A traditional Chinese medicine composition for acute pancreatitis, a pharmaceutical preparation and a preparation method and application thereof

Abstract

The application discloses a traditional Chinese medicine composition for acute pancreatitis, a medicine preparation and a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicines. The traditional Chinese medicine composition is prepared from the following raw medicinal materials in parts by weight: 35-45 parts of Astragalus membranaceus, 12-18 parts of Coptis chinensis, 10-15 parts of Pueraria lobata, 10-15 parts of Magnolia officinalis, and 12-18 parts of Lycium barbarum. Pharmacodynamic experiments show that the traditional Chinese medicine composition can significantly reduce the serum amylase, lipase, triglyceride and total cholesterol levels of model animals with acute pancreatitis, inhibit the release of inflammatory factors, reduce pathological damage of the pancreas, ileum and liver, and can play a synergistic treatment role through regulating the intestinal flora-bile acid metabolism axis. The traditional Chinese medicine composition has significant treatment effects on diabetes combined with acute pancreatitis and hyperlipidemic acute pancreatitis, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, and in particular relates to a traditional Chinese medicine composition, pharmaceutical preparation, preparation method and application for acute pancreatitis. Background Technology

[0002] Acute pancreatitis (AP) is a common digestive system disease, with a global incidence increasing annually, ranging from 4.9 to 73.4 per 100,000 AP cases per year. AP is caused by various factors, primarily characterized by local inflammation of the pancreas. Activation of pancreatic enzymes leads to autodigestion of pancreatic tissue, resulting in pancreatic hemorrhage and necrosis, and can also cause functional changes in other organs. With changes in modern lifestyles, hypertriglyceridemic acute pancreatitis (also known as hyperlipidemic acute pancreatitis, HTG-AP) is more common, accounting for 8% to 15% of AP cases. Diabetes mellitus (DM) is a chronic metabolic disease characterized by hyperglycemia. Type 1 diabetes is more common in children, caused by a relative or absolute deficiency of insulin, leading to metabolic disorders of the three major nutrients. Type 2 diabetes is predominant in middle-aged and elderly individuals, but it is now showing a trend towards affecting younger people. DM and AP are closely related and often occur together; studies have shown that the risk of AP in DM patients is 2 to 3 times higher than in the general population. Diabetes is an independent risk factor for the occurrence and severity of acute pancreatitis.

[0003] Currently, the clinical treatment of diabetes mellitus complicated with acute pancreatitis mainly relies on a comprehensive approach of Western medicine, including symptomatic support, blood glucose control, pancreatic enzyme inhibition, and anti-infection measures. While this approach can control acute symptoms to some extent, it still has many shortcomings and limitations. Western medicine treatment is mostly symptomatic, making it difficult to simultaneously address multiple goals such as pancreatic protection, pancreatic islet function repair, and correction of glucose and lipid metabolism disorders. Commonly used drugs such as metformin can regulate glucose and lipid metabolism, but their clinical use is contraindicated during acute pancreatitis attacks. Furthermore, current treatment methods cannot simultaneously improve metabolic disorders and local pancreatic inflammation, effectively repair the intestinal barrier, and regulate microecological imbalances, resulting in limited overall efficacy. Long-term use of insulin, enzyme inhibitors, antibiotics, and other drugs can easily lead to drug dependence, metabolic disorders, intestinal flora imbalance, drug resistance, and increased burden on the liver and kidneys.

[0004] The incidence of hypertriglyceridemia-associated pancreatitis (HTPG-AP) is on the rise globally, especially in China. Currently, it is the second most common cause of HTPG-AP after biliary pancreatitis. HTPG-AP is prone to severe illness, leading to rapid disease progression, high mortality, and frequent relapses even after treatment. Current clinical treatment for HTPG-AP mainly focuses on symptomatic support, rapid lipid-lowering, and organ protection using Western medicine, but lacks specific targeted drugs to address the underlying cause. While rapid lipid-lowering methods such as insulin, heparin, and plasma exchange can reduce triglycerides, they have not significantly improved the severity and mortality rates, and involve issues such as trauma, high costs, and the risk of complications. Treatment often focuses on controlling acute symptoms, failing to fundamentally correct lipid metabolism disorders. Patients have a high relapse rate in the medium to long term, and there is still a lack of effective means to prevent inflammatory storms and multi-organ damage during the severe stage. Overall, treatment is limited by the characteristics of "easy to treat symptoms, difficult to treat the root cause, strong emergency care, and weak chronic disease management." Summary of the Invention

[0005] In view of this, the present invention provides a traditional Chinese medicine composition for acute pancreatitis, its preparation method and application.

[0006] This invention provides a traditional Chinese medicine composition for acute pancreatitis, which is made from the following raw materials in parts by weight: Astragalus membranaceus 35-45 parts, Coptis chinensis 12-18 parts, Pueraria lobata 10-15 parts, Magnolia officinalis 10-15 parts, and Lycium barbarum 12-18 parts.

[0007] Furthermore, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 40 parts Astragalus membranaceus, 15 parts Coptis chinensis, 12 parts Pueraria lobata, 12 parts Magnolia officinalis, and 15 parts Lycium barbarum.

[0008] Furthermore, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 45 parts Astragalus membranaceus, 12 parts Coptis chinensis, 10 parts Pueraria lobata, 15 parts Magnolia officinalis, and 18 parts Lycium barbarum.

[0009] Furthermore, the traditional Chinese medicine composition is made from the following raw materials in parts by weight: 35 parts Astragalus membranaceus, 15 parts Coptis chinensis, 12 parts Pueraria lobata, 10 parts Magnolia officinalis, and 15 parts Lycium barbarum.

[0010] The present invention also provides a pharmaceutical preparation for treating acute pancreatitis, characterized in that the pharmaceutical preparation comprises the effective components of any one of the traditional Chinese medicine compositions according to claims 1-4.

[0011] Furthermore, the pharmaceutical preparation also includes excipients.

[0012] Furthermore, the dosage form of the pharmaceutical preparation is granules, tablets, capsules, or decoction.

[0013] The present invention also provides a method for preparing the pharmaceutical preparation, comprising the following steps: water extraction of the traditional Chinese medicine composition, wherein the water extraction method comprises the following steps: weighing each traditional Chinese medicine raw material according to the weight proportions, first decoction, soaking in cold water for 3 cm above the surface of the herbs for 30 min, then decocting for 30 min, filtering the residue and collecting the juice; second decoction, soaking in cold water for 2 cm above the surface of the herbs for 30 min, filtering the residue and collecting the juice, mixing the two decoctions to obtain an aqueous extract; and preparing the aqueous extract into the dosage form of the pharmaceutical preparation.

[0014] The present invention also provides the use of traditional Chinese medicine compositions in the preparation of drugs for the prevention or treatment of hyperglycemia, hyperlipidemia or acute pancreatitis.

[0015] Furthermore, the acute pancreatitis includes acute pancreatitis complicated by diabetes, acute pancreatitis due to hyperlipidemia, acute pancreatitis due to cholesteatosis, and acute pancreatitis of biliary origin.

[0016] Traditional Chinese medicine (TCM) possesses advantages in treating metabolic and inflammatory diseases due to its multi-component, multi-target, and holistic regulatory effects. However, few existing TCM formulas combine these effects with comprehensive benefits such as lipid-lowering, blood sugar-lowering, anti-inflammatory, and pancreatic-protective properties. Diabetes mellitus complicated by acute pancreatitis falls under the category of "spleen deficiency" or "spleen and heart pain" in traditional Chinese medicine, with the disease location being the pancreas. The original text of the *Nan Jing* (Classic of Difficult Issues), Chapter 42, states that "the spleen weighs two jin and three liang, is three inches wide and five inches long, and contains half a jin of pancreas, which governs blood circulation, warms the five internal organs, and stores the mind." Modern medical practitioners, based on systemic anatomical research, clearly identify the pancreas as the "pancreas." Its dysfunction is rooted in yin deficiency, manifested by dryness and heat, with frequent symptoms of both qi and yin deficiency, and qi stagnation and blood stasis throughout the course of the disease. Treatment of this disease consistently adheres to the holistic concept and syndrome differentiation principles of TCM, with the core treatment method revolving around "nourishing yin and clearing heat, tonifying qi and nourishing yin, and promoting blood circulation and removing blood stasis."

[0017] The traditional Chinese medicine composition provided by this invention takes replenishing qi and nourishing yin as its essence, nourishing yin and clearing heat as its symptoms, and promoting blood circulation and removing blood stasis throughout, combining the three methods into one.

