Preparation method for extracting compound polysaccharide from Erchen decoction and application of compound polysaccharide in improvement of obesity type 2 diabetes mellitus

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and discloses a preparation method for extracting compound polysaccharide from Erchen decoction, an application of the compound polysaccharide in improvement of obesity type 2 diabetes mellitus, and a pharmacological action and an application of the compound polysaccharide in improvement of obesity T2DM. According to the method, the Erchen decoction polysaccharide is extracted, and the monosaccharide composition, molecular weight, bonding structure, surface morphological structure, crystal form and crystallinity, molecular structure, characteristic functional group and absorption spectrum curve of the Erchen decoction polysaccharide are detected and analyzed after impurity removal, ion exchange purification and gel purification. Through structural characterization analysis, the sugar chain of the Erchen decoction polysaccharide is obtained. Besides, pharmacological experiments show that the Erchen decoction polysaccharide has relatively good activity, slight side effects and good druggability in the aspect of improving the obesity T2DM function, and can be used for preparing the medicine for improving the obesity T2DM function. The compound can be combined with acceptable carriers or excipients in the pharmaceutical field or the food industry to be prepared into various dosage forms such as granules, capsules and tablets.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a method for preparing compound polysaccharides extracted from Er Chen Tang and their application in improving obesity-related type 2 diabetes. The invention further relates to the pharmacological effects and applications of polysaccharides extracted from the classic formula Er Chen Tang, specifically to the structural characterization of Er Chen Tang polysaccharides and their use as active ingredients in improving obesity-related type 2 diabetes. Background Technology

[0002] Diabetes is one of the most serious public health problems in the 21st century. According to the International Diabetes Federation (IDF), the number of people with diabetes worldwide will rise to 643 million by 2030 and 783 million by 2045, of which more than 90% have type 2 diabetes mellitus (T2DM) (International Diabetes Federation. IDFDiabetes Atlas, 10th edn. Brussels, Belgium: International DiabetesFederation, 2021.). The Lancet points out that China currently has the largest number of overweight / obese adults in the world, reaching 402 million (Global, regional, and national prevalence of adult overweight and obesity, 1990-2021, with forecasts to 2050: a forecasting study for the Global Burden of Disease Study 2021 [J]. Lancet, 2025, 405(10481): 813-38.). Excessive fat accumulation can cause insulin resistance and β-cell dysfunction, leading to the occurrence of T2DM (Luo Jintong, Zhang Lijing, Zhan Libin, et al. Effect of Shenling Baizhu Powder on pancreatic β-cell dedifferentiation in obese type 2 diabetic ZDF rats [J]. Chinese Journal of Traditional Chinese Medicine, 2023, 38(12): 6034-6039.). Currently, about three-quarters of obese patients in my country suffer from type 2 diabetes mellitus (T2DM). With the accelerating aging of the population, the prevalence of T2DM combined with obesity is gradually increasing. Overweight / obese T2DM patients have lower rates of achieving target glycated hemoglobin levels and higher risks of stroke, coronary heart disease, and death (Zhang Fengjie, Cao Liang, Zhu Xiaoliang. Distribution of gut microbiota in obese patients with type 2 diabetes and its correlation with glucose and lipid metabolism and inflammatory markers [J]. Chinese Journal of Microecology, 2024, 36(02): 180-4+90.). Therefore, obese T2DM has become a pressing public health issue. A systematic and in-depth investigation into the pathogenesis of obese T2DM and the active exploration of practical and effective prevention and treatment strategies are of paramount scientific significance and urgent practical need.

[0003] The classic formula Er Chen Tang originates from the Song Dynasty's *Taiping Huimin Heji Jufang*. It is primarily used to treat phlegm-dampness syndrome caused by spleen deficiency leading to dampness accumulation (Zhao Tian, ​​Zhan Libin. Research progress on the mechanism of action of Er Chen Tang in metabolic diseases [J]. World Science and Technology - Modernization of Traditional Chinese Medicine, 2021, 23(04):998-1005). Er Chen Tang consists of six Chinese herbs: Pinellia ternata, Citrus reticulata peel, Poria cocos, Glycyrrhiza uralensis, Zingiber officinale, and Prunus mume. Pinellia ternata and Citrus reticulata peel are used in equal amounts, working synergistically to dry dampness, resolve phlegm, regulate qi, and relieve stagnation. Poria cocos acts as an adjuvant to strengthen the spleen and eliminate dampness. Zingiber officinale assists Pinellia ternata, while Citrus reticulata peel helps to reduce phlegm and counteract the toxicity of Pinellia ternata. A small amount of Prunus mume is used to astringe lung qi, and Glycyrrhiza uralensis is used to strengthen the spleen, harmonize the middle jiao, and moderate the imbalance of the other herbs in the formula. As a fundamental formula in traditional Chinese medicine for treating phlegm syndrome, Er Chen Tang has the effects of drying dampness, resolving phlegm, regulating qi, and harmonizing the stomach. Based on its definite efficacy and wide range of applications, this formula can be used to treat various types of phlegm syndrome and plays an important role in clinical practice (Yang Menghan, Li Yuanyuan, Zheng Xiujuan, et al. Effects of Er Chen Tang on serum leptin and hypothalamic LepR, POMC and NPY expression in mice with metabolic syndrome phlegm syndrome model [J]. Journal of Traditional Chinese Medicine, 2025, 66(09):948-954.DOI:10.13288 / j.11-2166 / r.2025.09.013.).

