Traditional Chinese medicine composition for treating post-infection irritable bowel syndrome as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention belongs to the field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine composition for treating post-infection irritable bowel syndrome as well as a preparation method and application thereof. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 10 to 15 parts of rhizoma atractylodis macrocephalae, 10 to 20 parts of radix paeoniae alba, 3 to 7 parts of pericarpium citri reticulatae, 5 to 15 parts of radix saposhnikoviae, 10 to 20 parts of folium microcotis, 10 to 20 parts of plumeria rubra, 5 to 15 parts of kapok, 25 to 35 parts of rhizoma smilacis glabrae and 5 to 15 parts of flos albiziae. Animal experiments show that the traditional Chinese medicine composition can effectively improve diarrhea phenotypes and depressive symptoms of rats with PI-IBS liver depression and spleen deficiency syndromes, relieve high sensitivity of viscera, recover a tight junction structure and a cell desmosome structure of colon, up-regulate cell TJ protein expression, recover Th1 / Th2 immune imbalance of blood, reduce the level of proinflammatory factors (TNF-alpha) in colon tissue, and improve the immunity of the rats. And the levels of LPS, DAO and D-LA in serum are up-regulated. Therefore, the traditional Chinese medicine composition disclosed by the invention can be used for effectively treating the liver depression and spleen deficiency syndrome of the irritable bowel syndrome after infection.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine, specifically relating to a traditional Chinese medicine composition for treating post-infectious irritable bowel syndrome, its preparation method, and its application. Background Technology

[0002] Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder in clinical practice. Post-infective irritable bowel syndrome (PI-IBS) is a common subtype of IBS, characterized by persistent abdominal pain, diarrhea, and other discomfort symptoms even after the relief of acute gastrointestinal infection symptoms. It falls under the category of diarrhea-predominant irritable bowel syndrome (IBS-D). Current research has clarified the specific pathogen-induced role of PI-IBS. However, the pathological mechanism of persistent abdominal pain and abnormal defecation after the acute gastrointestinal infection process has not been fully elucidated, resulting in a lack of clear and effective treatment and imposing a heavy economic burden on families and society.

[0003] Currently, clinical treatment for PI-IBS mainly focuses on symptomatic relief, such as using antidiarrheal drugs like montmorillonite powder and loperamide to relieve diarrhea symptoms; using atropine and pinaverium bromide to relieve intestinal smooth muscle spasms and abdominal pain symptoms; and using antidepressants like amitriptyline to relieve depressive symptoms. However, Western medicine treatments only address the symptoms and not the root cause, and the clinical treatment results are often unsatisfactory.

[0004] Traditional Chinese medicine (TCM) possesses unique advantages in treating IBS due to its multi-target efficacy and minimal side effects. IBS falls under the TCM categories of "abdominal pain," "diarrhea," and "constipation," making it one of the diseases where TCM has a strong advantage in prevention and treatment. In recent years, research reports on TCM interventions for IBS and other functional gastrointestinal disorders have been frequently published internationally. Currently, TCM compositions used clinically to treat IBS include Si Ni San, Chai Hu Shu Gan San, Shen Ling Bai Zhu San, and Tong Xie Ning granules. However, for PI-IBS, these TCM compositions often fail to achieve satisfactory results. Therefore, it is necessary to develop a TCM composition that can effectively treat PI-IBS, targeting its unique pathogenesis, to meet clinical needs. Summary of the Invention

[0005] The first objective of this invention is to provide a traditional Chinese medicine composition for treating post-infectious irritable bowel syndrome; the second objective of this invention is to provide a method for preparing the traditional Chinese medicine composition; and the third objective of this invention is to provide the application of the traditional Chinese medicine composition.