[0018] Astragalus membranaceus (Huang Qi) in this formula is sweet and slightly warm in nature, and enters the spleen and lung meridians. Its functions are: tonifying qi and raising yang, consolidating the exterior and stopping sweating, and promoting diuresis and reducing edema. A large dose of Astragalus membranaceus is used to greatly tonify the qi of the spleen and lungs. When qi is abundant, body fluids are naturally generated. Qi can guide blood, invigorate blood circulation, and unblock the meridians, ensuring the distribution of body fluids and the resolution of blood stasis, fundamentally addressing the underlying deficiency of both qi and yin, and the persistent problem of qi stagnation and blood stasis. Lycium barbarum (Gou Qi) in this formula is sweet and neutral in nature, and enters the liver and kidney meridians. Its function is to nourish the liver and kidneys, benefit essence, and improve eyesight. Combined with Astragalus membranaceus, one tonifies qi, and the other nourishes yin, working together to achieve the effect of tonifying qi and nourishing yin. These are the principal herbs.

[0019] The formula contains kudzu root, which is sweet and pungent in taste, cool in nature, and enters the spleen, stomach, and lung meridians. Its most notable functions are threefold: relieving muscle tension and reducing fever, generating fluids and quenching thirst, and raising yang to stop diarrhea. It also invigorates stomach qi and assists astragalus and wolfberry in distributing body fluids. Coptis root is bitter in taste, cold in nature, and enters the heart, spleen, stomach, liver, and gallbladder meridians. Its functions are clearing heat and drying dampness, purging fire and detoxifying. Its heat-clearing and fire-purging power specifically treats the superficial manifestation of "yin deficiency and fire excess" in this disease. Combined with kudzu root, one ascends and the other descends, one moistens and the other dries, working together to achieve the effect of nourishing yin and clearing heat. These two are assistant herbs. Magnolia bark is bitter and pungent in taste, warm in nature, and enters the spleen, stomach, lung, and large intestine meridians. Its functions are drying dampness and resolving phlegm, lowering qi and relieving fullness. Its functions are threefold: first, to dry dampness and prevent the cloying effects of Astragalus and Lycium; second, to regulate Qi and ensure that the entire formula is nourishing without causing stagnation and that the Qi mechanism is smooth; and third, to assist Pueraria lobata in ascending clear Qi and Coptis chinensis in descending turbid Qi, thus ensuring that the entire formula ascends and descends in an orderly manner.

[0020] In summary, Astragalus membranaceus "expels stagnant blood between the five internal organs" and "regulates blood vessels and promotes circulation in the meridians"; Lycium barbarum nourishes liver and kidney yin; Pueraria lobata relieves muscle tension, clears heat, and generates fluids to quench thirst; Coptis chinensis clears heat and drains fire; Magnolia officinalis lowers qi and relieves fullness, serving as an adjuvant. The five herbs combined work synergistically to invigorate qi and nourish yin, clear heat, and promote blood circulation. The entire formula prioritizes tonification as the principal ingredient and clearing as the secondary ingredient, heavily utilizing Astragalus membranaceus to support the body's vital energy, reflecting the principle of "diabetes as the root cause and pancreatitis as the manifestation, addressing both the root and the branch, and combining tonification and purgation." It employs clearing, regulating, tonifying, and nourishing methods: Coptis chinensis clears heat, Magnolia officinalis regulates qi, Astragalus membranaceus invigorates qi, Lycium barbarum nourishes yin, and Pueraria lobata generates fluids. This formula treats both the excess heat and qi stagnation of pancreatitis and the deficiency of both qi and yin in diabetes, making it suitable for diabetes complicated by acute pancreatitis, where the body's vital energy is already deficient, damp-heat qi stagnation is present, and the bowel qi is obstructed.

[0021] Meanwhile, current TCM treatment for hyperlipidemic acute pancreatitis (HTG-AP) focuses on purging and eliminating pathogens as its core principle. Combined with the pathogenesis of this disease—characterized by the intermingling of lipid turbidity, phlegm stasis, and heat toxins—it incorporates methods such as clearing heat and detoxifying, eliminating dampness and resolving phlegm, and promoting blood circulation and removing blood stasis, forming a comprehensive treatment plan with multiple pathways and methods. In this invention's formula, Astragalus membranaceus tonifies Qi and strengthens the exterior, promoting the elimination of toxins and pus; Coptis chinensis clears heat and dries dampness, purging fire and detoxifying; Magnolia officinalis dries dampness and eliminates phlegm, lowering Qi and relieving fullness; Pueraria lobata relieves muscle tension and reduces fever, promoting body fluids and quenching thirst; and Lycium barbarum nourishes the liver and kidneys, benefiting essence and improving eyesight. The entire formula works synergistically to "clear heat and detoxify, regulate the bowels and lower Qi, strengthen the body's resistance and eliminate toxins," regulating metabolism and inhibiting inflammatory responses.

[0022] The beneficial effects of the traditional Chinese medicine composition for treating acute pancreatitis of this invention are as follows: I. The herbal composition of this invention is based on tonifying as the principal ingredient and clearing as the secondary ingredient, with a heavy use of Astragalus membranaceus to support the body's vital energy. It embodies the principle of "treating diabetes as the root cause and pancreatitis as the manifestation, addressing both the root cause and the symptoms, and combining tonifying and purging methods." It uses clearing, regulating, tonifying, and nourishing herbs simultaneously: Coptis chinensis clears heat, Magnolia officinalis regulates qi, Astragalus membranaceus tonifies qi, Lycium barbarum nourishes yin, and Pueraria lobata generates fluids. It treats both the excess heat and qi stagnation in pancreatitis and the deficiency of both qi and yin in diabetes. It is suitable for diabetes complicated with acute pancreatitis, where the body's vital energy is already deficient, damp-heat qi stagnation occurs, and the bowel qi is obstructed.

[0023] II. The herbal composition of this invention can significantly reduce blood glucose in mice, increase insulin secretion, reduce serum amylase and lipase activity, alleviate pancreatic inflammation, and simultaneously downregulate TC, TG, and LDL-C, improving lipid metabolism abnormalities in DA mice; reduce pancreatic islet atrophy and acinar structure destruction in DA mice, reduce interstitial inflammatory cell infiltration, and lower pancreatic pathological damage scores; improve ileal villus atrophy, fusion, and epithelial shedding in DA mice, reduce intestinal mucosal inflammatory cell infiltration, and lower intestinal pathological damage scores; correct bile acid metabolism disorders and construct a "microbiota-bile acid" interaction chain; significantly upregulate the mRNA and protein expression of farnesol X receptor (FXR) and G protein-coupled bile acid receptor 5 (TGR5) in the liver, pancreas, and ileum of DA mice; promote the secretion of glucagon-like peptide-1 (GLP-1) by intestinal L cells. GLP-1 promotes insulin secretion and inhibits glucagon release in a glucose concentration-dependent manner, while synergistically achieving a dual effect of metabolic regulation and inflammation suppression with FXR / TGR5.

[0024] Third, the traditional Chinese medicine composition of this invention for treating diabetes mellitus complicated with acute pancreatitis and hyperlipidemic acute pancreatitis has synergistic effects among its components, with definite curative effect, high safety, and excellent clinical application prospects. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a statistical chart showing the effect of this traditional Chinese medicine composition on improving pancreatic, ileal, and liver tissue damage in HLAP mice.

[0027] Figure 2 The effects of this traditional Chinese medicine composition on improving serum lipid, enzyme, and systemic inflammation levels in HLAP (groups: normal control group (CON), hyperlipidemic acute pancreatitis group (MOD), positive control group (fenofibrate), and traditional Chinese medicine treatment group (ZKF) are the traditional Chinese medicine compositions of this invention; Figures A and B show the serum enzyme levels of each group: mouse amylase and lipase levels, Figures C and D show the serum lipid levels of each group: mouse TC and TG levels, and Figures E and F show the systemic inflammation levels of each group: mouse serum IL-1β and IL-6 inflammatory factors).

[0028] Figure 3The following is a statistical chart showing the effect of ZKF on the body weight of DA mice (Note: CON is the normal control group, AP is the acute pancreatitis group, DA is the diabetic acute pancreatitis model group, ZKF is the traditional Chinese medicine treatment group of this invention, and MET is the metformin positive control group; Figure A is the experimental design flowchart, and Figures B and C show the dynamic changes and increases in body weight of mice in each group over 32 days; compared with the DA group, * P<0.05 ** P<0.01 *** P<0.001).

[0029] Figure 4 Statistical graphs showing the effects of ZKF on blood glucose and enzymes in DA mice (Note: Figure A shows the dynamic changes in blood glucose in each group of mice, Figure B shows the serum insulin level in each group of mice, Figure C shows the lipase level in each group of mice, Figure D shows the amylase level in each group of mice; compared with the DA group). * P<0.05 ** P<0.01 *** P<0.001).