[0004] Erchen Decoction is widely used in clinical treatment and basic research for obesity and related metabolic diseases. It possesses weight-loss, anti-inflammatory, and antioxidant effects, and significantly improves insulin sensitivity and alleviates glucose and lipid metabolism disorders (ErchenDecoction Ameliorates Lipid Metabolism by the Regulation of the Protein CAV-1 and the Receptors VLDLR, LDLR, ABCA1, and SRB1 in a High-Fat Diet Rat Model[J]. Evid Based Complement Alternat Med, 2018, 2018: 5309490.). Erchen Decoction also shows significant effects in improving pancreatic function. Related studies have confirmed that it can upregulate the expression level of cyclin-dependent kinase 5 regulatory subunit-related protein 1 analog 1 in the liver, viscera, and subcutaneous adipose tissue of metabolically disordered mice. This mechanism of action helps improve pancreatic islet cell function, promotes insulin secretion, and thus optimizes glucose tolerance, providing a positive impact on glycemic regulation (Gao BZ, Chen JC, Liao LH, et al. Erchen Decoction Prevents High Fat Diet Induced Metabolic Disorders in C57BL / 6 Mice. Evid Based Complement Alternat Med, 2015: 501272). Other studies have confirmed that Erchen Decoction reduces body weight in ZDF rats, improves insulin resistance and lipid metabolism, and decreases free fatty acid (FFA) concentration (Zhao T, Zhan L, Zhou W, et al. The Effects of Erchen Decoction on Gut Microbiota and Lipid Metabolism Disorders in Zucker Diabetic Fatty Rats [J]. Front Pharmacol, 2021, 12: 647529.).Er Chen Tang treatment can improve lipid metabolism disorder in HFD-induced obese rats and increase the total SCFAs level (Zhang L, Chen N, Zhan L, et al. Erchen Decoctionalleviates obesity-related hepatic steatosis via modulating gut microbiota-driven butyric acid contents and promoting fatty acid β-oxidation[J]. J Ethnopharmacol, 2023, 317: 116811.).

[0005] Polysaccharides are a crucial class of macromolecules within cells, consisting of numerous monosaccharide molecules linked together by glycosidic bonds to form long-chain polymer structures. Studies have shown that polysaccharides contained in traditional Chinese medicine have the effect of controlling blood sugar. For example, Coptis chinensis polysaccharide and Astragalus membranaceus polysaccharide have significant effects on improving blood sugar levels and lipid metabolism in type 2 diabetes mellitus (Jiang S, Wang Y, Ren D, et al. Antidiabetic mechanism of Coptis chinensis polysaccharide through its antioxidant property involving the JNK pathway [J]. Pharm Biol, 2015, 53(7): 1022-9.;Liu S, Wang L, Zhang Z, et al. The potentialofastragalus polysaccharide for treating diabetes and its action mechanism[J]. Front Pharmacol, 2024, 15: 1339406.). Other studies have found that Achyranthes bidentata polysaccharide increases the level of gut microbiota-derived short-chain fatty acids (SCFAs) and activates the GLP-1 / GLP-1R / cAMP / PKA / CREB / INS pathway, thereby regulating glucose, amino acid, and lipid metabolism in T2DM mice (Xia T, He W, Luo Z, et al. Achyranthes bidentata polysaccharide ameliorates type 2 diabetes mellitus by gut microbiota-derived short-chain fatty acids-induced activation of the GLP-1 / GLP-1R / cAMP / PKA / CREB / INS pathway [J]. Int J Biol Macromol, 2024, 270(Pt 2): 132256.). As a classic Chinese medicine compound, Er Chen Tang embodies the scientific connotation of the theory of Chinese medicine compatibility through its multi-component and multi-target integrated action system (Zhu Boran, Zhan Libin. Exploring the material basis and mechanism of action of Er Chen Tang in preventing and treating obesity based on the integrated pharmacology platform of Chinese medicine [J]. Chinese Pharmaceutical Journal, 2019, 54(07): 536-541.).Compared to single-herb polysaccharides, compound polysaccharides can produce a synergistic effect by integrating the bioactive properties of various single-herb polysaccharides (Su Fuqin, Cui Hongxia, Liu Jicheng. Immune synergistic effect of compound polysaccharides [J]. New Drugs and Clinical Pharmacology of Traditional Chinese Medicine, 2004, (05):317-9.). Therefore, we need to explore whether the polysaccharides in Er Chen Tang can be the main pharmacologically active substances of Er Chen Tang and to clarify their specific potential pharmacologically active substances through polysaccharide content determination and structural characterization.

[0006] The effective components and structural characterization of Er Chen Tang, which exert this effect, are of positive significance for interpreting the compatibility changes and applications of the formula, but systematic research is still lacking. A literature search revealed no reports on the structural characterization of Er Chen Tang polysaccharides or their effects on improving type 2 diabetes mellitus (T2DM). Summary of the Invention

[0007] To address the problems existing in the prior art, the purpose of this invention is to provide a structural characterization and new application of a polysaccharide extracted from Er Chen Tang. Specifically, the Er Chen Tang polysaccharide is a neutral polysaccharide with an absolute molecular weight (Mn) of 518.325 kDa, a molecular weight (Mw) of 755.258 kDa, a polydispersity (Mw / Mn) of 1.457, and a monosaccharide molar ratio of 100% dextran content. Its sugar chain structure consists of a main chain formed by interconnected →4)-α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→), with the branches mainly composed of α-D-Glcp-(1→ linked to the O-6 positions of the sugar residue →4,6)-α-D-Glcp-(1→). The sugar chain is... The study verified that the polysaccharide has the function of improving type 2 diabetes caused by obesity, and can be used to prepare anti-diabetic drugs and health products, showing significant therapeutic effects on type 2 diabetes caused by obesity. The results indicate that polysaccharides are the main active ingredient in Er Chen Tang for improving type 2 diabetes caused by obesity, and pharmacological experiments have shown that Er Chen Tang polysaccharides have the effect of improving type 2 diabetes caused by obesity.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0009] This invention discloses a polysaccharide called Erchentang, characterized in that it is a neutral and homogeneous polysaccharide. The absolute weight-average molecular weight (Mw) of the Erchentang polysaccharide is 755.258 kDa, the number-average molecular weight (Mn) is 518.325 kDa, and the molecular weight distribution (Mw / Mn) is 1.457. The monosaccharide composition of the Erchentang polysaccharide contains 100% glucan. Its sugar chain structure consists of a main chain formed by interconnected →4)-α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→), and side chains formed by α-D-Glcp-(1→ linked to the O-6 positions of the sugar residue →4,6)-α-D-Glcp-(1→), as shown in Formula 1. .

