibrosis. This suggests that AMPs effectively intervene in DKD by inhibiting the expression of Fn1, Colage-1, and TGFB1, thereby alleviating renal interstitial fibrosis and improving the progression of DKD.

[0111] The mRNA levels of colonic tight junction molecules Zo-1, E-cadherin, Ocln, Cldn2 (CLDN-2), and Cldn4 (CLDN-4) in each treatment group are as follows: Figure 6 As shown, Figure 6 This image shows the mRNA levels of colonic tight junction molecules Zo-1, E-cadherin, Ocln, Cldn2, and Cldn4. The results indicate that the mRNA expression levels of Zo-1, E-cadherin, Ocln, Cldn2, and Cldn4 were significantly decreased in the kidney tissue of DKD-FMT mice. Treatment with AMPs, AMPs-CM, and LOS all increased the expression levels of Zo-1 and Occludin. These results suggest that AMPs and AMPs-CM can repair damage to the intestinal mucosal barrier and restore the expression levels of tight junction proteins in DKD mice, with AMPs showing significantly higher efficacy than AMPs-CM.

[0112] Example 4

[0113] Effects of AMPs on renal function (24h-urine protein, urine protein / creatinine ratio, and serum creatinine level) in DKD mice

[0114] Mice subjected to different drug treatments as described in Example 2 were used in the experiment. After each mouse was placed in a metabolic cage for 24 hours, urine samples were collected from the cages, centrifuged at 3000 rpm for 5 min, and after removing the sediment, stored at -80°C for later use. Serum creatinine and urine protein were measured in mice according to the manufacturer's instructions using a creatinine assay kit and a urine protein quantification test kit. The results are as follows: Figure 7 As shown, Figure 7The graph shows the effects of AMPs on biochemical parameters in DKD mice. In the graph, A represents the urinary protein level (Albumiuria) of each group of mice, B represents the urinary protein / creatinine ratio (Urine Albumin / Creatinine Ratio) of each group of mice, and C represents the serum creatinine content (Serum creatinine) of each group of mice.

[0115] 24-hour urine protein, urine protein / creatinine (UACR) ratio, and serum creatinine level (Scr) are important indicators commonly used in clinical practice to assess kidney function. Abnormally elevated levels of these indicators reflect the degree of damage to glomerular filtration function. Figure 7 The results showed that, compared with the CON group, the 24-hour urinary protein level in the DKD group was significantly increased, while administration of AMPs (high and low doses) and LOS significantly reduced the abnormal increase in 24-hour urinary protein. Similarly, administration of AMPs and LOS reduced serum creatinine in DKD mice, and renal function was improved. These results indicate that the renal protective effect of AMPs on DKD mice can improve renal function indicators (24-hour urinary protein, urinary protein / creatinine ratio, and serum creatinine level) in DKD mice.

[0116] Example 5

[0117] Effects of AMPs on blood glucose in DKD mice

[0118] (1) Mice treated with different administration methods as described in Example 2 were used in the experiment. Mouse body weight was recorded weekly, and random blood glucose levels were recorded every two weeks. The effects of Atractylodes macrocephala polysaccharide on body weight and blood glucose levels in DKD mice were recorded after 8 weeks. The results are as follows: Figure 8 As shown, Figure 8 The figure shows the effect of Atractylodes macrocephala polysaccharide on body weight and blood glucose in DKD mice. In the figure, A represents the change in body weight of mice over 8 weeks, and B represents the fasting blood glucose of mice in week 8.

[0119] The results showed that the DKD group experienced a significant decrease in body weight compared to the CON group, while there were no significant differences among the MET, AMPs-L, and AMPs-H groups. Figure 8 Figure B shows the blood glucose curve. The results indicate that compared with the normal control group, the blood glucose level of mice in the model control group was significantly increased, while the blood glucose level of mice in the AMPs group and MET group was significantly decreased compared with the model control group. Therefore, it can be concluded that AMPs can significantly reduce fasting blood glucose in diabetic nephropathy mice.