[0030] Figure 5 Statistical graph showing the effects of ZKF on blood lipids TC, TG, HDL-C, and LDL-C in DA mice (Note: compared with the DA group, * P<0.05 ** P<0.01 *** P<0.001).

[0031] Figure 6 Statistical graphs showing ZKF's effect on improving pancreatic pathological damage and inhibiting inflammatory response in DA mice (Note: Figure A is a HE-stained pathological section of pancreatic tissue (×200), Figure B is the pancreatic tissue pathological damage score, Figures C to G are the mRNA expression of pancreatic inflammation-related genes NF-κB, NLRP3, IL-1β, TNF-α, and IL-6, and Figures H and I are the detection of serum inflammatory factors TNF-α and IL-6 levels; compared with the DA group, * P<0.05 ** P<0.01 *** P<0.001).

[0032] Figure 7 Statistical charts showing the improvement of intestinal barrier integrity in DA mice by ZKF (Note: Figure A is a HE-stained pathological section of ileum tissue (×200), Figure B is the pathological damage score of ileum tissue, and Figures C to E are the mRNA expression of intestinal barrier-related genes MUC2, Occludin-1, and ZO-1; compared with the DA group, * P<0.05 ** P<0.01 *** P<0.001).

[0033] Figure 8 Figure 1 shows the statistical effects of ZKF on gut microbiota diversity and structure (Note: Figure A is the Venn plot of gut microbiota OTU numbers, Figure B is the PCA analysis of microbiota β diversity, and Figure C is the Simpson and Chao1 analysis of microbiota α diversity indices; compared with the DA group, * P<0.05 ** P<0.01 *** P<0.001).

[0034] Figure 9 Figure 1 shows the relative abundance of eight key bacterial genera in the intestines of mice in each group (Note: Figures A to D represent the key bacterial genera Akkermansia, Lactococcus, Bacteroides, and Lactobacillus; compared with group DA, ...). * P<0.05 ** P<0.01 *** P<0.001).

[0035] Figure 10 Figure 1 shows the PCA analysis of serum bile acid metabolism profiles (Note: Figure A shows the CON group and DA group; Figure B shows the ZKF group and DA group).

[0036] Figure 11 Figure 1 shows the serum bile acid metabolome characteristics (Note: Figure A is glycodeoxycholic acid (GCDCA), Figure B is glycocholic acid (GCA), Figure C is glycodeoxycholic acid (GHDCA), Figure D is glycochenodeoxycholic acid (GDCA), Figure E is glycodeoxycholic acid-3S (GDCA-3S), Figure F is deoxycholic acid-3S (DCA-3S), Figure G is lithocholic acid (LCA); compared with the DA group, * P<0.05 ** P<0.01 *** P<0.001).

[0037] Figure 12 A heatmap showing the correlation between bile acids and gut microbiota levels (Note: * P<0.05).

[0038] Figure 13 Statistical graphs showing the activation effects of ZKF on the bile acid receptor FXR / TGR5 pathway and GLP-1 secretion in DA mice (Note: Figures A to D show FXR / TGR5 mRNA expression in the liver and pancreas; Figures E to G show FXR / TGR5 protein expression and grayscale analysis in the pancreas; Figures H and I show FXR / TGR5 mRNA and protein expression in the ileum; Figures J to L show FXR / TGR5 protein expression and grayscale analysis in the ileum; Figures M and N show GLP-1 immunofluorescence staining and quantitative analysis in the ileum; compared with the DA group). * P<0.05 ** P<0.01*** P<0.001). Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that an article or device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such an article or device. Without further limitation, an element defined by the phrase "comprising..." does not exclude the presence of other identical elements in the article or device that includes said element.

[0041] Unless otherwise specified, all raw materials and excipients of the traditional Chinese medicine composition of the present invention can be obtained commercially. Any traditional Chinese medicine composition within any range described in the present invention, including end values ​​and any values ​​between end values ​​and any sub-ranges formed by end values ​​or any values ​​between end values, can achieve the effects described in the present invention.

[0042] The term "parts by weight" in this invention specification refers to the proportion of each raw material in the traditional Chinese medicine composition. Depending on the actual application, "1 part by weight" in this invention can be "1 g", "1 kg" or any other arbitrary weight.

[0043] Preparation example: The preparation method of the pharmaceutical formulation for treating acute pancreatitis in this invention is as follows: Weigh out each herbal ingredient according to the specified weight proportions. For the first decoction, submerge the herbs in cold water to a depth of 3 cm, soak for 30 minutes, then decoct for another 30 minutes. Filter out the residue and collect the liquid. For the second decoction, submerge the herbs in cold water to a depth of 2 cm, then decoct for another 30 minutes. Filter out the residue and collect the liquid. Combine the two decoctions to obtain the aqueous extract, which is the drug preparation for treating acute pancreatitis. In all test cases, the experimental group (e.g., ZKF) used the herbal composition prepared according to the components and dosages of Example 1, following the method of this preparation example.

[0044] The components and dosages of Examples 1-7 are shown in Table 1: Table 1

[0045] The components and dosages of Comparative Examples 1-4 are shown in Table 2: Table 2

[0046] Comparative Example 5: Reference (Li Huifen. Treatment of 20 cases of severe pancreatitis with oral Chinese medicine plus intraperitoneal perfusion of ligustrazine [J]. Chinese Practical Medicine, 2025, 20 (1): 112-114.) Formula: The prescription is as follows: Astragalus membranaceus 20 parts, Bupleurum chinense, Magnolia officinalis, Citrus aurantium 15 parts each, Aucklandia lappa, Paeonia lactiflora, Corydalis yanhusuo, Coptis chinensis, Picrorhiza scrophulariiflora, Lonicera japonica, Prunus persica, Carthamus tinctorius, Salvia miltiorrhiza 12 parts each, Rheum palmatum (added later) and Glauber's salt (added separately) 6 parts each.

[0047] Test Example 1: This test case is a study on the effect of the traditional Chinese medicine composition of the present invention on mice with hyperlipidemic acute pancreatitis.

[0048] This invention utilizes a mouse model of hyperlipidemia induced by poloxamer 407 (P407) combined with a mouse model of hyperlipidemic acute pancreatitis induced by cephalosporin (Cer) to verify the therapeutic effect and mechanism of action of the traditional Chinese medicine composition of this invention. The experimental design, methods, and results are as follows: 1.1 Experimental Design: Establishment of a mouse model of hyperlipidemic acute pancreatitis (HLAP): Thirty-two male C57BL / 6J mice were randomly divided into four groups of eight each: a control group (CON group), a hyperlipidemic acute pancreatitis model group (MOD group), a fenofibrate positive drug group (for lowering triglycerides), and a traditional Chinese medicine composition treatment group (ZKF group). The control group received normal food and water. The MOD group received normal food and water, and received intraperitoneal injection of P407 (300 mg / kg, intraperitoneal injection, every one day for 28 days). On day 29, mice received intraperitoneal injection of fenofibrate (Cae 150 μg / kg, once every hour for a total of 10 times), and were sacrificed 24 hours later. Mice in the positive drug group and the traditional Chinese medicine group received normal food and water, and received P407 injection (300 mg / kg, intraperitoneal injection, every one day for 28 days). From day 14 to 28, mice were administered either the positive drug fenofibrate (Example 1) or the traditional Chinese medicine composition by gavage (15.21...). On day 29, the animals were injected intraperitoneally with Cae (150 μg / kg, once every hour for a total of 10 times). They were then sacrificed 24 hours later.

[0049] 1.2 Experimental Methods: 1.2.1 HE staining of pancreas, ileum, and liver tissues: HE staining of pancreatic, ileal, and liver tissues. First, paraformaldehyde-fixed pancreatic and ileal tissues were dehydrated using a gradient ethanol series, and then embedded in paraffin. The tissue blocks were cut into 4 μm thick sections using a paraffin microtome. After slight air drying, the sections were baked at 60°C for 1 h. For HE staining, the sections were dewaxed with xylene and then hydrated with a gradient ethanol series. They were then stained with hematoxylin for 5 min and eosin for 2 min. The stained sections were dehydrated again with a gradient ethanol series and finally mounted with neutral resin. After mounting and solidification, the tissue morphology was observed under an optical microscope, and images were acquired and evaluated.

[0050] The pancreas was assessed using the Schmidt score, evaluating edema, inflammatory infiltration, acinar necrosis, and hemorrhage, with each item scored from 0 to 4 points, for a total score of 0 to 16 points. The ileum was assessed using the intestinal mucosal injury score, evaluating villous structure, crypt damage, and inflammatory infiltration, with a total score of 0 to 8 points. The liver was assessed for edema, inflammatory infiltration, hepatocellular degeneration, and necrosis, with a total score of 0 to 12 points.