[0006] According to a first aspect of the present invention, a traditional Chinese medicine composition for treating post-infectious irritable bowel syndrome is provided, comprising, by weight, the following raw materials: 10-15 parts of Atractylodes macrocephala, 10-20 parts of Paeonia lactiflora, 3-7 parts of Citrus reticulata peel, 5-15 parts of Saposhnikovia divaricata, 10-20 parts of Brussels sprouts, 10-20 parts of Plumeria rubra, 5-15 parts of Bombax ceiba, 25-35 parts of Smilax glabra, and 5-15 parts of Albizia julibrissin.

[0007] This invention posits that spleen and stomach weakness and liver dysfunction exist throughout the entire pathogenesis of diarrhea-predominant irritable bowel syndrome (IBS-D), with liver stagnation and spleen deficiency being the core pathogenesis. Post-infectious irritable bowel syndrome (PI-IBS) not only exhibits the typical core symptoms of IBS-D such as abdominal pain and diarrhea, but also displays unique clinical heterogeneity due to its infectious etiology. After infection, the body fails to completely eliminate the pathogen, and the remaining pathogens struggle against the body's vital energy, leading to a persistent pathological state of "struggle between the body's resistance and pathogenic factors." This state causes recurrent and prolonged clinical symptoms. This struggle between the body's resistance and pathogenic factors makes the treatment of PI-IBS more complex, requiring further elimination of latent pathogens in addition to soothing the liver and strengthening the spleen. Therefore, this invention believes that the pathogenesis of PI-IBS can be summarized as a complex syndrome of "liver stagnation and spleen deficiency" coexisting with "unresolved pathogenic factors," in which dampness plays a crucial role as a key pathological factor.

[0008] Dampness-related diseases are characterized by their heaviness, turbidity, and stickiness. Their pathological evolution is mainly reflected in three aspects: First, dampness easily obstructs the middle jiao, affecting the ascending and descending of qi in the spleen and stomach, leading to qi stagnation and dampness obstruction, clinically manifested as symptoms such as abdominal discomfort and abdominal pain; Second, dampness easily damages spleen yang, causing the spleen to lose its ability to transport and transform, resulting in the retention of water and dampness internally, and the inability to distinguish between clear and turbid substances, leading to diarrhea; Third, dampness has the characteristic of being lingering and difficult to cure, and can transform into heat or cold, or combine with external pathogens to cause disease. This is the pathological basis for the recurrent symptoms of PI-IBS.

[0009] Therefore, this invention posits that dampness is the latent pathogenic factor in PI-IBS, and liver stagnation and spleen deficiency are the key pathological mechanisms of PI-IBS. Based on liver stagnation and spleen deficiency, and disharmony between the liver and spleen, there is also a state of struggle between pathogenic factors and the body's resistance, with lingering pathogenic factors and latent pathogenic factors remaining internally.

[0010] Based on the above understanding, and targeting the core pathogenesis of PI-IBS with liver stagnation, spleen deficiency, and internal dampness, this invention uses Atractylodes macrocephala as the chief ingredient to invigorate the spleen, replenish qi, and dry dampness to stop diarrhea. The assistant ingredients are Paeonia lactiflora to soothe the liver and relieve urgency, Saposhnikovia divaricata to soothe the liver and regulate the spleen, and Citrus reticulata to regulate qi and harmonize the middle jiao, assisting Atractylodes macrocephala in restoring spleen function. Together, they harmonize the liver and spleen and treat "painful diarrhea." The adjuvant ingredients, Smilax glabra, together with Brussels sprouts, Plumeria rubra, and Bombax ceiba, form a powerful dampness-removing combination, targeting the key pathogenesis of lingering damp-heat in the intestines after infection. The high dosage of Smilax glabra highlights its central role in clearing intestinal damp-toxins and repairing the intestinal mucosal barrier. Albizia julibrissin, which soothes the liver and relieves stagnation, and regulates brain-gut axis dysfunction, also serves as an adjuvant. The entire formula is meticulously formulated, addressing both the root cause and symptoms, achieving the combined effects of invigorating the spleen, soothing the liver, and removing dampness to stop diarrhea. This invention, while invigorating the spleen and soothing the liver, enhances the function of eliminating dampness and pathogens, allowing dampness to be expelled through urination and defecation. The formula not only addresses the core pathogenesis of liver stagnation and spleen deficiency, but also takes into account the struggle between pathogenic factors and the body's resistance, allowing dampness to be expelled. It has been used in clinical practice for a long time with remarkable efficacy.