[0120] (2) Mice with different drug treatments obtained in Example 2 were subjected to an oral glucose tolerance test (OGTT). The effect of AMPs on glucose tolerance in DKD mice was detected 30 minutes after gavage administration of glucose solution. The results are as follows: Figure 9 As shown, Figure 9 The graph shows the effect of AMPs on glucose tolerance in DKD mice. In the graph, A is a line graph of oral glucose tolerance test (Blood glucose) for each group of mice, and B is the area under the curve (AUC) of OGTT.

[0121] like Figure 9 The results showed that blood glucose levels in all mice peaked 30 minutes after gavage administration of glucose solution, and then gradually decreased. Compared with the normal control group, the AUC value of the oral glucose tolerance test (OGTT) in the model control group mice was significantly increased, indicating a significant decrease in their glucose tolerance. AMP treatment improved oral glucose tolerance in DKD mice and reduced the AUC value of the OGTT.

[0122] Example 6

[0123] Effects of AMPs on Kidney Injury in DKD Mice

[0124] The mice with different drug treatments obtained in Example 2 were used in the experiment.

[0125] (1) Sample collection

[0126] Eight weeks after drug administration, mice were anesthetized and euthanized. Blood was collected, allowed to stand for 30 minutes, centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected and stored at -80°C. Mice were euthanized by cervical dislocation after blood collection. The internal organs were exposed, and the gastrointestinal tract was gently separated with a cotton swab, revealing both kidneys. The kidneys were carefully cut off, placed in pre-chilled PBS to remove surface blood and hair, and the renal capsule was gently peeled off with forceps, avoiding damage to the renal cortex. After removing the renal capsule, the kidneys were removed, excess water was absorbed on filter paper, and both kidneys were weighed, arranged, and photographed to record their morphology. Half of the right kidney tissue was placed in an embedding cassette and immersed in tissue fixative for paraffin section preparation; the remaining kidney tissue was placed in EP tubes, quickly frozen in liquid nitrogen, and then transferred to a -80°C freezer for subsequent assays.

[0127] (2) Preparation of tissue sections

[0128] ① Tissue fixation: After soaking in fixative for 24 hours, remove the embedding cassette and rinse with running water for 30 minutes to remove excess tissue fixative.

[0129] ②Tissue dehydration: Add the appropriate amount of dehydration reagent to the automatic dehydrator, set the dehydration program, place the tissue sample, and start dehydration.

[0130] ③ Tissue Embedding: After dehydration, remove the tissue. Turn on the paraffin embedding machine and freezing stage beforehand. Once the paraffin in the embedding machine has melted, use forceps to place the kidney tissue into the embedding mold. Press the wax dispensing button on the embedding machine; the molten wax will flow out, covering the tissue in the mold. Transfer the mold to the freezing stage to cool and solidify. After the wax block has completely solidified, remove the embedding mold from the tissue wax block. During embedding, keep the tissue centered and avoid air bubbles. Store the embedded wax block in a dry environment at room temperature.

[0131] ④ Sectioning: Before sectioning, the paraffin block was pre-frozen on a freezing stage to increase its hardness. The section thickness was 10 μm. When a paraffin section containing tissue was cut and the cut surface was smooth, the section thickness was adjusted to 4 μm. The cut section was then picked up with forceps and placed in a slide flattening machine. After the tissue sections were flattened, they were picked up with a glass slide, labeled with the corresponding number, placed in a slide box, and dried in an oven at 37 degrees Celsius. After drying, the paraffin sections were stored at room temperature in a dry environment for pathological examination. The appearance and pathological manifestations of the kidneys of DKD mice were observed.

[0132] (3) After pathological examination, kidney H&E staining was performed using the hematoxylin-eosin (H&E) method.

[0133] Place the paraffin sections into a slide rack and put them into a 65℃ oven for 1 hour.