[0051] 1.2.2 Serum lipid and enzyme tests: Follow the instructions to measure lipid metabolism indicators in mouse serum, including total cholesterol (TC) and triglyceride (TG) levels; at the same time, measure the activities of α-amylase (AMS) and lipase (LPS) in mouse serum.

[0052] 1.2.3 ELISA Analysis: The expression levels of interleukin-1β (IL-1β) and interleukin-6 (IL-6) in mouse serum were detected according to the experimental procedure described in the ELISA kit.

[0053] 1.2.4 Real-time quantitative PCR analysis: Real-time quantitative PCR analysis comprises three main steps: tissue RNA extraction, reverse transcription, and PCR amplification. Total RNA was extracted from mouse pancreatic tissue using Trizol reagent. Following the instructions of the Evo M-mLV reverse transcription premix kit, the extracted RNA was reverse transcribed into complementary DNA (cDNA). One μg of cDNA was diluted three-fold with RNase-free water for subsequent PCR detection. The expression of the target genes IL-1 and IL-6 in pancreatic tissue was detected, using β-actin as an internal control. Primers were designed and synthesized by Shanghai Sangon Biotech.

[0054] Experimental data were statistically analyzed and plotted using GraphPad Prism 9.5.1 software. One-way ANOVA was used for comparisons among multiple groups, with a p-value less than 0.05 considered statistically significant. All independent experiments were repeated at least three times to ensure the reliability and repeatability of the results. Data are presented as mean ± standard deviation (mean ± SD) and used for plotting.

[0055] 1.3 Experimental Results: 1.3.1 The traditional Chinese medicine composition of this invention significantly improves the damage to the pancreas, ileum, and liver tissues in HLAP mice: (1) Pancreas: In the normal group (C), the pancreatic tissue structure was intact, with clear capsule and lobular septa. Acinar structures were clearly visible, and no obvious pancreatic tissue edema, congestion, or hemorrhage points were observed. Acinar cells were tightly and neatly arranged, with no inflammatory cell infiltration. In the model group (M), significant tissue edema, congestion, and extensive acinar necrosis were visible under the microscope. A large number of neutrophils diffusely infiltrated. Nuclear dissolution, rupture, or disappearance of acinar cells was observed, with completely blurred cell outlines, and the pathological score was significantly higher than that of the normal group. In the treatment group (Z) of the traditional Chinese medicine composition in Example 1 of this invention, the pancreatic lobular structure was basically maintained. Compared with the model group, the acinar cell arrangement gradually returned to neatness, and the range and density of inflammatory cell infiltration were significantly reduced compared to the model group. The area of ​​cell edema and necrosis was significantly reduced, and the pathological score was significantly lower.

[0056] (2) Ileum: In the normal group (C), the ileum tissue structure was intact, with normal mucosa, muscularis propria, and serosa. The intestinal villi were tightly and neatly arranged, the epithelial cells were intact, and no gaps or detachment between the epithelium and lamina propria were observed. There was no edema in the lamina propria, and no obvious inflammatory cell infiltration was observed. In the model group (M), a large number of intestinal villi were disordered and structurally damaged, with decreased villi height and shortening and thickening. The gap between the mucosal epithelium and lamina propria was significantly widened. Extensive congestion and edema were observed in the lamina propria, accompanied by a large number of inflammatory cell infiltrations, and the pathological score was significantly higher than that of the control group. Compared with the model group (M), the degree of pathological damage in the ileum tissue of the treatment group (Z) of the traditional Chinese medicine composition in Example 1 of this invention was significantly improved. Microscopically, the intestinal villi were more neatly arranged, the villi structure was gradually more complete, and the height was increased. The gap between the mucosal epithelium and lamina propria was significantly reduced. Inflammatory cell infiltration in the lamina propria and interstitium was also significantly reduced, edema and congestion were significantly alleviated, and the pathological score was significantly lower than that of the model group.

[0057] (3) Liver: In the normal group (C), the liver tissue structure was intact, and the lobule outlines were clearly defined. The nuclei were clearly centered. No fat vacuoles, inflammatory cell infiltration, or necrotic foci were observed. In the model group (M), hepatocytes showed disordered arrangement, diffuse fatty degeneration within the hepatocytes, and the cytoplasm was filled with small lipid droplets and vacuoles. Macrovesicular fatty degeneration was observed in some areas. Scattered neutrophil infiltration was visible, and the pathological score was significantly elevated. In the treatment group (Z) with the herbal composition of this invention, some hepatocytes still showed fatty degeneration, but the distribution range of lipid droplets was significantly reduced compared to the model group. Inflammatory cell infiltration was significantly reduced compared to the model group, and no obvious necrotic lesions, fatty degeneration, or inflammatory damage were observed. The pathological score was significantly lower than that of the model group.

[0058] The above pathological images are shown in [the image]. Figure 1 .

[0059] 1.3.2 The traditional Chinese medicine composition of this invention significantly improves serum lipid, enzyme, pancreatic local inflammation, and systemic inflammation levels in HLAP mice: (1) In terms of enzymology, the serum amylase and lipase levels in the model group mice were significantly higher than those in the control group, confirming severe damage to pancreatic acinar cells; compared with the model group, the serum amylase and lipase levels in the treatment group of the traditional Chinese medicine composition in Example 1 were significantly lower (P<0.01), while the positive control drug fenofibrate group had no significant effect, suggesting that the traditional Chinese medicine composition of the present invention can effectively alleviate acute damage to pancreatic exocrine function (e.g., Figure 2 (A and 2B).

[0060] (2) Regarding blood lipids, compared with the control group, the serum TC and TG levels of the model group mice were significantly increased. After intervention with the traditional Chinese medicine composition of Example 1 of this invention, the serum TC and TG levels of the mice showed a significant decrease (P<0.001). However, fenofibrate in the positive control group only had a significant effect on reducing TG, and had no statistically significant effect on reducing TC levels. This indicates that the traditional Chinese medicine composition of this invention can effectively improve HLAP-related lipid metabolism disorders (such as...). Figure 2 (C and 2D).

[0061] (4) Regarding systemic inflammation, serum IL-1β and IL-6 levels were significantly elevated in the model group, while their levels significantly decreased after intervention with the herbal composition in Example 1 of this invention (P<0.001). The positive control group also showed a significant reduction in inflammatory factor levels. These results indicate that the herbal composition of this invention can effectively inhibit HLAP-induced local and systemic inflammatory responses (such as...). Figure 2 E and 2F).

[0062] Experiments have shown that the herbal composition of this invention can reduce the levels of lipase and amylase, TC, TG, and can also reduce the level of systemic inflammation. It has significant efficacy in the treatment of hyperlipidemic acute pancreatitis and is superior to the positive control drug fenofibrate treatment group in terms of reducing serum enzymes and comprehensively reducing blood lipids.

[0063] Test Example 2: This test case is an experimental study of the effects of the traditional Chinese medicine composition of the present invention on rats with hyperlipidemic acute pancreatitis.

[0064] 2.1 Experimental Design: Forty-eight SPF-grade SD rats, weighing (220±20) g, were randomly divided into eight groups of six rats each after one week of acclimatization. These groups were: sham-operated group, model group, the herbal composition group of Example 1 (10.5 g / kg), the herbal composition group of Comparative Example 1 (10.5 g / kg), the herbal composition group of Comparative Example 2 (10.5 g / kg), the herbal composition group of Comparative Example 3 (10.5 g / kg), the herbal composition group of Comparative Example 4 (10.5 g / kg), and the herbal composition group of Comparative Example 5 (10.5 g / kg). Except for the sham-operated group, the rats in the other groups were first fed a basal diet for two weeks, followed by a high-fat diet for eight weeks.

[0065] After successfully establishing the hyperlipidemia model, except for the sham-operated group, all other groups used a retrograde injection of 5% sodium taurocholate solution into the pancreatic-biliary duct to replicate the rat model of acute pancreatitis. After thorough anesthesia, the rats were fixed in a supine position with the left chest and abdomen exposed and hair removed. After disinfection with povidone-iodine, an incision was made 1 cm below the diaphragm through the midline of the abdominal wall to enter the abdomen. The subhepatic space was carefully exposed, and the confluence of the pancreatic-biliary duct into the duodenum to form papillae and the common bile duct at the porta hepatis were identified and confirmed. The common bile duct was temporarily clamped at the confluence of the hepatic ducts below the porta hepatis. The pancreatic-biliary duct was then inserted into the lateral wall of the duodenum along the papillae, punctured, and continued to be advanced antegradely for 0.5-1.0 cm parallel to the pancreatic-biliary duct. 5% sodium taurocholate was slowly injected retrogradely using a micro-infusion pump, and the needle was left in place for 4 minutes after injection. After suturing the small opening, the intestine was repositioned, and the abdomen was closed layer by layer.