[0011] The raw materials for this invention are sourced as follows: Atractylodes macrocephala: The rhizome of Atractylodes macrocephala Koidz., a plant in the Asteraceae family.

[0012] White peony root: The root of Paeonia lactiflora Pall., a plant in the Paeoniaceae family.

[0013] Chenpi: The dried, mature peel of the citrus fruit and its cultivated varieties, belonging to the Rutaceae family.

[0014] Saposhnikovia root: The root of Saposhnikovia divaricata, a plant in the Apiaceae family.

[0015] Microcos paniculata L., a plant in the Tiliaceae family, is a dried leaf.

[0016] Frangipani: The flower of Plumeria rubra L. var. acutifolia (Poir.) Bailey, a plant in the Apocynaceae family.

[0017] Kapok flower: The flower of the kapok tree (Gossampinus malabbarica (DC.) Merr.) of the Bombacaceae family.

[0018] Smilax glabra Roxb., a plant in the Liliaceae family, is a dried rhizome.

[0019] Albizia flower: The flower or bud of the Albizia julibrissin plant, a legume.

[0020] In some embodiments, the raw material composition by weight includes: 12 parts of Atractylodes macrocephala, 15 parts of Paeonia lactiflora, 5 parts of Citrus reticulata peel, 10 parts of Saposhnikovia divaricata, 15 parts of Brussels sprout leaves, 15 parts of Plumeria rubra, 9 parts of Bombax ceiba, 30 parts of Smilax glabra, and 10 parts of Albizia julibrissin flowers.

[0021] In some implementations, Atractylodes macrocephala is stir-fried, and Paeonia lactiflora is stir-fried. Stir-fried Atractylodes macrocephala enhances the spleen-strengthening and dampness-drying effects compared to raw Atractylodes macrocephala, while stir-fried Paeonia lactiflora neutralizes the cooling properties of the herb, making it more effective in improving diarrhea symptoms.

[0022] In some implementations, pharmaceutically acceptable excipients may also be added.

[0023] In some embodiments, the dosage form of the traditional Chinese medicine composition is a decoction, granules, powder, tablets, pills, or capsules.

[0024] According to a second aspect of the present invention, a method for preparing the above-mentioned traditional Chinese medicine composition for treating post-infectious irritable bowel syndrome is provided, comprising the following steps: Soak the raw materials in 4-8 times their weight of water for 20-40 minutes, then boil over high heat for 30-40 minutes, then simmer over low heat for 30-40 minutes. Filter the mixture and repeat the boiling process on the residue 1-2 times. Combine the filtrates to obtain the final product.

[0025] According to a third aspect of the present invention, the use of the above-described traditional Chinese medicine composition for treating post-infectious irritable bowel syndrome in the preparation of a medicament for treating post-infectious irritable bowel syndrome is provided.

[0026] According to a fourth aspect of the present invention, the above-described traditional Chinese medicine composition for treating post-infectious irritable bowel syndrome is provided for use in the preparation of a medicament for treating liver stagnation and spleen deficiency syndrome of post-infectious irritable bowel syndrome.

[0027] The beneficial effects of this invention include: Animal experiments have shown that the traditional Chinese medicine composition of this invention can effectively improve the diarrhea phenotype and depressive symptoms in rats with PI-IBS liver stagnation and spleen deficiency syndrome, reduce visceral hypersensitivity, restore the tight junction structure and desmosome structure of the colon, upregulate cellular TJ protein expression, restore the blood Th1 / Th2 immune imbalance, reduce the level of pro-inflammatory factors (TNF-α) in colonic tissue, and upregulate the levels of LPS, DAO, and D-LA in serum. This suggests that the traditional Chinese medicine composition of this invention can effectively treat liver stagnation and spleen deficiency syndrome in post-infectious irritable bowel syndrome. Attached Figure Description

[0028] Figure 1 This is a flowchart of the preparation process of Trichinella spiralis suspension in animal experiments.