[0134] Dewaxing of sections: Xylene I 20 min → Xylene II 20 min → Anhydrous ethanol I 15 min → Anhydrous ethanol II 15 min → 95% ethanol 10 min → 90% ethanol 5 min → 80% ethanol 5 min;

[0135] Soak in tap water for 5 minutes;

[0136] Immerse in hematoxylin staining solution for 6 minutes, then rinse with running water for 10 minutes;

[0137] Differentiate with 1% hydrochloric acid and ethanol, rinse with running water for 15 minutes to restore blue color;

[0138] Immerse in eosin dye for 1 minute, then rinse with tap water.

[0139] Dehydration: 75% ethanol 2s → 85% ethanol 2s → 95% ethanol 2s → anhydrous ethanol 2s → xylene 2s → xylene 2s;

[0140] Air dry, then seal with neutral resin.

[0141] The appearance and pathological findings of the kidneys in DKD mice are as follows: Figure 10 As shown, Figure 10The images show the morphology of mouse kidneys, where A is a representative image of the kidneys of each group, B is the kidney weight / body weight ratio (Kidney index), and C is the kidney H&E staining.

[0142] Figure 10 The results showed that the kidneys of DKD mice exhibited significant swelling and weight increase, with a marked increase in the kidney index (kidney weight / body weight * 100%), while administration of AMPs and LOS reversed this trend. Figure 10 (AB). H&E staining of kidney sections revealed lesions in the glomeruli and tubulointerstitium of DKD mice. H&E staining results showed that DKD mice exhibited pathological features including glomerular swelling and deformation, tubular edema and vacuolar changes, accompanied by extensive inflammatory cell infiltration. Compared with the DKD group, AMPs and LOS administration significantly improved renal pathological features and significantly reduced inflammatory cell infiltration. Figure 10 The results (C) indicate that AMPs can alleviate kidney damage in DKD.

[0143] Example 7

[0144] Effects of AMPs on Kidney Inflammation in DKD Mice

[0145] To investigate whether AMPs inhibit renal tubular interstitial inflammation in DKD mice, mice with different drug treatments obtained in Example 2 were used in the experiment, and the expression of CD68 (macrophage marker protein) in the proximal tubules of the kidneys of diabetic mice was detected.

[0146] 1. Immunohistochemistry staining:

[0147] The paraffin slices obtained in Example 5 were placed in a glass slide holder and placed in a 65°C oven for 1 hour.

[0148] Dewaxing of sections: Xylene I 20 min → Xylene II 20 min → Anhydrous ethanol I 15 min → Anhydrous ethanol II 15 min → 95% ethanol 10 min → 90% ethanol 5 min → 80% ethanol 5 min;

[0149] Rinse with tap water for 5 minutes;

[0150] High temperature and high pressure repair: Prepare sodium citrate buffer solution, add water to the pressure cooker and bring to a boil, pour sodium citrate solution into a beaker, put in the tissue section, seal with aluminum foil, cover the pot and cook until steam is released, then time for 15 minutes.

[0151] After releasing the gas, remove the beaker and allow it to cool naturally to room temperature.

[0152] Soak in distilled water for 2 minutes, let stand at room temperature for 10 minutes with 3% hydrogen peroxide, and wash for 5 minutes 3 times with PBS buffer (pH=7.2-7.4).

[0153] Incubation with primary antibody: First, blot dry with filter paper, block with 1% BSA serum for 1 hour, and place at room temperature. During this period, prepare primary antibody working solution (FN / CD68:1% BSA = 1:500), shake off the blocking solution, wash with PBS for 5 minutes × 3 times, add primary antibody dilution solution to completely cover the tissue surface, and incubate overnight at 4°C.

[0154] The next day, warm to room temperature for 30 minutes, then wash with PBS 3 times for 5 minutes each time.