[0066] Two weeks before the rats finished their high-fat diet, the drug was administered, with each group of rats being gavaged with the corresponding dose of the traditional Chinese medicine composition for two weeks. Finally, a pancreaticobiliary model was established by retrograde injection of 5% sodium taurocholate solution into the pancreatic duct.

[0067] 2.2 Experimental Methods: Lipid metabolism parameters, including total cholesterol (TC) and triglyceride (TG) levels, were detected in the serum of rat Z.

[0068] 2.2.1 ELISA method for detecting IL-6 and IL-1β levels in rats: The levels of interleukin-1β (IL-1β) and interleukin-6 (IL-6) in rat serum were detected according to the experimental procedure described in the ELISA kit.

[0069] 2.2.2 GraphPad Prism 9.5.1 software was used for data statistical analysis and graph plotting. One-way ANOVA was used for comparisons among multiple groups, and a p-value less than 0.05 was considered statistically significant. All independent experiments were repeated at least three times to ensure the reliability and repeatability of the experimental results. Data are presented in the form of mean ± standard deviation (mean ± SD) and used for graphing.

[0070] 2.3 Experimental Results: 2.3.1 Comparison of TC and TG levels in rats of different groups: Table 3 Comparison of TC and TG levels in rats of different groups

[0071] Note: Compared with the sham surgery group: * P<0.05, ** P<0.01; compared with the model group, # P < 0.05 ## P < 0.01; compared with Example 1 group, △ P<0.05, △△ P<0.01.

[0072] Compared with the sham surgery group, the model group showed significantly higher levels of TC and TG.

[0073] Compared with the model group, the TC level and TG level in Example 1 group were significantly lower. Compared with Comparative Examples 1-5, the TC level and TG level in Example 1 group were even lower. The results are shown in Table 3.

[0074] 2.3.2 Comparison of serum IL-6 and IL-1β levels in different groups of rats Table 4 Comparison of serum IL-6 and IL-1β levels in rats of different groups

[0075] Note: Compared with the sham surgery group: * P<0.05, ** P<0.01; compared with the model group, # P < 0.05 ## P < 0.01; compared with Example 1 group, △ P<0.05, △△ P<0.01.

[0076] Compared with the sham surgery group, the model group showed significantly higher levels of IL-6 and IL-1β.

[0077] Compared with the model group, the IL-6 level and IL-1β level were significantly reduced in the Example 1 group. Compared with Comparative Examples 1-5, the IL-6 level and IL-1β level were even lower in the Example 1 group. The results are shown in Table 4.

[0078] The results showed that the traditional Chinese medicine composition of the present invention has a good therapeutic effect on rats with hyperlipidemic acute pancreatitis.

[0079] 2.4 Analysis of Experimental Results Compared with Example 1, Comparative Example 1 only replaced Astragalus with Codonopsis pilosula; Comparative Example 2 only replaced Coptis chinensis with Scutellaria baicalensis; Comparative Example 3 only replaced Magnolia officinalis with Citrus aurantium; Comparative Example 4 was different from Example 1 in that it did not contain Pueraria lobata and its dosage was increased to include Magnolia officinalis and Lycium barbarum. Comparative Example 5 adopted the formula recorded in the literature, specifically: 20 parts Astragalus membranaceus, 15 parts each of Bupleurum chinense, Magnolia officinalis, and Citrus aurantium, 12 parts each of Aucklandia lappa, Paeonia lactiflora, Corydalis yanhusuo, Coptis chinensis, Picrorhiza scrophulariiflora, Lonicera japonica, Prunus persica, Carthamus tinctorius, and Salvia miltiorrhiza, and 6 parts each of Rheum palmatum (added later) and Glauber's salt (added separately), prepared according to the method of Example 1. The results showed that the efficacy of Comparative Examples 1-5 in treating hyperlipidemic acute pancreatitis was not as good as that of Example 1.

[0080] Comparative Examples 1-4 showed significantly inferior effects in improving indicators related to hyperlipidemic acute pancreatitis compared to Example 1. This suggests that the combination of the five herbs—Astragalus membranaceus, Coptis chinensis, Pueraria lobata, Magnolia officinalis, and Lycium barbarum—in the predetermined ratio in Example 1 can achieve a better therapeutic effect through synergistic effects among the herbs. Substituting, deleting, or altering the ratio of any herb will significantly weaken the overall efficacy, confirming that the formula has a clear synergistic effect.

[0081] The formulation of Comparative Example 5 differs from the traditional Chinese medicine composition of the present invention. The effect of Comparative Example 5 in improving hyperlipidemic acute pancreatitis is not as good as that of the traditional Chinese medicine composition of Example 1, suggesting that the traditional Chinese medicine composition of the present invention has a better therapeutic effect in treating hyperlipidemic acute pancreatitis.

[0082] Experimental results show that the herbal composition of the present invention is significantly superior to the comparative formulations in improving lipid metabolism disorders, reducing serum amylase and lipase activity, and inhibiting inflammatory factors in rats with hyperlipidemic acute pancreatitis, thus confirming that the composition has a synergistic effect.

[0083] Test Example 3 This test case is an experimental study of the effects of the traditional Chinese medicine composition of the present invention on diabetic mice complicated with acute pancreatitis.

[0084] This invention utilizes streptozotocin (STZ)-induced diabetic mouse models and STZ combined with ceramide (Cer)-induced diabetic mouse models complicated with acute pancreatitis, with metformin as a positive control, to verify the therapeutic effect and mechanism of action of the herbal composition in Example 1. The experimental design, methods, and results are as follows: 3.1 Experimental Design: Establishment of a mouse model of diabetes mellitus complicated with acute pancreatitis (DA): Forty male C57BL / 6J mice were randomly divided into five groups of eight each: control group (CON), acute pancreatitis group (AP), diabetes mellitus complicated with acute pancreatitis group (DA), traditional Chinese medicine treatment group (ZKF, the traditional Chinese medicine composition in Example 1), and metformin treatment group (MET). Mice in the DA, ZKF, and MET groups were fasted for 12 hours and then injected intraperitoneally with 50 mg / kg streptozotocin (STZ) for 5 consecutive days to induce a streptozotocin model. The CON group was injected with an equal volume of physiological saline. The model was considered successful one week after the model was established when the mean fasting blood glucose level was ≥11.1 mmol / L for 3 consecutive days. Subsequently, the ZKF group was administered the herbal composition of this invention by gavage at 12.35 g / kg / d, the MET group was administered metformin at 80 mg / kg / d by gavage, and the CON, DA, and AP groups were administered an equal volume of pure water by gavage. All treatments were continued for 4 weeks. During this period, blood glucose and body weight were measured at fixed times each week, and food intake and water intake were recorded. An oral glucose tolerance test (OGTT) was performed in the third week after drug administration.

[0085] After the diabetes model was successfully established, the ZKF group was administered the herbal composition of this invention at 12.35 g / kg / d by gavage, the MET group was administered metformin at 80 mg / kg / d by gavage, and the CON, AP, and DA groups were administered an equal volume of pure water by gavage for 2 weeks. During this period, blood glucose and body weight were measured at fixed times every week, and food intake and water intake were recorded. After 2 weeks of administration, acute pancreatitis was induced in the AP, DA, ZKF, and MET groups by intraperitoneal injection of spirulina extract (50 μg / kg, 6 times a day, 1 hour apart, for 2 consecutive days). The ZKF group continued to be administered the drug, and the CON group was injected with an equal volume of 0.9% NaCl solution. Blood glucose and body weight of the mice were measured again before sampling.

[0086] Construction and administration of DA mouse model Figure 3 As shown.

[0087] 3.2 Experimental Methods: 3.2.1 Mouse body weight and fasting blood glucose (FBG) detection: Each week, mice were fasted for 6 hours at a fixed time (9:00 AM) with free access to water to ensure a fasting state. Weight was then measured. During measurement, the mice were gently placed on a balance pan, and the weight was recorded after the reading stabilized. This was repeated twice, and the average was taken to minimize error. Blood glucose was then measured using the tail tip prick method. The tail tip was disinfected with an alcohol swab, and a drop of blood was gently pricked with a lancet and placed on a blood glucose test strip. The FBG value (mmol / L) displayed on the glucometer was then read. The bedding of the mice needed to be changed during the fasting period to prevent hidden food from affecting the accuracy of blood glucose readings. The entire procedure needed to be quick and gentle to minimize stress on the mice and its impact on blood glucose levels.