[0029] Figure 2 It is a flowchart of animal modeling and drug administration in animal experiments.

[0030] Figure 3 This is a comparison of the body weights of rats in different groups during an animal experiment.

[0031] Figure 4 This is a comparison of fecal water content in different groups of rats at week 6 in an animal experiment.

[0032] Figure 5 This is a comparison of the sucrose preference index of rats in different groups at week 6 in animal experiments.

[0033] Figure 6 This is a comparison of the open field test results of rats in different groups during week 6 in animal experiments.

[0034] Figure 7 This describes the visceral motor responses of rats in each group during week 6 of an animal experiment.

[0035] Figure 8 This shows the HE staining results of rats in each group at week 6 in animal experiments.

[0036] Figure 9 This is an ultrastructural observation (transmission electron microscopy) of the colonic epithelial cells and intercellular connections of rats in each group during week 6 of an animal experiment.

[0037] Figure 10 This refers to the changes and ratios of Th1 and Th2 cells in the blood PBMCs of rats in each group during week 6 of an animal experiment.

[0038] Figure 11 These are the results of detecting the levels of TNF-α and IL-10 in the colon tissue of rats in each group during week 6 of an animal experiment.

[0039] Figure 12 These are the levels of LPS, DAO, and D-LA in the serum of rats in each group during week 6 of an animal experiment.

[0040] Figure 13 This shows the expression of intestinal mucosal barrier-related proteins in each group of rats at week 6 in animal experiments. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. The materials involved in the following embodiments are all available from commercial sources. Unless otherwise specified, the experimental methods employed are conventional methods known in the art.

[0042] Example 1 The traditional Chinese medicine decoction for treating post-infectious irritable bowel syndrome in this embodiment is prepared from the following raw materials: 12g of stir-fried Atractylodes macrocephala, 15g of stir-fried Paeonia lactiflora, 5g of Citrus reticulata peel, 10g of Saposhnikovia divaricata, 15g of Brussels sprout leaves, 15g of Plumeria rubra flower, 9g of Bombax ceiba flower, 30g of Smilax glabra rhizome, and 10g of Albizia julibrissin flower.

[0043] Its preparation method includes the following steps: Add the raw materials to 500ml of water and soak for 30 minutes. Then boil over high heat for 35 minutes and simmer over low heat for 40 minutes. Collect 150mL of decoction. Add another 500ml of water to the dregs and continue to decoct until 150mL remains. Combine the decoctions obtained from the two decoctions to obtain the final product.

[0044] The traditional Chinese medicine decoction prepared in this embodiment is a single dose, which is approximately 300 mL.

[0045] Example 2 The traditional Chinese medicine decoction for treating post-infectious irritable bowel syndrome in this embodiment is prepared from the following raw materials: 12g of stir-fried Atractylodes macrocephala, 15g of stir-fried Paeonia lactiflora, 5g of Citrus reticulata peel, 10g of Saposhnikovia divaricata, 15g of Brussels sprout leaves, 15g of Plumeria rubra flower, 9g of Bombax ceiba flower, 30g of Smilax glabra rhizome, and 10g of Albizia julibrissin flower.

[0046] Its preparation method includes the following steps: After preparing the medicinal juice according to the method in Example 1, the juice was concentrated to 1.21 g crude drug / mL using a rotary evaporator. It was then stored at -80°C for later use.

[0047] Example 3 The traditional Chinese medicine decoction for treating post-infectious irritable bowel syndrome in this embodiment is prepared from the following raw materials: 15g of Atractylodes macrocephala, 20g of Paeonia lactiflora, 6g of Citrus reticulata peel, 15g of Saposhnikovia divaricata, 20g of Brussels sprout leaves, 20g of Plumeria rubra flower, 15g of Bombax ceiba flower, 35g of Smilax glabra rhizome, and 15g of Albizia julibrissin flower.