[0155] Secondary antibody incubation: Prepare secondary antibody dilution solution with 1% BSA in advance, incubate at room temperature for 1 hour, then remove the working solution of secondary antibody from the slide, and wash 3 times for 5 minutes with PBS buffer (pH=7.2-7.4);

[0156] Color development: Transfer to a dark environment and develop the color with DAB colorimetric reagent. Pay attention to controlling the color development time to avoid overdevelopment and false positive areas.

[0157] Stain with hematoxylin for 30 seconds, then run in water for 5 minutes; differentiate with 1% hydrochloric acid ethanol differentiation solution, then rinse with water for 15 minutes.

[0158] Dehydrate, air dry, and seal with neutral resin.

[0159] The results are as follows Figure 11 As shown, Figure 11 The figure shows the effect of AMPs on kidney inflammation in DKD mice. In the figure, A is the CD68 immunohistochemical staining of mouse kidneys; B is the statistical analysis of the positive area of ​​CD68.

[0160] The results showed that AMP administration significantly reduced CD68 expression in the kidneys of DKD mice.

[0161] 2. Western Blot Assay

[0162] Sample preparation: Using tweezers, place two grinding beads into an EP tube. Carefully cut approximately 20 mg of collected mouse kidney / colon tissue from an ultra-low temperature freezer and place it into the corresponding EP tube. Add 500 μL of lysis buffer (RIPA:PMSF = 100:1). Homogenize using a tissue homogenizer (60 Hz, 2 min × 3 times). The homogenizer module should be pre-cooled at -20°C before use. After homogenization, allow the homogenate to stand at room temperature for 20 minutes. Centrifuge at 4°C, 12000 rpm for 25 minutes. Transfer the supernatant to a new EP tube as the tissue protein stock solution. Quantitative analysis using BCA: Prepare a working solution (A:B = 50:1). Add the protein stock solution and BCA working solution sequentially. Incubate at 35°C for 30 minutes, then measure the absorbance using a microplate reader. The concentration of the stock protein was calculated using a linear equation and balanced. The solution was then transferred to a new EP tube, and 10% bromophenol blue loading buffer (supernatant:loading buffer = 4:1) was added and mixed thoroughly. Finally, the tubes were placed in a 95°C metal water bath for denaturation for 10 minutes to obtain the working protein solution, which was then stored at -80°C.

[0163] Sample pretreatment: Before the experiment, take the above samples out of the -80℃ freezer and put them into a 95℃ metal bath water bath for 3 minutes to reheat, then vortex them briefly.

[0164] Gel preparation: Mount the long and short glass plates onto the dispensing rack, ensuring a tight fit between the two plates to prevent leakage during dispensing. According to the required gel concentration, mix polyacrylamide, ultrapure water, SDS, 10% APS, and TEMED in the desired proportions, and operate on ice to prepare the separating gel. Pour this gel between the two plates using a 5mL pipette. Then, quickly press anhydrous ethanol onto the gel surface using a 200μL pipette. Let it stand at room temperature for 30 minutes to solidify, then discard the anhydrous ethanol. Prepare a stacking gel according to the same proportions (using the same reagents) and fill the top layer of the separating gel. Insert a clean comb to avoid air bubbles.

[0165] Electrophoresis: Fix the prepared electrophoresis gel into the electrophoresis cartridge, carefully place it into the electrophoresis box, and pour 1× electrophoresis buffer to the mark. Remove the comb from each gel. First, add 5 μL of marker to the first lane, then slowly add 10 μL of protein working solution to the subsequent lanes. After loading the samples, start electrophoresis at a constant voltage of 80 volts for 30 minutes. When all lanes are level, adjust the voltage to 120 volts and continue electrophoresis until near the bottom. Electrophoresis is then complete.