[0088] 3.2.2 Oral glucose tolerance test (OGTT): In model mice, oral glucose tolerance tests were performed in the third week of drug intervention. Before the test, the mice were fasted for 12 hours but allowed free access to water. Fasting blood glucose (0 min) was measured at 9:00 am the next day. Subsequently, the mice were given 5% glucose solution by gavage. Blood glucose levels were measured at 15 min, 30 min, 60 min, 90 min and 120 min after gavage. Blood glucose changes in each group of mice were recorded. Blood glucose change curves over time were plotted and the area under the curve (AUC) was calculated to evaluate the mice's ability to regulate blood glucose.

[0089] 3.2.3 HE staining of pancreatic and ileal tissues HE staining of pancreatic and ileal tissues. First, paraformaldehyde-fixed pancreatic and ileal tissues were dehydrated using a gradient ethanol series, and then embedded in paraffin. The tissue blocks were cut into 4 μm thick sections using a paraffin microtome. After slight air drying, the sections were baked at 60°C for 1 h. For HE staining, the sections were dewaxed with xylene and then hydrated with a gradient ethanol series. They were then stained with hematoxylin for 5 min and eosin for 2 min. The stained sections were dehydrated again with a gradient ethanol series and finally mounted with neutral resin. After mounting and solidification, the tissue morphology was observed under an optical microscope, and images were acquired and evaluated.

[0090] The scoring dimensions mainly cover four core damage indicators of pancreatic tissue, with a total score of 6 points. Each dimension is assigned a reverse value, as detailed below: (1) Integrity of pancreatic acini structure (2 points): Normal (no damage): 2 points (acini are neatly arranged, without edema or necrosis); Mild damage: 1.5 points (minor acinar edema, mild disordered arrangement); Moderate damage: 1 point (partial acini necrosis, significant edema); Severe damage: 0 points (numerous acinar necrosis, structural disintegration).

[0091] (2) Degree of inflammatory cell infiltration (1.5 points): Normal (no damage): 1.5 points (no inflammatory cell infiltration); Mild injury: 1 point (a small number of scattered inflammatory cells infiltrating); Moderate injury: 0.5 points (numerous inflammatory cell aggregation and infiltration); Severe injury: 0 points (diffuse inflammatory cell infiltration, accompanied by tissue congestion and edema).

[0092] (3) Degree of bleeding and edema (1.5 points): Normal (no damage): 1.5 points (no bleeding, no edema); Mild injury: 1 point (minor interstitial edema, no obvious bleeding); Moderate injury: 0.5 points (moderate edema, a few punctate hemorrhages); Severe injury: 0 points (severe edema, diffuse hemorrhage).

[0093] (4) Degree of pancreatic duct injury (1 point): Normal (no damage): 1 point (pancreatic duct structure intact, no dilation, no accumulation of secretions); Mild injury: 0.75 points (mild dilation of the pancreatic duct); Moderate injury: 0.5 points (significant dilation of the pancreatic duct and accumulation of a small amount of secretions); Severe injury: 0 points (ruptured pancreatic duct, leakage of secretions).

[0094] 3.2.4 Serum lipid and enzyme tests: Lipid metabolism parameters in the serum of two batches of mice were measured, including total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL). C) Low-density lipoprotein cholesterol (LDL-C) levels; simultaneously, serum α-cholesterol levels in each group of mice were measured. Amylase (AMS) and lipase (LPS) activities.

[0095] 3.2.5 ELISA Analysis The expression levels of insulin, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in the serum of mice in each group were detected according to the experimental procedure described in the ELISA kit.

[0096] 3.2.6 Detection of intestinal microbiota structure and abundance using 16S rDNA Total DNA was extracted from cecal contents samples of Model 2 mice using CTAB technology, and DNA purity and concentration were then assessed by agarose gel electrophoresis and Nanodrop. PCR amplification was performed targeting the V3-V4 variable region of the 16S rRNA gene. After purification, sequencing libraries were constructed using the Illumina platform, and the two ends of the sequenced data were split according to the barcode information. Bioinformatics analysis was performed using the QIIME2 workflow, including Alpha diversity, Beta diversity (PCA / PCoA), and LEfSe differential species analysis.

[0097] 3.2.7 Targeted metabolomics quantitative detection of bile acids: (1) Sample pretreatment for standard curve: The external standard mixed standard stock solution was serially diluted to the required concentration with 15% methanol (MeOH); 60 μL of each gradient standard curve solution was accurately transferred to a 1.5 mL Eppendorf tube, and 240 μL of extraction reagent containing 50 ng / mL internal standard (BA-IS) was added. The mixture was vortexed at 1500 rpm for 3 min and centrifuged at 12000 rpm for 10 min; 240 μL of the supernatant was transferred to a 1.5 mL Eppendorf tube and vacuum dried for 2.5 h; after drying, 80 μL of 15% methanol was added to reconstitute the solution, and the mixture was vortexed at 1500 rpm for 3 min and centrifuged at 12000 rpm for 10 min; all the supernatant was transferred to a sample vial for LC-MS / MS analysis.

[0098] (2) Pretreatment of mouse serum samples: Take the mouse serum stored at -80℃, accurately transfer 60 μL of serum into a 1.5 mL Eppendorf tube, add 240 μL of extraction reagent containing 50 ng / mL BA-IS, vortex at 1500 rpm for 3 min, and centrifuge at 12000 rpm for 10 min; carefully transfer 240 μL of supernatant into a new 1.5 mL Eppendorf tube, and vacuum dry for 2.5 h; after drying, add 80 μL of 15% methanol to reconstitute, vortex at 1500 rpm for 3 min, and centrifuge at 12000 rpm for 10 min; transfer all supernatant to a sample vial for LC-MS / MS detection.

[0099] (3) LC-MS / MS analysis conditions: This study used a Sciex 4500 MD liquid chromatography-mass spectrometry (LC-MS) system to quantitatively detect 70 bile acids in mouse serum, with isotope-labeled internal standard method for quantification. LC employed an ACE Excel PFPC18 column (2 μm, 2.1 × 100 mm) at a constant temperature of 40 °C; injection volume was 3 μL; total flow rate was 0.4 mL / min; and maximum pressure limit was 66 MPa. Gradient elution was performed using 2 mM ammonium acetate aqueous solution (phase A) and acetonitrile (phase B) as the mobile phase. Mass spectrometry was performed using a Turbo V electrospray ionization source in negative ion mode with selected reaction monitoring (SRM) data acquisition; ionization voltage was -4.5 kV; ion source temperature was 550 °C; curtain gas was 35 psi; nebulizer gas (GS1) was 50 psi; and auxiliary gas (GS2) was 50 psi.

[0100] 3.2.8 Immunofluorescence: Mouse ileal tissue was fixed in 4% paraformaldehyde and then paraffin-embedded and sectioned (2-5 μm) according to steps 2.3 above. After baking fixation, the sections were dewaxed with xylene and hydrated with graded ethanol. Heat-induced antigen retrieval was performed using citrate buffer to expose the antigenic epitopes. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide solution, followed by blocking nonspecific binding sites with 5-10% normal serum. The tissue was then incubated with primary antibody overnight at 4°C, followed by incubation with HRP or fluorescently labeled secondary antibody at room temperature for 30-60 min. Finally, the localization and expression level of glucagon-like peptide-1 (GLP-1) were observed and evaluated under a fluorescence microscope.

[0101] 3.2.9 Western Blot Analysis: Western blotting was used to detect the expression of FXR and TGR5 in pancreatic and ileal tissues. Briefly, total protein was extracted from pancreatic and ileal tissues using a protein lysis buffer containing phosphatase inhibitors, protease inhibitors, and benzyl sulfonyl fluoride (PMSF) in a specific ratio. The protein concentration of each sample was measured at 562 nm using a micro-spectrophotometer with a BCA protein assay kit. The protein concentration was adjusted to a uniform level with 5× protein loading buffer, and the mixture was heated in a 100°C metal bath for 10 min to denature the protein. After aliquoting, the protein was stored at -80°C. The loading volume was 50 µg of protein per well. The standard Western blotting procedure was as follows: proteins were first separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to pre-cut polyvinylidene fluoride (PVDF) membranes. After transfer, the PVDF membranes were blocked with 5% skim milk for 1 h, followed by washing three times with washing buffer for 10 min each time. The protein was then incubated overnight at 4°C with the corresponding primary antibody, washed again, and then incubated with the secondary antibody at room temperature for 1 hour. Finally, the target protein bands were visualized using a Tanon 5200 fully automated chemiluminescence imaging system, with β-actin used as an internal control. The obtained band images were analyzed for grayscale using ImageJ software.