[0048] Its preparation method is the same as that of Example 1.

[0049] Example 4 The traditional Chinese medicine decoction for treating post-infectious irritable bowel syndrome in this embodiment is prepared from the following raw materials: 10g of Atractylodes macrocephala, 10g of Paeonia lactiflora, 3g of Citrus reticulata peel, 5g of Saposhnikovia divaricata, 10g of Brussels sprout leaves, 10g of Plumeria rubra flower, 5g of Bombax ceiba flower, 25g of Smilax glabra rhizome, and 5g of Albizia julibrissin flower.

[0050] Its preparation method is the same as that of Example 1.

[0051] To verify the therapeutic effect of the traditional Chinese medicine composition of the present invention on post-infectious irritable bowel syndrome, the following animal experiments were conducted.

[0052] I. Laboratory Animals Thirty-six 7-week-old SPF-grade male Wistar rats were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. (Certificate No. A202409077) and housed in the SPF-grade laboratory of the Animal Experiment Center of Guangzhou University of Chinese Medicine. The temperature was 22±2℃, the humidity was 50±10%, and the light and dark cycle was 12h. The rats had free access to food and water and were kept in an acclimatization environment for one week before the formal experiments began.

[0053] II. Grouping Thirty-six Wistar rats were randomly divided into six groups using a random number table: a normal control group (Control group), a model group (PI-IBS group), a low-dose group of traditional Chinese medicine composition (MTXYF-L group), a medium-dose group of traditional Chinese medicine composition (MTXYF-M group), a high-dose group of traditional Chinese medicine composition (MTXYF-H group), and a positive control drug compound glutamine group (CGEC group), with six rats in each group.

[0054] III. Modeling Methods A combined model of PI-IBS (liver stagnation and spleen deficiency syndrome) was constructed using a trichinella infection method and restraint stress. After acclimatization, rats in all groups except the control group were administered 1 mL of trichinella suspension via gavage once daily. Restraint stress was then introduced every other day for 6 weeks, with the daily restraint time gradually increasing from 30 minutes to 3 hours. Restraint stress involved placing the rats in a restraint bag that restricted their limbs but did not impair their breathing, and then removing them from the restraint bag and placing them back into their cages after the designated time.

[0055] The preparation process of Trichinella spiralis suspension is as follows: Figure 1 As shown in the figure, steps 1-3: Microscopic examination of the diaphragm of the breeding rat to confirm infection with Trichinella spiralis; step 4: Digestion of the breeding rat muscle; step 5: Washing the precipitate; step 6: Collection of the suspension; and steps 7-8: Microscopic examination of the suspension. The specific steps for preparing Trichinella spiralis suspension are as follows: (1) Sample collection After euthanizing the C57BL / 6 mice by enucleating their eyeballs to collect blood, their fur, fat, and internal organs were removed. Microscopic examination of the diaphragm revealed a large number of Trichinella cysts. The masseter muscle, diaphragm, temporalis muscle, and other muscles of the mice were then cut off and minced into meat paste.

[0056] (2) Sample digestion The digestive solution was prepared at a ratio of 10 mL of 36% concentrated hydrochloric acid + 1 L of distilled water + 10 g of pepsin. The minced meat and digestive solution were mixed in a beaker at a ratio of 20 mL of digestive solution to 1 g of minced meat sample. The beaker was sealed with sealing film to prevent splashing during digestion. The beaker was then placed in a 37°C constant-temperature shaker and digested at 180 rpm for 3 hours.

[0057] (3) Sample filtration Use a 60-mesh (250μm aperture) stainless steel sieve placed on a 1000mL beaker to filter out the digestion residue; rinse the residue with distilled water until the beaker is filled to its maximum capacity, then let it stand at room temperature for 1 hour. After the experiment, sterilize the filtered residue and used equipment by boiling at high temperature and then clean them.