[0010] This invention also discloses a method for preparing the above-mentioned Er Chen Tang polysaccharide, characterized by comprising the following steps: (1) The medicinal materials of Er Chen Tang were extracted with water, the extracts were combined, concentrated and precipitated with ethanol, and the precipitate was collected to obtain crude polysaccharide of Er Chen Tang; (2) Dissolve the crude polysaccharide and perform protein removal, fat removal and decolorization treatment; (3) The polysaccharide elution peak was collected by ion exchange column chromatography. (4) Further purification was carried out using a gel chromatography column. The homogeneous fraction was collected, dialyzed, concentrated, and dried to obtain Er Chen Tang polysaccharide.

[0011] Furthermore, the medicinal materials of Er Chen Tang mentioned in step (1) include Pinellia ternata, Citrus reticulata peel, Poria cocos, Glycyrrhiza uralensis, Zingiber officinale and Prunus mume. The extraction method is water extraction, the number of extractions is 2, the amount of water added each time is 8 times the amount of medicinal material, and the extraction time is 0.5h.

[0012] Furthermore, in step (2), papain and complex protease are used to enzymatically decompose the protein, chloroform-n-butanol is used to remove fat, and macroporous resin AB-8 is used for decolorization.

[0013] Furthermore, in step (3), the ion exchange column is an anion exchange column, the eluent is a NaCl gradient solution, and the 0.1~0.3M NaCl elution fraction is collected.

[0014] Furthermore, in step (4), the gel chromatography column is a dextran gel column, the elution buffer is water or buffer, and a single elution peak is collected.

[0015] The present invention also discloses a pharmaceutical composition, characterized in that it comprises the polysaccharide of Er Chen Tang as described in claim 1 and a pharmaceutically acceptable carrier or excipient.

[0016] Furthermore, its dosage form is at least one of granules, capsules, and tablets.

[0017] The present invention also discloses a health food, characterized in that it comprises the Er Chen Tang polysaccharide as described in claim 1 and food science acceptable excipients.

[0018] This invention also discloses the application of the above-mentioned Er Chen Tang polysaccharide, or the Er Chen Tang polysaccharide prepared by any of the above-mentioned methods, or the above-mentioned pharmaceutical composition, or the above-mentioned health food in the preparation of a drug or health food for improving obesity-related type 2 diabetes.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0020] 1. This invention is the first to investigate the molecular weight, monosaccharide composition, infrared and ultraviolet absorption spectra, surface morphology, crystal morphology and crystallinity, bonding structure, and molecular structure of Erchentang polysaccharide. Specifically, the absolute molecular weight (Mn) of Erchentang polysaccharide is 518.325 kDa, Mw is 755.258 kDa, and the polydispersity (Mw / Mn) is 1.457; the monosaccharide molar ratio is 100% dextran; the infrared absorption bands at 3600–3200 cm⁻¹ are characteristic peaks of the -OH stretching vibration, and at 3301.95 cm⁻¹ are also characteristic peaks of OH stretching vibration. The ultraviolet spectrum of the polysaccharide shows that, compared to the blank control, the sample has no significant absorption in the wavelength range of 200–400 nm, indicating that the sample contains almost no impurities such as pigments, proteins, and nucleic acids. Under a magnification of 5K, the surface of the polysaccharide sample is rough, with spherical structures of varying particle sizes attached. A broad diffraction peak was observed at 20° (2θ), indicating the amorphous polymeric structure of the polysaccharide. Methylation analysis revealed it to be a neutral polysaccharide with a linkage mode of t-Glc(p), a derivative name of 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol, an RT of 12.279, mass-to-charge ratios of 87, 102, 118, 129, 145, 161, 162, 205, and a relative molar ratio (%) of 8.03. Its sugar chain structure consists of the main chain formed by the interconnection of →4)-α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→), with the branches mainly composed of α-D-Glcp-(1→ linked to the O-6 positions of the sugar residue →4,6)-α-D-Glcp-(1→). .

[0021] 2. This invention explores for the first time the new pharmacological effects of Er Chen Tang polysaccharide, namely, the new use of Er Chen Tang polysaccharide in improving obesity type 2 diabetes mellitus.

[0022] 3. This invention is the first to apply Er Chen Tang polysaccharide to the preparation of drugs and health foods that improve the function of obesity type 2 diabetes mellitus (T2DM). It can be formulated into dosage forms such as granules, capsules and tablets with pharmaceutically or food-acceptable carriers or excipients. Attached Figure Description

[0023] Figure 1 The effects of Er Chen Tang polysaccharide on body weight and abdominal circumference in obese type 2 diabetes mellitus (T2DM) rats. A: Body weight of rats in each group from the start to the end of the experiment; B: Body weight of rats in each group at the end of drug administration; C: Abdominal circumference of rats in each group from the start to the end of the experiment; D: Abdominal circumference of rats in each group at the end of drug administration; Compared with group C, ### P <0.001, ## P <0.01, # P <0.05; compared with group M, *** P<0.001,** P <0.01, * P <0.05.