[0102] 3.2.10 Real-time quantitative PCR analysis: Real-time quantitative PCR analysis comprises three main steps: tissue RNA extraction, reverse transcription, and PCR amplification. Total RNA was extracted from mouse pancreatic and ileal tissues using Trizol reagent. Following the instructions of the Evo M-mLV reverse transcription premix kit, the extracted RNA was reverse transcribed into complementary DNA (cDNA). One μg of cDNA was diluted three-fold with RNase-free water for subsequent PCR detection. The expression of target genes NLRP3, NF-κB, TNF-α, IL-1β, IL-6, FXR, and TGR5 in pancreatic tissue and the expression of FXR, TGR5, ZO-1, MUC2, and Occludin-1 in ileal tissue were detected, with β-actin used as an internal control. Primers were designed and synthesized by Shanghai Sangon Biotech.

[0103] 3.2.11 Statistical Analysis: For metabolomics data, we first preprocessed the raw metabolomics data for statistical analysis to ensure the reliability and accuracy of the results. Metabolites with missing values ​​exceeding 50% of the total sample and metabolites with small peak values ​​were removed. For the remaining metabolites, missing values ​​were initially imputed using half the minimum value. Unimpeded missing values ​​were first estimated and imputed using the K-Nearest Neighbor (KNN) algorithm. The processed datasets were then merged and standardized for statistical analysis. GraphPad Prism 9.5.1 software (GraphPad Software Inc., United States) was used for statistical analysis and graphing. The p-value was calculated using a t-test, and the Benjamini-Hochberg method was used to control the false discovery rate (FDR) to obtain the corrected q-value. A p-value less than 0.05 and a q-value less than 0.05 were used as the criteria for statistical significance. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed using the MetaboAnalyst online platform (http: / / www.metaboanalyst.ca) to construct a discriminant model between groups and screen for metabolites with differential enrichment between groups. Heatmaps were generated to visually display the changes in metabolite expression abundance between groups and the clustering characteristics of samples. Box plots and other results were visualized using GraphPad Prism 9.5.1 software.

[0104] Non-metabolomics data were statistically analyzed and plotted using GraphPad Prism 9.5.1 software. One-way ANOVA was used for comparisons among multiple groups, with a p-value less than 0.05 considered statistically significant. All independent experiments were repeated at least three times to ensure the reliability and reproducibility of the results. Data are presented as mean ± standard deviation (mean ± SD) and used for plotting.

[0105] 3.3 Experimental Results: 3.3.1 Improvement of glucose and lipid metabolism disorders in diabetic and DA mice: like Figure 3 , Figure 4 and Figure 5 As shown, in the DA model: the traditional Chinese medicine group in this implementation case can significantly reduce blood glucose in mice, increase insulin secretion, reduce serum amylase and lipase activity (alleviating pancreatic inflammation), and downregulate TC, TG and LDL-C, thereby improving lipid metabolism abnormalities in DA mice.

[0106] 3.3.2 Inhibition of local and systemic inflammatory responses in the pancreas: like Figure 6As shown, at the pathological level: In this case, the traditional Chinese medicine group can alleviate pancreatic islet atrophy and acinar structure destruction in the pancreatic tissue of DA mice, reduce interstitial inflammatory cell infiltration, and lower the pancreatic pathological damage score. At the molecular level: The traditional Chinese medicine group can significantly inhibit the activation of the NLRP3 / NF-κB inflammatory pathway in the pancreatic tissue of DA mice, downregulate the mRNA and protein expression of downstream pro-inflammatory factors TNF-α, IL-1β and IL-6, reduce serum TNF-α and IL-6 levels, and alleviate systemic inflammatory state.

[0107] 3.3.3 Repairing the integrity of the intestinal barrier: like Figure 7 As shown, at the pathological level: the traditional Chinese medicine group can improve ileal villus atrophy, fusion, and epithelial shedding in DA mice, reduce intestinal mucosal inflammatory cell infiltration, and lower intestinal pathological damage scores. At the molecular level: The traditional Chinese medicine group can upregulate the mRNA expression of intestinal barrier-related genes MUC2 (mucin), Occludin-1, and ZO-1 (tight junction protein), enhance intestinal barrier function, and block bacterial and endotoxin translocation.

[0108] 3.3.4 Reshaping the gut microbiota structure and regulating microbiota diversity: like Figure 8 , Figure 9 As shown, the traditional Chinese medicine group can increase the Simpson index (community dominance) and Chao1 index (community richness) of gut microbiota in DA mice, and improve gut microbiota dysbiosis. Regulate genus abundance: increase Akkermansia (Ackermania) Lactobacillus (Lactobacillus) Bacteroides The abundance of beneficial bacteria such as Bacteroides was reduced. Enterococcus The abundance of pro-inflammatory bacteria such as Enterococcus restores the bacterial community structure to a healthy state.

[0109] 3.3.5 Correcting bile acid metabolism disorders and constructing a "microbiota-bile acid" interaction chain: like Figure 10 , Figure 11 and Figure 12 As shown, the traditional Chinese medicine group can improve the serum bile acid metabolism profile of DA mice, downregulate the levels of pro-inflammatory and cytotoxic bile acids (such as lithocholic acid LCA), and upregulate the levels of anti-inflammatory and protective bile acids (such as glycine-bound bile acids such as glycine-deoxycholic acid GCDCA and glycine-deoxycholic acid GHDCA). Correlation analysis confirmed that the beneficial bacteria regulated by the traditional Chinese medicine group ( Akkermansia, Bacteroides It showed a significant positive correlation with anti-inflammatory bile acids and a significant negative correlation with pro-inflammatory bile acids.

[0110] 3.3.6 Activate the FXR / TGR5-GLP-1 signal axis (core operating mechanism): like Figure 13 As shown, the traditional Chinese medicine group can significantly upregulate the mRNA and protein expression of farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 5 (TGR5) in the liver, pancreas and ileum tissues of DA mice. It promotes the secretion of glucagon-like peptide-1 (GLP-1) by intestinal L cells. GLP-1 promotes insulin secretion and inhibits glucagon release in a glucose concentration-dependent manner, while synergistically achieving a dual effect of metabolic regulation and inflammation suppression with FXR / TGR5.

[0111] In summary, the herbal composition of this invention exerts a comprehensive therapeutic effect on a mouse model of diabetes complicated with acute pancreatitis by simultaneously improving glucose and lipid metabolism disorders, inhibiting inflammatory responses, and repairing the intestinal barrier through the signaling axis of "reshaping intestinal flora - correcting bile acid metabolism - activating FXR / TGR5 - promoting GLP-1 secretion".

[0112] Test Example 4: This test case is an experimental study of the effects of the traditional Chinese medicine composition of the present invention on diabetic rats with acute pancreatitis.

[0113] 4.1 Experimental Design: Thirty-six type 2 diabetes mellitus (T2DM) rats, weighing approximately 180 g, were fasted for 12 hours prior to surgery but allowed free access to water. The rats were anesthetized with intraperitoneal injection of pentobarbital solution (2 mg / 100 g body weight), and a T2DM combined with acute pancreatitis (AP) model was established by retrograde injection of 3.5% sodium taurocholate (1 mL / kg body weight) along the pancreatic duct at a rate of 0.2 mL / min.

[0114] The criteria for successful animal model preparation are: within 24 hours of injection of 3.5% sodium taurocholate, rats show obvious AP symptoms, namely, rats exhibit anxiety, irritability, and vomiting; and the pancreatic tissue sections of rats show tissue edema, hemorrhage, indistinct tissue boundaries, and severe tissue infiltration.

[0115] After successful modeling, the rats in the modeling group were randomly divided into the T2DM combined AP model group, Example 1 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group and Comparative Example 5 group, with 6 rats in each group.

[0116] In Example 1, rats were administered the drug solution of Example 1 at 12.35 g / kg / d via gavage. Comparative Examples 1-3 and 5 were administered the drug solutions of Comparative Examples 1-3 and 5 at 12.35 g / kg / d via gavage, respectively. The model group rats were administered an equal volume of 0.9% NaCl solution via gavage. All groups of rats were administered the drug once daily for 28 consecutive days.

[0117] 4.2 Experimental Methods: 4.2.1 Detection of serum amylase and lipase in rats: Two mL of venous blood was collected from each group of rats after 48 hours of administration of 3.5% sodium taurocholate or physiological saline. The blood was centrifuged at 1000g for 10 min, and the serum was collected and aliquoted into sterile EP tubes. Serum amylase and lipase were detected using a fully automated biochemical analyzer.