[0058] (4) Sample precipitation and rinsing Use a 50mL syringe to slowly draw the supernatant along the wall of the beaker until 80% of the supernatant is removed. Transfer the remaining precipitate to a 500mL beaker, add distilled water to the maximum volume, and let it stand for 40 minutes. Then, use the syringe to slowly draw off the supernatant along the wall of the beaker until 80% of the supernatant is removed. Transfer the remaining precipitate to a smaller beaker, let it stand, and then draw off the supernatant. Repeat this process five times until the remaining precipitate is relatively clear. Collect the insect bodies and titrate to a volume of 10mL to obtain the concentrated insect body solution.

[0059] (5) Counting and gavage 10 mL of the concentrated worm body solution was stirred and mixed to obtain a Trichinella spiralis suspension. While mixing, 10 μL of the Trichinella spiralis suspension was pipetted onto a glass slide, covered with a coverslip, and the worms were counted under a microscope. The counted Trichinella spiralis suspension was titrated to 1000 worms / mL to obtain a Trichinella spiralis suspension. 1 mL of the Trichinella spiralis suspension was administered orally to each rat model by gavage.

[0060] Rats in the model group, low-dose group of traditional Chinese medicine composition, medium-dose group of traditional Chinese medicine composition, high-dose group of traditional Chinese medicine composition, and positive drug compound glutamine group were modeled according to the above modeling method, while rats in the normal group were fed normally. IV. Administration Two weeks after the initial restraint stress, drug intervention was initiated, with each group receiving the intervention according to the following dosing regimen: Control group: The same volume of 0.9% saline was administered by gavage for 2 consecutive weeks.

[0061] Model group (PI-IBS group): Administered an equal volume of 0.9% saline by gavage for 2 consecutive weeks.

[0062] Low-dose group of traditional Chinese medicine composition (MTXYF-L group): The traditional Chinese medicine decoction of Example 2 was administered by gavage at a dose of 5.45 g crude drug / kg / d for 2 consecutive weeks.

[0063] Medium-dose group of traditional Chinese medicine composition (MTXYF-M): The traditional Chinese medicine decoction of Example 2 was administered by gavage at a dose of 10.89 g crude drug / kg / d for 2 consecutive weeks.

[0064] High-dose group of traditional Chinese medicine composition (MTXYF-H): The traditional Chinese medicine decoction of Example 2 was administered by gavage at a dose of 21.78 g crude drug / kg / d for 2 consecutive weeks.

[0065] The positive control group (CGEC group) was administered compound glutamine aqueous solution by gavage at a dose of 0.1152 g / kg / day for two consecutive weeks. The compound glutamine aqueous solution was prepared by dissolving compound glutamine enteric-coated capsules (purchased from the pharmacy of Guangdong Provincial Hospital of Traditional Chinese Medicine, specification 320mg / capsule) in distilled water to obtain a concentration of 0.0035g / mL.

[0066] Animal experimental modeling and drug administration flowchart as follows Figure 2 As shown.

[0067] V. Evaluation Indicators 1. General condition and behavioral testing The body weight of rats in each group was measured, and their behavioral characteristics were observed, including fecal water content, open space, sucrose consumption, and visceral motor responses.

[0068] (1) Sugar water preference index The sucrose consumption study included an adaptation training phase and a testing phase. In the adaptation training phase, each cage of rats was given a 1% (w / v) sucrose solution on the first day. After 24 hours, one bottle of sucrose solution was replaced with pure water. After 12 hours, the sucrose solution and pure water were swapped, and adaptation continued for another 12 hours. After adaptation, the rats were fasted and deprived of water for 23 hours before the testing phase began. In the testing phase, each cage of rats was given one 200mL bottle of 1% (w / v) sucrose solution and one 200mL bottle of pure water, and the weights were recorded. After one hour of free drinking, both bottles were removed, and the rats were weighed again, recording the remaining sucrose solution and pure water volumes.