[0024] Figure 2 This study investigated the effects of Er Chen Tang polysaccharide on food intake and water consumption in obese type 2 diabetes mellitus (T2DM) rats. A: Food intake of rats in each group from the start to the end of the experiment; B: Water consumption of rats in each group from the start to the end of the experiment; D: Average water consumption per rat in each group at the end of drug administration; Compared with group C, ### P <0.001, ## P <0.01, # P <0.05; compared with group M, *** P <0.001,** P <0.01, * P <0.05.

[0025] Figure 3 The effect of Er Chen Tang polysaccharide on glucose metabolism in obese type 2 diabetes mellitus (T2DM) rats. A: Fasting blood glucose weekly after administration; BC: Comparison of OGTT values ​​and area under the curve for each group after administration; EF: Comparison of ITT values ​​and area under the curve for each group after administration; Compared with group C, ### P <0.001, ## P <0.01, # P <0.05; compared with group M, *** P <0.001,** P <0.01, * P <0.05.

[0026] Figure 4 The effect of Er Chen Tang polysaccharide on lipid metabolism in obese type 2 diabetes mellitus (T2DM) rats. A: Serum TC level in rats after drug administration; B: TG level in rats after drug administration; C: LDL level in rats after drug administration; D: HDL level in rats after drug administration; Compared with group C, ### P <0.001, ## P <0.01, # P <0.05; compared with group M, *** P <0.001,** P <0.01, * P <0.05.

[0027] Figure 5 The effects of Er Chen Tang polysaccharide on serum AST, ALT, UREA, and CREA levels in obese type 2 diabetes mellitus (T2DM) rats. A: Serum AST level in rats after drug administration; B: ALT level in rats after drug administration; C: UREA level in rats after drug administration; D: CREA level in rats after drug administration; Compared with group C, ### P<0.001, ## P <0.01, # P <0.05; compared with group M, *** P <0.001,** P <0.01, * P <0.05.

[0028] Figure 6 The results of gel elution of Er Chen Tang polysaccharides and the purity of the polysaccharides.

[0029] Figure 7 The molecular weight of Er Chen Tang polysaccharide is shown in Figure A. Sample image; Figure B. Absolute molecular weight analysis diagram; Figure C. Molecular configuration diagram. Figure 8 This is the monosaccharide composition of Er Chen Tang polysaccharides. Chromatographic comparison and percentage of polysaccharide composition are shown.

[0030] Figure 9 Infrared scanning determination of polysaccharides in Er Chen Tang.

[0031] Figure 10 Ultraviolet (UV) scanning determination of polysaccharides from Er Chen Tang. A: Absorption spectrum of blank control; B: Absorption spectrum of Er Chen Tang polysaccharides; C: Absorption spectra of Er Chen Tang polysaccharides and blank control. Figure 11 Scanning electron microscopy (SEM) and X-ray diffraction (XRD) analysis of Erchentang polysaccharide. A: Surface morphology of Erchentang polysaccharide at 1000x magnification; B: Surface morphology of Erchentang polysaccharide at 500x magnification; C: XRD pattern of Erchentang polysaccharide.

[0032] Figure 12 Methylation analysis of Erchentang polysaccharides. A: Total ion chromatogram of Erchentang polysaccharides; B: Secondary mass spectrum of Erchentang polysaccharides.

[0033] Figure 13NMR analysis of polysaccharides from Erchentang. A: One-dimensional proton NMR (¹H NMR), note: the labels in the figure indicate the position of the hydrogen sugar residue (linkage type, link at position 1 is omitted). B: One-dimensional carbon NMR (¹³C NMR), note: the labels in the figure indicate the position of the carbon sugar residue (linkage type, link at position 1 is omitted). C: 5.3. Hydrogen-hydrogen correlation spectrum (COSY), note: the labels in the figure indicate the adjacent hydrogen number of the sugar residue (linkage type, link at position 1 is omitted). D: 5.4. Hydrogen-hydrogen correlation spectrum (NOESY), note: the labels in the figure indicate the sugar residue (linkage type, link at position 1 is omitted) H-linked hydrogen number - linked sugar residue (linkage type, link at position 1 is omitted) H-linked hydrogen number. E: 5.5. Carbon-hydrogen correlation spectrum (HSQC), note: the labels in the figure indicate the position of the hydrogen and carbon sugar residue (linkage type, link at position 1 is omitted). F: 5.6. Carbon-Hydrogen Correlation Spectrum (HMBC), Note: The labels in the figure correspond to: H / C linked hydrogen / carbon numbers of sugar residues (linkage type, link at position 1 omitted) - C / H linked carbon / hydrogen numbers of connected sugar residues (linkage type, link at position 1 omitted). G: DEPT-135 spectrum, Note: The labels in the figure correspond to: Carbon-sugar residue number (linkage type, link at position 1 omitted). H: 5.8. Total Correlation Spectrum (TOCSY), Note: The labels in the figure correspond to: Anomalous hydrogen numbers of sugar residues – Hydrogen numbers of the same sugar residues with coupled signals. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0035] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0036] Example 1: Preparation of polysaccharides from Er Chen Tang.

[0037] The herbs for Er Chen Tang (each dose of Er Chen Tang includes 15g of prepared Pinellia ternata, 15g of dried tangerine peel, 9g of Poria cocos, 4.5g of prepared licorice root, 7 slices of fresh ginger, and 1 dried plum—totaling 43.5g) were soaked in pure water at a volume eight times that of the raw herbs for 2 hours. The solution was then heated until boiling, and then distilled over low heat for 0.5 hours. After distillation, the solution was filtered through a cloth to obtain the first decoction, while retaining the dregs. Eight times the volume of the raw herbs were added to the remaining dregs, and the solution was heated to boiling and then distilled over low heat for 0.5 hours. The solution was then filtered through a cloth to obtain the second decoction. The two decoctions were combined and concentrated under reduced pressure until the solution volume was reduced to one times the volume of the raw herbs. Anhydrous ethanol was slowly added to the concentrated solution while continuously stirring to adjust the ethanol concentration to 70%. The solution was left to stand overnight at 0–4°C, and the precipitate was then collected. The supernatant was poured off, and the precipitate was repeatedly sonicated with anhydrous ethanol. After aspiration, the polysaccharide component was obtained, and after freeze-drying, the Er Chen Tang polysaccharide was obtained.