[0166] Transfer: Pre-cut PVDF membranes to the size of the electrophoresis gel, mark the upper left corner of each membrane with a pencil, and pre-activate them by soaking in anhydrous methanol for 15 minutes. Pour pre-cooled 1× transfer buffer into the transfer tray, and prepare the transfer clamp and the necessary clean filter paper. Remove the electrophoresis gel after electrophoresis, remove the top layer of stacking gel and excess gel after removing bromophenol blue, transfer it to the transfer tray for rinsing, then clamp the PVDF membrane and gel tightly together, and cut out a piece of gel the same size as the membrane. Transfer the tightly fitted membrane and gel to the transfer clamp, with three layers of clean filter paper and a sponge pad on each side, close the clamp, and place it in the transfer tank. Add the pre-cooled 1× transfer buffer. To maintain a low temperature during the transfer process, add ice packs to the transfer box, place it in a 4°C freezer, use a constant current of 300 mA, and transfer for 90 minutes.

[0167] Blocking: After the transfer is complete, use tweezers to remove the membrane and place it in the washing box. Rinse three times with 1×TBST for 5 minutes each time. During this time, weigh 5g of skim milk powder, dissolve it in 1×TBST as a solvent, and bring the volume to 50mL to prepare the blocking milk solution. Pour the blocking milk solution into the membrane to completely cover it and incubate at room temperature for 1 hour.

[0168] Primary antibody incubation: After blocking, discard the blocking milk solution, remove the membrane with forceps, place it in a clean membrane washing box, add an appropriate amount of 1×TBST, and rinse 3 times for 5 minutes each time. During this period, prepare the primary antibody incubation solution by adding primary antibody dilution buffer (protein antibody = 1:1000) to a 50mL centrifuge tube. Use forceps to firmly place the membrane in the 50mL centrifuge tube, ensuring the protein side is in contact with the primary antibody incubation solution. Incubate overnight at 4°C by rotation.

[0169] Secondary antibody incubation: The next day, remove the membrane with tweezers and place it in a clean membrane washing box. Pour in an appropriate amount of 1×TBST and rinse three times for 5 minutes each time. During this period, prepare a secondary antibody incubation solution using a 50 ml centrifuge tube by adding 5% skim milk powder to the secondary antibody at a ratio of 1:5000. Use tweezers to firmly place the membrane in the centrifuge tube, ensuring that the protein-containing side is inward and in contact with the secondary antibody incubation solution. Incubate at room temperature by rotating for 1 hour.

[0170] Chemiluminescence development: Prepare and mix the chemiluminescence solution. First, immerse the membrane in an opaque box for 1 minute to develop the color. Then, place it in a chemiluminescence analyzer for exposure, photographing and saving.

[0171] The mRNA expression levels of pro-inflammatory cytokines Ccl2, Il-1β, and the inflammasome Nlrp3 in the kidney were detected, and the results are as follows: Figure 12 As shown, Figure 12The image shows the expression of MCP-1 and NLRP3 in the kidneys of DKD mice as detected by Western Blot (GAPDH is the internal control, and protein expression indicates the fold change in the expression of the corresponding protein).

[0172] The protein expression levels of pro-inflammatory cytokines MCP-1 and inflammasome NLRP3 were detected using Western blot technology. Figure 12 MCP-1 and Nlrp3 were significantly upregulated in DKD mice and significantly reduced after AMP administration. These results indicate that AMPs can alleviate inflammation in the kidneys of DKD mice.

[0173] Example 8

[0174] Effects of AMPs on renal fibrosis in DKD mice

[0175] As kidney disease (DKD) progresses, renal fibrosis often occurs, leading to irreversible loss of kidney function and accelerating the progression of renal failure. Collagen fibers are widely distributed in the body and primarily reflect the degree of damage and fibrosis in the kidneys. To clarify the effect of AMPs on renal fibrosis in DKD mice, the following experiments were conducted.

[0176] 1. Masson staining was performed on mouse kidney tissue sections (obtained in Example 6), and the positive area was statistically analyzed using Image J.

[0177] Masson staining:

[0178] Place the paraffin sections into a slide rack and put them into a 65℃ oven for 1 hour.