[0069] Sugar water preference (%) = (Sugar water consumption) / [(Sugar water consumption) + (Pure water consumption)] * 100% (2) Open field experiment The open field experimental setup consists of two parts: an open field reaction chamber and an automatic data acquisition device. The rat reaction chamber has a base length of 100cm and is divided into 25 small squares of 4*4cm each. The border is white, and the bottom is black. A digital camera at the top collects and records the rats' movement trajectories within the chamber. Before each experiment, to eliminate the influence of rat odor and other exogenous factors, the open field reaction chamber is wiped clean with 75% alcohol. The experiment begins after the alcohol has evaporated. The rat is placed in the center of the chamber, and its movement trajectory is recorded for 5 minutes. After the experiment, the inside of the chamber is cleaned again to prevent residual odors from affecting the test results of the next rat. The next rat is replaced after the previous one has been tested, until all tests are completed.

[0070] (3) Visceral motor response Before the experiment, rats in each group were fasted for 12 hours but allowed free access to water. The skin on the abdomen and inner thighs of each rat was shaved beforehand. Gas anesthesia was initiated using a gas anesthesia machine combined with isoflurane at a concentration of 5% isoflurane. After successful induction of anesthesia, 0.9-1.1% isoflurane was used to maintain anesthesia. Before the computer program started, red / green recording electrodes were shallowly inserted into the external oblique muscles of the left / right inguinal ligaments, and a white electrode was inserted into the thigh muscles to record electromyographic activity. After recording began, colon and rectum dilation was performed. The distal 8cm of the infant catheter (at the colon-rectum junction) was marked with a marker, and the infant catheter was repeatedly aspirated to a vacuum using a 2ml syringe. A small amount of pure water was injected to inflate the infant catheter, ensuring the balloon was just filled with pure water without dilation. Preliminary experiments showed that after connecting the pressure device, every 20mmHg corresponded to a 0.2mL syringe mark. After applying glycerin to the distal end of the catheter, it was inserted into the rat's anus to the marked position. Electromyographic responses were measured at four pressure ranges: 20, 40, 60, and 80 mmHg, by repeatedly injecting air using a 2 mL syringe. Each dilation lasted 20 seconds, with a 2-minute interval between stimulations. Electromyographic signals were recorded using a BiopacSystems EMG 100c and then digitized using Acknowledge. Electromyographic data processing: The net value at the corresponding pressure was calculated by subtracting the baseline area of ​​the curve before dilation from the area of ​​the curve after each dilation.

[0071] 2. Morphological observation (1) HE staining After sacrificing rats, a 0.5 cm segment of the colon was filled with 4% paraformaldehyde and fixed for 48 h. After dehydration, the sections were embedded in paraffin wax to a thickness of 3 mm. The sections were baked at 60°C for 3 h, then sequentially immersed in xylene I, II, and III for 15 min each time. After dewaxing, the sections were sequentially immersed in anhydrous ethanol, 95% ethanol, 85% ethanol, 75% ethanol, and pure water for 5 min each. Hematoxylin staining was applied for 5 min, followed by rinsing with tap water for 2 min. Differentiation was performed with 1% hydrochloric acid-ethanol solution for 3 s, followed by rinsing with tap water for 2 min. The sections were then soaked in PBS for 5 min for blue inversion. Eosin staining was performed for 20 min, followed by rinsing for 2 min, and then 75% ethanol, 85% ethanol, and 95% ethanol for 1 min each, followed by dehydration with anhydrous ethanol for 5 min. Clearing was performed with xylene I and II for 10 min each. The sections were mounted with neutral resin and fixed overnight. Images were acquired using an upright microscope imaging system. (2) Transmission electron microscopy Preliminary tissue fixation: Tissue blocks were immersed in 2.5% glutaraldehyde (4°C, pH 7.4) overnight for fixation; Re-fixation and dehydration: First, rinse three times with 0.1 mol / L PBS buffer for 15 min each time to completely remove residual glutaraldehyde. Then fix with 1% osmium tetroxide (OsO4) solution at 4℃ for 2 h to complete gradient ethanol dehydration, each stage for 15 min. Infiltration and embedding: The mixture was incubated with epoxy resin for 2 h and then resin embedding polymerization was carried out at 60℃ for 48 h. Ultramicrosection preparation: The surface of the sample block is roughly trimmed to expose the target tissue area as much as possible. Ultrathin sections (approximately 100 nm thick) are obtained using an ultramicrotome. Carbon-coated copper mesh is used for retrieval to ensure that the sections are dry and clean. Counterstaining of sections: First, stain with 2% uranyl acetate aqueous solution in the dark for 30 min, then rinse with double-distilled water; Second, stain with lead citrate for 5 min. In this step, NaOH easily absorbs CO2, so care should be taken to avoid CO2 causing lead carbonate precipitation and contamination of the sections. After rinsing with double-distilled water, air dry to complete the counterstaining.