[0038] Example 2: Er Chen Tang polysaccharide improves obesity and type 2 diabetes.

[0039] I. Animal grouping and sample collection.

[0040] After one week of acclimatization, 10 rats were randomly selected as the blank control group (Control group) using a random number table. The remaining rats were fed a high-fat diet to obtain obese rats. Obese rats were induced by a single rapid intraperitoneal injection of 1% STZ at a dose of 30 mg / kg body weight. Obese T2DM rats were selected based on fasting blood glucose (FBG) and random blood glucose (RBG). Obese T2DM rats were randomly divided into the obese T2DM model group (Obese T2DM group), the low-dose Er Chen Tang polysaccharide group (ECP-L group), the medium-dose Er Chen Tang polysaccharide group (ECP-M group), the high-dose Er Chen Tang polysaccharide group (ECP-H group), and the positive control liraglutide group (Liraglutide group, L group) using a random number table. After modeling and grouping, the blank control group continued to be fed a normal diet; the other groups continued to be fed a high-fat diet. Gavage treatment began the day after modeling, with a single dose of 1 mL / 100 g (body weight was measured before gavage daily), and the treatment cycle was 4 weeks, once daily. Based on the preliminary screening results of the optimal effective dose of Er Chen Tang decoction (4.57 g / kg), this model will be used in subsequent experiments to study the effect of Er Chen Tang polysaccharides at this dose on obese type 2 diabetes mellitus (T2DM). The amount of raw medicinal material was converted into the corresponding multiple based on the polysaccharide yield. The obese T2DM model group and the blank control group were given the same volume of ultrapure water by gavage for 4 weeks, once a day.

[0041] At the end of the experiment, after fasting for 12 hours, all rats were anesthetized with isoflurane, blood was drawn from the abdominal aorta, tissues were collected and stored in a -80°C refrigerator until testing.

[0042] II. Indicator Testing.

[0043] 1. General condition of rats: During the administration period, the weight, abdominal circumference, body length, food intake and water intake of rats in each group were recorded weekly.

[0044] 2. Blood glucose monitoring: Fasting blood glucose (FBG) should be monitored weekly during the course of drug administration.

[0045] 3. OGTT and ITT. OGTT and ITT were performed at the end of week 4 of treatment. OGTT: After fasting for 12 hours, rats were administered 50% glucose solution at 2 g / kg body weight via gavage (equivalent to 0.4 mL / 100 g body weight). Blood glucose levels were measured by tail vein sampling before glucose administration (0 min) and at 30, 60, 90, and 120 min after administration. ITT: After one day of acclimatization following OGTT, rats were fasted for 6 hours and then injected intraperitoneally with conventional short-acting insulin (0.5 U / kg body weight). Blood glucose levels were measured by tail vein sampling before injection (0 min) and at 15, 30, 60, 90, and 120 min after injection.

[0046] 4. Lipid metabolism and related indicators of liver and kidney function. Serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CR), and urea (UREA) were automatically measured using a fully automated biochemical analyzer.

[0047] 5. Statistical Methods. Data were analyzed using Prsim 9.0 software. Values ​​are expressed as mean ± standard deviation. One-way ANOVA was used to compare the means of multiple groups. P <0.05 indicates a statistically significant difference.

[0048] III. Experimental Results.

[0049] 1. Effects of Er Chen Tang polysaccharide on body weight and abdominal circumference in obese T2DM rats.

[0050] Changes in body weight and abdominal circumference of rats in each group during the experiment are as follows: Figure 1 As shown. Compared with the blank control group, the rats showed an increasing trend in body weight after 4 weeks of high-fat diet feeding. After intraperitoneal injection of 1% STZ solution, the body weight of rats in groups M, L, and ECP decreased. After administration, the body weight of rats in each group was lower than that in group M; at week 4 of administration, the body weight of rats in group L ( P <0.001), ECP group ( P Rats with <0.05% weight loss showed a significant decrease in body weight.

[0051] After 4 weeks of high-fat diet feeding, the abdominal circumference of rats fed the high-fat diet was found to be higher than that of group C; after drug administration, the abdominal circumference of rats in all drug-treated groups tended to decrease compared to group M. At week 4 of drug administration, group L ( P <0.0001), ECP group ( P <0.001) Significant reduction in abdominal circumference in rats.

[0052] 2. Effects of Er Chen Tang polysaccharides on food intake and water consumption in obese type 2 diabetes mellitus (T2DM) rats The changes in food intake and water consumption of rats in each group during the experiment are as follows: Figure 2 As shown. After intraperitoneal injection of 1% STZ solution, the food intake of rats in group M and each of the treatment groups decreased before administration, with group L showing a significant decrease in food intake compared to before administration ( P <0.0001). In the absence of drug treatment, the food intake of rats in group M was increased compared to group C. After drug administration, the food intake of rats in group M and each drug-treated group recovered. The food intake of each drug-treated group was decreased compared to group M. P <0.01).

[0053] Based on the water intake results, it was found that after intraperitoneal injection of 1% STZ solution, the water intake of rats in groups M and ECP was higher than that in group C, exhibiting obvious characteristics of type 2 diabetes mellitus (T2DM) with polydipsia. After administration, compared with group M, the water intake of rats in groups L, ECP-L, and ECP-H was significantly decreased. P <0.01).