[0179] Dewaxing of sections: Xylene I 20 min → Xylene II 20 min → Anhydrous ethanol I 15 min → Anhydrous ethanol II 15 min → 95% ethanol 10 min → 90% ethanol 5 min → 80% ethanol 5 min;

[0180] Soak in tap water for 5 minutes;

[0181] Stain with the prepared Weigert iron hematoxylin for 5 minutes, then rinse with running water for 10 minutes.

[0182] Differentiate with 1% hydrochloric acid and ethanol, then rinse with running water for 10 minutes;

[0183] Restore blue color using Masson's blueing solution;

[0184] Stain with Ponceau S acid fuchsin solution for 5 minutes, then rinse briefly with distilled water;

[0185] Stain with phosphomolybdic acid solution for 2 min, then treat with prepared weak acid working solution (distilled water: glacial acetic acid solution = 2:1) for 1 min;

[0186] Pour off the supernatant, do not wash the section with water, directly add aniline blue staining solution and stain for 5 minutes;

[0187] Treat with weak acid solution for 1 min;

[0188] Dehydration: 75% ethanol 2s → 85% ethanol 2s → 95% ethanol 2s → anhydrous ethanol 2s → xylene 2s → xylene 2s;

[0189] Air dry, then seal with neutral resin.

[0190] Masson staining results of mouse kidneys as follows Figure 13 As shown, Figure 13 The figure shows the effect of AMPs on renal fibrosis in DKD mice. A represents Masson staining of mouse kidneys, and B represents the statistical analysis of the positive area of ​​Masson staining (Fibrotic area represents the fibrotic area).

[0191] The results showed that, compared with the CON group, the DKD group had significant collagen deposition, with a marked increase in collagen fibers in the interglomerular and tubular spaces, and glomerular sclerosis. Compared with the DKD group, the collagen fibers in the interglomerular and tubular spaces of each treatment group were significantly reduced, indicating that AMPs can improve the fibrosis level in the kidneys of DKD mice.

[0192] 2. Fibronectin plays a key role in renal tubulointerstitial fibrosis by promoting extracellular matrix remodeling, enhancing cell adhesion and migration, activating fibroblasts, and regulating inflammatory responses, leading to the formation and accumulation of fibrous tissue. Immunohistochemical staining (the method used in Example 7) was used to show the expression of Fibronectin protein, and the positive area was statistically analyzed using Image J.

[0193] Immunohistochemical staining results as follows Figure 14 As shown, Figure 14 The figure shows the effect of AMPs on renal tubulointerstitial fibrosis in DKD mice. In the figure, A is the CD68 immunohistochemical staining of mouse kidneys, and B is the statistical analysis of the positive area of ​​FN.

[0194] The results showed that the expression level of Fibronectin protein in the DKD group was significantly higher than that in the control group, and the expression level in each treatment group was significantly reduced, indicating that AMPs can improve the fibrosis level of the kidneys in DKD mice.

[0195] 3. Fibrosis-related genes in the kidneys were detected by q-PCR and Western Blot (the method used in Example 7).

[0196] The results are as follows Figure 15 As shown, Figure 15 The image shows the expression of FN, Collage-1 and TGF-β1 in the kidneys of DKD mice detected by Western blotting, with GAPDH as an internal standard for statistical analysis.

[0197] Western blot analysis showed that ( Figure 15 The protein expression of FN, Collage-1, and TGF-β1 was also significantly increased. After treatment with AMPs and LOS, the expression levels of FN, Collage-1, and TGF-β1 all decreased. Increased FN, Collage-1, and TGF-β1 are key markers of renal interstitial fibrosis. AMPs effectively intervened in DKD by inhibiting the expression of FN, Collage-1, and TGF-β1, thereby alleviating renal interstitial fibrosis and improving the progression of DKD.

[0198] The results showed that treatment with high doses of AMPs (300 mg / kg and 100 mg / kg) and low doses of AMPs in diabetic nephropathy mice significantly repaired the pathological features of kidney tissue, reduced kidney fibrosis, and decreased the expression of CD68, a macrophage marker in the kidney.