[0118] 4.2.2 Histopathological evaluation of pancreatic tissue: Pancreatic head tissue was collected, fixed with 100 mL / L formaldehyde solution, routinely dehydrated, embedded, sectioned, stained with hematoxylin and eosin (HE), and pathologically scored for pancreatic damage.

[0119] 4.2.3 GraphPad Prism 9.5.1 software was used for data statistical analysis and graph plotting. One-way ANOVA was used for comparisons among multiple groups, and a p-value less than 0.05 was considered statistically significant. All independent experiments were repeated at least three times to ensure the reliability and repeatability of the experimental results. Data are presented in the form of mean ± standard deviation (mean ± SD) and used for graphing.

[0120] 4.3 Experimental Results: 4.3.1 Comparison of amylase and lipase levels in rats of different groups: Table 5 Comparison of amylase and lipase levels in rats of different groups

[0121] Note: Compared with the model group, * P < 0.05 ** P < 0.01; compared with Example 1 group, # P < 0.05 ## P < 0.01.

[0122] Compared with the model group, the amylase activity and lipase activity in the Example 1 group were significantly reduced. Compared with comparative groups 1-3 and 5, the amylase activity and lipase activity in the Example 1 group were even lower. The results are shown in Table 1.

[0123] 4.3.2 Comparison of pathological scores among rats in different groups Table 6 Comparison of pathological scores of rats in each group

[0124] Note: Compared with the model group, * P < 0.05 ** P < 0.01; compared with Example 1 group, # P < 0.05 ## P < 0.01.

[0125] Compared with the model group, the pathology score of the Example 1 group was significantly lower. Compared with comparative groups 1-3 and 5, the pathology score of the Example 1 group was even lower. The results are shown in Table 2.

[0126] The results showed that the traditional Chinese medicine composition of the present invention has a good therapeutic effect on a rat model of diabetes mellitus complicated with acute pancreatitis.

[0127] 4.4 Analysis of Experimental Results Comparative Example 1 consisted of: 40 parts Codonopsis pilosula, 15 parts Coptis chinensis, 12 parts Pueraria lobata, 12 parts Magnolia officinalis, and 15 parts Lycium barbarum. Compared with Example 1, only Codonopsis pilosula replaced Astragalus membranaceus. Comparative Example 2 consisted of: 40 parts Astragalus membranaceus, 15 parts Scutellaria baicalensis, 12 parts Pueraria lobata, 12 parts Magnolia officinalis, and 15 parts Lycium barbarum. Compared with Example 1, only Scutellaria baicalensis replaced Coptis chinensis. Comparative Example 3 consisted of: 40 parts Astragalus membranaceus, 15 parts Coptis chinensis, 12 parts Pueraria lobata, 12 parts Citrus aurantium, and 15 parts Lycium barbarum. Compared with Example 1, only Citrus aurantium replaced Magnolia officinalis. Comparative Example 5 used the formula recorded in the literature, specifically: 20 parts Astragalus membranaceus, 15 parts each of Bupleurum chinense, Magnolia officinalis, and Citrus aurantium, 12 parts each of Aucklandia lappa, Paeonia lactiflora, Corydalis yanhusuo, Coptis chinensis, Picrorhiza scrophulariiflora, Lonicera japonica, Prunus persica, Carthamus tinctorius, and Salvia miltiorrhiza, and 6 parts each of Rheum palmatum (added later) and Glauber's salt (added separately). It was prepared according to the method of Example 1. The results showed that the efficacy of comparative groups 1-3 and 5 in treating diabetes mellitus complicated with acute pancreatitis was not as good as that in Example 1.

[0128] The formulation of Comparative Example 5 differs from the traditional Chinese medicine composition of the present invention. The effect of Comparative Example 5 in improving diabetes mellitus complicated with acute pancreatitis is not as good as that of the traditional Chinese medicine composition of Example 1, suggesting that the traditional Chinese medicine composition of the present invention has a better therapeutic effect in treating diabetes mellitus complicated with acute pancreatitis.

[0129] The results showed that the traditional Chinese medicine composition of the present invention could significantly reduce the serum amylase and lipase activities in diabetic rats with acute pancreatitis and alleviate pancreatic pathological damage. Its efficacy was significantly better than that of the comparative formulations, further verifying the clinical value of the present invention.

[0130] In summary, the herbal composition of this invention uses Astragalus membranaceus and Lycium barbarum as the principal herbs to invigorate Qi and nourish Yin, Coptis chinensis and Pueraria lobata as the assistant herbs to nourish Yin and clear heat, and Magnolia officinalis as the adjuvant herbs to promote Qi circulation and relieve fullness. Together, they achieve the effects of invigorating Qi and nourishing Yin, clearing heat and promoting blood circulation, and addressing both the root cause and the symptoms. Pharmacological experiments have confirmed that this composition can significantly reduce serum triglyceride, total cholesterol, low-density lipoprotein, blood glucose, amylase, and lipase levels in hyperlipidemic acute pancreatitis and diabetic acute pancreatitis models, increase insulin secretion, inhibit the NLRP3 / NF-κB inflammatory pathway and the expression of pro-inflammatory factors such as IL-1β, IL-6, and TNF-α, alleviate pathological damage to the pancreas, ileum, and liver, and repair the intestinal barrier. This invention upregulates ZO-1, Occludin-1, and MUC2, and achieves a synergistic effect of metabolic regulation and inflammation suppression by reshaping the gut microbiota (increasing beneficial bacteria such as Akkermansia and Lactobacillus), correcting the bile acid metabolism profile, activating FXR / TGR5 receptors, and promoting GLP-1 secretion. Comparative experiments show that replacing or deleting any of the herbs in the formula or using existing technologies to formulate the formula cannot achieve the same therapeutic effect, confirming that the formula has a significant synergistic effect. Therefore, the herbal composition of this invention has clear clinical application prospects and industrial value in the preparation of drugs for the prevention or treatment of diabetes mellitus complicated with acute pancreatitis and hyperlipidemic acute pancreatitis.

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A traditional Chinese medicine composition for acute pancreatitis, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: Astragalus membranaceus 35-45 parts, Coptis chinensis 12-18 parts, Pueraria lobata 10-15 parts, Magnolia officinalis 10-15 parts, and Lycium barbarum 12-18 parts; the acute pancreatitis is acute pancreatitis complicated with diabetes mellitus or acute pancreatitis caused by hyperlipidemia.

2. The traditional Chinese medicine composition for acute pancreatitis according to claim 1, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: Astragalus membranaceus 40 parts, Coptis chinensis 15 parts, Pueraria lobata 12 parts, Magnolia officinalis 12 parts, and Lycium barbarum 15 parts.

3. The traditional Chinese medicine composition for acute pancreatitis according to claim 1, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: Astragalus membranaceus 45 parts, Coptis chinensis 12 parts, Pueraria lobata 10 parts, Magnolia officinalis 15 parts, and Lycium barbarum 18 parts.

4. The traditional Chinese medicine composition for acute pancreatitis according to claim 1, characterized in that, The traditional Chinese medicine composition is made from the following raw materials in parts by weight: Astragalus membranaceus 35 parts, Coptis chinensis 15 parts, Pueraria lobata 12 parts, Magnolia officinalis 10 parts, and Lycium barbarum 15 parts.

5. A pharmaceutical preparation for treating acute pancreatitis, characterized in that, The pharmaceutical preparation comprises the traditional Chinese medicine composition for acute pancreatitis as described in any one of claims 1-4.

6. The pharmaceutical preparation for treating acute pancreatitis according to claim 5, characterized in that, The dosage form of the pharmaceutical preparation is granules, tablets, capsules, or decoction.

7. A method for preparing a pharmaceutical preparation for treating acute pancreatitis as described in claim 6, characterized in that, The preparation method includes the following steps: water extraction of the traditional Chinese medicine composition, wherein the water extraction method includes the following steps: weighing each traditional Chinese medicine raw material according to the weight parts, first decoction, cold water covering the surface of the medicine by 3 cm, soaking for 30 min, then decocting for 30 min, filtering the residue and taking the juice; second decoction, cold water covering the surface of the medicine by 2 cm, decocting for 30 min, filtering the residue and taking the juice, mixing the two decoctions to obtain an aqueous extract; and preparing the aqueous extract into the dosage form of the pharmaceutical preparation.

8. The use of the traditional Chinese medicine composition according to any one of claims 1-4 in the preparation of drugs for the prevention or treatment of diabetes mellitus complicated with acute pancreatitis, hyperlipidemic acute pancreatitis, cholesteatogenic acute pancreatitis, and biliary acute pancreatitis.

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