[0054] 3. Effects of Er Chen Tang polysaccharide on glucose metabolism in obese type 2 diabetes mellitus (T2DM) rats.

[0055] 3.1 Effect of Er Chen Tang polysaccharide on fasting blood glucose in obese type 2 diabetes mellitus (T2DM) rats.

[0056] Compared with group C, the FBG of T2DM rats injected with 1% STZ solution was significantly increased. FBG data of T2DM rats showed that the FBG of all rats was greater than 11.1 mM, indicating a good T2DM model establishment rate. With drug administration, compared with group M, the drug-treated group significantly reduced the FBG of obese T2DM rats ( P <0.001).

[0057] 3.2 Effects of Er Chen Tang polysaccharide on glucose tolerance and insulin tolerance in obese T2DM rats.

[0058] After the administration of the medication, glucose tolerance was improved in T2DM rats in the ECP-H group compared to the M group. Insulin tolerance was improved in T2DM rats in both the L group and the ECP-H group compared to the M group.

[0059] 3.3 Effects of Er Chen Tang polysaccharide on lipid metabolism in obese T2DM rats.

[0060] Compared with group C, the serum levels of TC, TG, and LDL-C in group M rats were increased, while the HDL-C level was significantly decreased. P <0.05. After the administration period, compared with group M, all treatment groups showed a decrease in TC, TG, and LDL-C levels, with the ECP-M and ECP-H groups showing a significant decrease in TC levels ( P <0.01), the ECDP group significantly reduced TG levels ( P <0.05; compared with group M, groups L and ECP-H significantly increased HDL-C levels ( P <0.05). These results indicate that Er Chen Tang polysaccharide can improve lipid metabolism in obese type 2 diabetes mellitus (T2DM) rats.

[0061] 3.4 Effects of Er Chen Tang polysaccharide on liver and kidney function in obese T2DM rats.

[0062] Compared with group C, the serum levels of AST, ALT, and UREA in group M rats were significantly increased (P<0.05). After the administration, compared with group M, the levels of AST, ALT, and UREA in all treatment groups decreased, with the LZ group and ECP group showing a significant decrease in AST levels. P <0.05); the L group and ECP-H group significantly reduced ALT levels (P<0.01); the L group, ECP-L group and ECP-H group significantly reduced UREA levels (P<0.05); P <0.05). These results indicate that Er Chen Tang polysaccharide can improve liver and kidney function damage in obese type 2 diabetes mellitus (T2DM) rats.

[0063] Example 3: Characterization of the results of Er Chen Tang polysaccharide.

[0064] I. Experimental Methods.

[0065] 1. The crude polysaccharide extract of Er Chen Tang was purified by removing protein, fat, and color. 600 mL–1 L of pure water was added to the crude polysaccharide extract solid to fully dissolve the polysaccharides. 0.4–0.6 g of papain and a complex protease were added separately, and the mixture was allowed to hydrolyze overnight. 1 / 4 volume of chloroform and n-butanol (4:1, v / v) were added to the aqueous phase, and the mixture was thoroughly mixed. The upper aqueous phase was collected. 1 / 4 volume of petroleum ether was added to the liquid phase, and the mixture was thoroughly mixed. The lower aqueous phase was collected. 1 / 2 volume of macroporous resin AB-8 was added to the lower aqueous phase, and the mixture was thoroughly mixed and allowed to adsorb overnight. The liquid was collected and dialyzed through a 3000 Da dialysis bag for 24–48 h to remove small molecule components. The polysaccharide solution was then precipitated with alcohol and dried.

[0066] 2. The crude polysaccharide from Er Chen Tang was purified using an ion exchange method. Approximately 5 g of the crude polysaccharide sample was dissolved in 200 mL of pure water. The solution was centrifuged at 10000 g for 10 min, and the supernatant was purified by passing it through an ion exchange column at a flow rate of 4 mL / min. A gradient elution was performed sequentially using pure water, 0.1 M, 0.2 M, and 0.3 M NaCl solutions, collecting 15 mL of each eluent in one tube. The total sugar content of the eluent in each collection tube was determined using the sulfuric acid-phenol method. Specifically, 100 μL of the diluted polysaccharide supernatant was added to 600 μL of sulfuric acid-phenol reagent (5% phenol solution: concentrated sulfuric acid = 1:5 (v / v), the same below), mixed thoroughly, and allowed to stand in the dark for 10 min. The absorbance was measured at 490 nm. An ion purification elution curve was plotted. Identify the elution peaks, combine the eluents from each collection tube corresponding to the same elution peak, concentrate them to 1 / 5 of the original volume by rotary evaporation, and dialyze through a 3000 Da dialysis bag for 24-48 hours to remove salt.

[0067] 3. Further purification of the Er Chen Tang polysaccharide was performed using gel chromatography. Approximately 1 g of the ion-purified polysaccharide sample was added to 20 mL of pure water. The sample was centrifuged at 10000 g for 10 min. The supernatant was then purified by gel chromatography at a flow rate of 1 mL / min. Elution was performed with 1.5 column volumes of pure water (actual elution conditions are detailed in document / 2. Data Processing Results). One 10 mL tube was collected, and all eluent was collected. The total sugar content of the eluent in each collection tube was determined using the sulfuric acid-phenol method. Specifically, 100 μL of the diluted polysaccharide supernatant was added to 600 μL of sulfuric acid-phenol reagent (5% phenol solution: concentrated sulfuric acid = 1:5 (v / v), the same below). The mixture was stirred and reacted in the dark for 10 min. The absorbance was measured at 490 nm. A gel purification elution curve was plotted. Identify the elution peaks, combine the eluates from all collection tubes corresponding to the same elution peak, and concentrate them to 1 / 5 of their original volume by rotary evaporation. Dialyze using a 3000 Da dialysis bag for 24–48 h to remove small molecule components. Freeze-dry the sample, and determine the purity of the polysaccharide after gel purification using the sulfuric acid-phenol method. The specific procedure is as follows: Weigh 2–5 mg of the freeze-dried polysaccharide sample, dissolve and dilute it with water, take 100 μL of the polysaccharide supernatant, add the sulfuric acid-phenol reagent, mix well, allow to stand in the dark for 10 min, and measure the absorbance at 490 nm.