[0199] Example 9

[0200] Effects of AMPs on intestinal mucosal barrier damage in DKD mice

[0201] FMT mouse models were established according to the method in Example 3, resulting in the CON-FMT, DKD-FMT, and AMPs-FMT (i.e., AMPs-H-FMT) mouse models. Gene expression analysis was performed after 8 weeks.

[0202] To further evaluate the impact of AMPs on intestinal integrity in DKD mice, H&E-stained tissue samples were analyzed. Zonula Occludens 1 (ZO-1) is a key component in the formation of tight junctions. Decreased expression or activity of ZO-1 interferes with the formation of tight junctions, hindering the intestinal mucosa from fulfilling its crucial protective function, thereby increasing the risk of harmful bacteria and toxins entering the bloodstream and causing enterogenic infections. Therefore, immunohistochemical staining was used to visualize ZO-1 protein expression, and the positive area was statistically analyzed using ImageJ.

[0203] Immunofluorescence staining:

[0204] Place the paraffin sections into a slide rack and put them into a 65℃ oven for 1 hour.

[0205] Dewaxing of sections: Xylene I 20 min → Xylene II 20 min → Anhydrous ethanol I 15 min → Anhydrous ethanol II 15 min → 95% ethanol 10 min → 90% ethanol 5 min → 80% ethanol 5 min;

[0206] Rinse with tap water for 5 minutes;

[0207] High temperature and high pressure repair: Prepare sodium citrate buffer solution, add water to the pressure cooker and bring to a boil, pour sodium citrate solution into a beaker, put in the tissue section, seal with aluminum foil, cover the pot and cook until steam is released, then time for 15 minutes.

[0208] After releasing the gas, remove the beaker and allow it to cool naturally to room temperature.

[0209] Soak in distilled water for 2 minutes, let stand at room temperature for 10 minutes with 3% hydrogen peroxide, and wash for 5 minutes 3 times with PBS buffer (pH=7.2-7.4).

[0210] Primary antibody incubation: First, blot dry with filter paper, block with 1% BSA serum for 1 hour, and incubate at room temperature. During this time, prepare the primary antibody working solution (FN / CD68:1% BSA = 1:500). Shake off the blocking solution, wash with PBS for 5 minutes × 3 times. Add the primary antibody dilution solution to completely cover the tissue surface, and incubate overnight at 4°C.

[0211] The next day, warm the product for 30 minutes and wash it three times with PBS for 5 minutes each time.

[0212] Secondary antibody incubation: Prepare secondary antibody dilution solution with 1% BSA in advance and incubate at room temperature for 1 hour. After removing the working solution of secondary antibody from the slide, wash 3 times for 5 minutes with PBS buffer (pH=7.2-7.4).

[0213] Transfer to a dark place and counterstain with DAPI for 10 minutes. Wash with PBS for 5 minutes each time, 3 times. Mount the slide and store at -20°C.

[0214] H&E-stained tissue samples and immunofluorescence staining results are as follows: Figure 16 As shown, Figure 16 The diagram shows the effect of AMPs on the intestinal barrier in DKD mice. A represents H&E staining of colonic tissue, B represents ZO-1 (red) immunofluorescence staining of colon, and C represents the statistical analysis of the positive area of ​​ZO-1.

[0215] In H&E stained tissue samples ( Figure 16In group A), the CON group showed normal colonic structure, while in the DKD group, the epithelial cell layer of the colon was destroyed, and the crypts and goblet cells were severely swollen and deformed. This phenomenon was significantly reversed after 8 weeks of gavage administration of AMPs and LOS. Immunofluorescence staining results showed ( Figure 16 In the DKD group (in BC), the expression of ZO-1 in the colon was inhibited, while the expression of ZO-1 increased after AMP treatment.