[0068] 4. The monosaccharide components of the purified Erchentang polysaccharide were analyzed and detected using a chromatographic system and an electrochemical detector. The specific steps were as follows: 1) Solid sample extraction: Take a clean chromatographic bottle, weigh an appropriate amount of polysaccharide sample, add 1 mL of 2M TFA acid solution, and heat at 121℃ for 2 hours. Purge with nitrogen and dry. Add 99.99% methanol for washing, then dry again, repeating the methanol washing 2-3 times. Dissolve in sterile water and transfer to a chromatographic bottle for analysis. 2) Liquid sample extraction: Take an appropriate amount of supernatant, concentrate by rotation or dry with nitrogen. Add 1 mL of 2M TFA solution and heat at 121℃ for 2 hours. Purge with nitrogen and dry. Add 99.99% methanol for washing, then dry again, repeating the methanol washing 2-3 times. Dissolve in sterile water and transfer to a chromatographic bottle for analysis. The monosaccharide components were analyzed and detected using an ion chromatography system and an electrochemical detector. The chromatographic data were processed using Chromeleon software.

[0069] 5. The molecular weight of the purified polysaccharide was determined using a gel permeation chromatography-differential light scattering (GPS-DSS) system. The sample was dissolved in a 0.1M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL, and filtered through a 0.45 μm filter before analysis. The concentration was determined by a differential detector based on refractive index, while the light scattering of macromolecules was detected using a DPS-DSS system. The molecular weight of each component was calculated using the Mark-Houwink equation.

[0070] 6. Characteristic functional groups of Er Chen Tang polysaccharides were analyzed using a Nicolet iZ-10 Fourier transform infrared spectrometer. A small amount of polysaccharide sample was weighed, mixed with 200 mg of potassium bromide, pressed into 1 mm thick sheets, and then analyzed by the instrument. The instrument resolution was 4.00 cm⁻¹, the scanning range was 4000–450 cm⁻¹, and the number of scans was 32. The sampling gain was 8.0; the moving mirror speed was 0.4747; the aperture was 80.00; the instrument used a DTGS KBr detector, a KBr beam splitter, and an infrared light source.

[0071] 7. Ultraviolet (UV) scanning analysis of the Er Chen Tang polysaccharide sample was performed using a full-wavelength spectral scan (200–1000 nm), and the UV absorption spectrum curve of the substance was plotted. A multi-functional microplate reader was used for quantitative analysis of the polysaccharide solution. A small amount of the polysaccharide sample was weighed and dissolved in pure water to prepare a 5 mg / mL polysaccharide solution. The scan start wavelength was 200 nm, the end wavelength was 1000 nm, and the scan interval was 1 nm. Pure water was used as a blank control, and measurements were performed under the same conditions.

[0072] 8. The surface morphology and structure of the polysaccharide sample of Er Chen Tang were observed using a high-resolution field emission scanning electron microscope. The polysaccharide sample was passed through a 100-mesh sieve, and a small amount was taken onto a conductive carbon tape. After gold sputtering, the sample was scanned and photographed using an electron microscope at a magnification of 500 to 10,000 times.

[0073] 9. The polysaccharides of Er Chen Tang were analyzed using an X'Pert Pro X-ray diffractometer. A copper target (Cu-Kα, λ = 0.15406 nm) was used with a power of 1600 W (40 kV × 40 mA). An I crystal scintillation counter was employed to measure the X-ray intensity. The scanning range was 5°–60°, with a step size of 0.02° and a scanning speed of 4° / min. The DS-SS-RS settings were 1 mm–1 mm–0.1 mm [divergent slit (DS), anti-scattering slit (SS), and receiving slit (RS)].

[0074] 10. Qualitative analysis of polysaccharide bonding was performed using gas chromatography-mass spectrometry (GC-MS). The specific steps were as follows: Take a small amount of sample (2-3 mg), dissolve it in 500 μL of DMSO, add 1 mg of NaOH, and incubate for 30 min. Add 50 μL of iodomethane solution and react for 1 h. Add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the washing with water three times. Pipette the lower dichloromethane phase and dry it under nitrogen. Add 100 μL of 2M TFA and react at 121 °C for 90 min. Evaporate to dryness at 30 °C. Add 50 μL of 2M ammonia and 50 μL of 1M NaBD4, mix well, and react at room temperature for 2.5 h. Add 20 μL of acetic acid to terminate the reaction, dry under nitrogen, wash twice with 250 μL of methanol, and dry under nitrogen. Add 250 μL of acetic anhydride, vortex to mix, and react at 100 °C for 2.5 h. Add 1 mL of water and let stand for 10 min. Add 500 μL of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and wash with water three times.

[0075] The lower layer of dichloromethane phase was removed and analyzed by GC-MS.

[0076] 11. The molecular structure of Er Chen Tang polysaccharide was analyzed by nuclear magnetic resonance (NMR). The specific steps included: (1) Dissolving an appropriate amount of purified polysaccharide in D2O to prepare a polysaccharide solution with a concentration greater than or equal to 40 mg / mL. (2) Transferring the dissolved solution to an NMR tube with an added volume of 0.5 mL. (3) Placing the NMR tube in a nuclear magnetic resonance spectrometer to scan one-dimensional 1H spectrum, 13C spectrum, DEPT135, and two-dimensional COSY, HSQC, HMBC, NOESY, and TOCSY spectra.

[0077] II. Experimental Results.

[0078] After impurity removal, ion exchange purification, and gel purification, the polysaccharide purity of Er Chen Tang was found to be 92.3%.

[0079] The monosaccharide composition of Er Chen Tang polysaccharide is 100% glucan content and 100% molar ratio glucan content.

[0080] The absolute molecular weight of Er Chen Tang polysaccharide is 518.325 kDa (Mn), 755.258 kDa (Mw), and 1.457 (Polydispersity (Mw / Mn)).

[0081] Characteristic functional groups of Erchentang polysaccharide. Infrared spectroscopy shows that the absorption bands at 3600–3200 cm⁻¹ are due to the stretching vibration of -OH, and at 3301.95 cm⁻¹ are also characteristic peaks of carbohydrates. The absorption peak at 2924.66 cm⁻¹ is attributed to the CH stretching vibration. There is an absorption peak at 1015.67 cm⁻¹, attributed to the CO stretching vibration.

[0082] Absorption spectrum curves of Er Chen Tang polysaccharides. The ultraviolet spectrum of the polysaccharides showed that, compared to the blank control, the sample had no significant absorption in the wavelength range of 200–400 nm, indicating that the sample contained almost no impurities such as pigments, proteins, and nucleic acids.

[0083] Surface structure analysis of Erchentang polysaccharide. At a magnification of 5K, the polysaccharide sample showed a rough surface with spherical structures of varying sizes attached. Crystallization morphology and crystallinity analysis of Erchentang polysaccharide. The absorption spectrum showed a broad diffraction peak at 20° (2θ), indicating the amorphous polymeric structure of the polysaccharide.

[0084] Methylation analysis of Erchentang polysaccharide. The bonding structure of Erchentang polysaccharide was analyzed by GC-MS. It was identified as a neutral polysaccharide with a t-Glc(p) linkage, derivative name 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol, RT value of 12.279, mass-to-charge ratio of 87, 102, 118, 129, 145, 161, 162, 205, and relative molar ratio (%) of 8.03.

[0085] The sugar chain structure of Erchentang polysaccharide. Its sugar chain structure consists of the main chain formed by the interconnection of →4)-α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→), with the branches mainly composed of α-D-Glcp-(1→ linked to the O-6 positions of the sugar residue →4,6)-α-D-Glcp-(1→. The sugar chain is... .

[0086] This invention involves the large-scale preparation of Er Chen Tang polysaccharide, and for the first time, it explores whether Er Chen Tang polysaccharide has the function of improving obesity type 2 diabetes mellitus and characterizes its structure. Through extensive experimental research, it was found that Er Chen Tang polysaccharide has a good effect on improving obesity type 2 diabetes mellitus.

[0087] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A polysaccharide of Erchen Decoction, characterized in that, It is a neutral homogeneous polysaccharide, the absolute weight average molecular weight Mw of the Erchen Decoction polysaccharide is 755.258 kDa, the number average molecular weight Mn is 518.325 kDa, and the molecular weight distribution Mw / Mn is 1.457; the content of glucan in the monosaccharide composition of the Erchen Decoction polysaccharide is 100%, and the sugar chain structure is formed by the mutual connection of →4)-alpha-D-Glcp-(1→ and →4,6)-alpha-D-Glcp-(1→ to form a backbone, and the branch is composed of alpha-D-Glcp-(1→ connected at the O-6 position of the sugar residue →4,6)-alpha-D-Glcp-(1→, and the structure is shown as formula 1: .

2. The method for preparing the polysaccharide of Erchen Decoction according to claim 1, characterized in that, It comprises the following steps: (1) Extracting Erchen decoction medicinal materials with water, combining the extract, adding ethanol after concentration, collecting the precipitate to obtain crude polysaccharide of Erchen decoction; (2) Dissolving the crude polysaccharide, removing protein, removing fat and decolorizing; (3) Purifying by ion exchange column chromatography, collecting polysaccharide elution peak; (4) Further purifying by gel chromatography column, collecting uniform components, dialysis, concentration and drying to obtain polysaccharide of Erchen decoction.

3. The production method according to claim 2, characterized by, The medicinal materials of Erchen decoction in step (1) include Fashanxia, Chenpi, Fuling, Zhigancao, Shengjiang and Wumei, the extraction method is water extraction, the extraction times is 2 times, the water amount is 8 times of the medicinal material quality, and the extraction time is 0.5 h.

4. The preparation method according to claim 2, characterized in that, In step (2), papain and complex protease are used for enzymatic deproteinization, chloroform-n-butanol method is used for fat removal, and AB-8 macroporous resin is used for decolorization.

5. The preparation method according to claim 2, characterized in that, In step (3), the ion exchange column is a cation exchange column, the eluent is a NaCl gradient solution, and the 0.1-0.3 M NaCl elution part is collected.

6. The preparation method according to claim 2, characterized in that, In step (4), the gel chromatography column is a dextran gel column, the eluent is water or buffer, and the single elution peak is collected.

7. A pharmaceutical composition, characterized by, It comprises the polysaccharide of Erchen decoction of claim 1 and a pharmaceutically acceptable carrier or excipient.

8. The pharmaceutical composition of claim 7, wherein, Its dosage form is at least one of granules, capsules and tablets.

9. A health food, characterized by, It comprises the polysaccharide of Erchen decoction of claim 1 and a food acceptable auxiliary.

10. The use of the polysaccharide of Erchen decoction of claim 1, or the polysaccharide of Erchen decoction prepared by the method of claims 2-6, or the pharmaceutical composition of any one of claims 7-8 or the health food of claim 9 in the preparation of a drug or health food for improving obesity and type 2 diabetes.

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