[0216] Intestinal tight junction proteins are a group of proteins that form tight junctions between intestinal epithelial cells. They are essential for maintaining intestinal barrier function and regulating intestinal permeability. These proteins prevent harmful substances and pathogens from entering the bloodstream by controlling the opening and closing of intercellular spaces, while allowing the absorption of nutrients. Genes related to tight junction factors in the colon were detected by q-PCR and Western blotting.

[0217] The results are as follows Figure 17 As shown, Figure 17 The expression levels of colonic tight junction molecules (ZO-1 and Occludin) are plotted, with GAPDH as an internal standard for statistical analysis.

[0218] Western Blot analysis Figure 17 The results showed that the protein expression of ZO-1 and Occludin was significantly reduced. After treatment with AMPs and LOS, the expression levels of both ZO-1 and Occludin increased. AMPs were able to repair damage to the intestinal mucosal barrier in DKD mice and restore the expression levels of tight junction proteins.

[0219] In summary, this invention established a diabetic nephropathy model using intraperitoneal injection of STZ combined with a high-fat diet, and investigated the pharmacological effects of AMPs on diabetic nephropathy. The study showed that AMPs significantly improved fasting blood glucose and glucose tolerance in STZ-induced diabetic mice, indicating that AMPs have a certain hypoglycemic effect and are expected to be used as a hypoglycemic drug for treating diabetes. Regarding the therapeutic effect on diabetic nephropathy, the study showed that after AMP treatment, various renal injury indicators in STZ-induced diabetic mice, such as kidney-related physiological and biochemical indicators (kidney-to-body weight ratio, 24-hour urinary protein, serum creatinine), renal hypertrophy, renal fibrosis, and inflammatory response, were significantly improved, indicating that AMPs have a good therapeutic effect on diabetic nephropathy and can improve and delay the progression of kidney damage, fibrosis, and inflammation in diabetic nephropathy. Crucially, AMP treatment can also repair the colonic mucosal barrier function and restore the expression of intestinal epithelial marker proteins, providing a new treatment strategy for diabetic nephropathy and / or diabetic intestinal diseases by regulating the gut microbiota.

[0220] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of Atractylodes macrocephala polysaccharide in the preparation of drugs for treating diabetes-related diseases, characterized in that, The Atractylodes macrocephala polysaccharide is used to prepare a drug for treating a mixed disease of diabetic nephropathy and diabetic intestinal disease. The diabetic nephropathy is characterized by renal function impairment, inflammation, and renal fibrosis. The inflammatory factors in the inflammation are Ccl2, Il-1β, and Nlrp3. The indicators of renal fibrosis are fibronectin, collagen I, and transforming growth factor β1. The diabetic intestinal disease described herein is intestinal mucosal damage, which is defined as: suppressed expression of tight junction proteins in the intestinal mucosa or intestinal flora dysbiosis. The tight junction proteins include... Zo-1 , E-cadherin , Ocln , Cldn2 and Cldn4 One or more of the following; The Atractylodes macrocephala polysaccharide was prepared by a water extraction and alcohol precipitation method, specifically as follows: Step 1): Extract Atractylodes macrocephala and ultrapure water at a ratio of 1g:20mL at 90℃ for 2 hours, centrifuge at 4000rpm for 20 minutes, and collect the supernatant and residue. Step 2): Following the method in Step 1), the residue is extracted and centrifuged a second time. The supernatants from the two extractions are combined and concentrated to 1 / 3 of the original volume at 60°C to obtain a concentrated solution. Step 3): Add 3 times the volume of 95% ethanol aqueous solution to the concentrate, precipitate overnight at 4°C, centrifuge at 4000 rpm for 10 min, collect the precipitate, wash the precipitate twice with 95% ethanol aqueous solution, freeze dry to obtain Atractylodes macrocephala polysaccharide.

2. The application according to claim 1, characterized in that, The Atractylodes macrocephala polysaccharide is composed of 83.93% glucose, 7.19% arabinose, 5.47% galactose, 3.00% galacturonic acid and 0.41% glucuronic acid.

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