[0072] Transmission electron microscopy image observation and acquisition: Transmission electron microscopy equipment was used to locate and photograph the colonic epithelial cells and the state of intercellular connections.

[0073] 3. Peripheral immune detection Flow cytometry was used to detect peripheral blood mononuclear cells (PBMCs) in each group of rats. The flow cytometry data analysis in this section was performed using Flowjo 10.9.0 and CytExpert software.

[0074] 4. Detection of intestinal mucosal inflammatory factors Take an appropriate amount of colon tissue, grind it with homogenizing medium to prepare a 10% tissue homogenate, centrifuge the homogenate at 4000 r / min for 10 min, take the supernatant, and determine the content of TNF-α and IL-10 in colon tissue according to the ELISA kit instructions.

[0075] 5. Intestinal permeability testing Lipopolysaccharide (LPS), diamine oxidase (DAO), and D-lactic acid (DLA) are markers of intestinal mucosal damage, indirectly reflecting changes in intestinal mucosal permeability and the degree of intestinal barrier damage. The levels of LPS, DAO, and D-LA in rat serum were detected using enzyme-linked immunosorbent assay (ELISA). A 96-well plate was set up according to the standard experimental procedure: (1) Blank control wells: 100 μL of PBS buffer was added; (2) Standard wells: 100 μL of serially diluted standard solutions (concentration range: 0-1000 pg / mL) were added sequentially; (3) Sample wells: 100 μL of 1:10 diluted serum sample was added. After incubating the reaction plate at 37℃ for 60 minutes, the absorbance (OD value) of each well was measured at 450 nm using an ELISA reader. A standard curve was plotted with the standard concentration as the x-axis and the corresponding OD value as the y-axis (R < 0.05). 2 >0.99). Calculate the actual concentrations of LPS, DAO, and D-LA in each sample based on the standard curve equation.

[0076] 6. Western Blot detection of intestinal mucosal barrier-related proteins (1) Protein extraction: Weigh 50 mg of rat colon tissue, clean it, and place it in a homogenization tube. Cut the colon tissue into small pieces with small scissors, and add 500 L of protein lysis buffer and 3 magnetic beads to each homogenization tube. Homogenize twice with a tissue homogenizer at 5000 rpm for 30 s, with a 2 min interval on ice between homogenizations. After homogenization, sonicate each sample for 5 s at 30% power, repeating 5 times. Let stand on ice for 10 min. Centrifuge at 4℃ for 14000 rpm for 15 min. After centrifugation, transfer the supernatant to a 1.5 mL Eppendorf tube and place it on ice. Protein lysis buffer preparation: Dissolve 10 mL RIPA + 1 tablet of PhosStop phosphatase inhibitor + 1 tablet of cOmpete™ protease inhibitor by vortexing.

[0077] (2) Protein quantification: Protein quantification was performed using the Thermo Fisher BCA kit. Standard curve A-1, BCA reaction solution, and protein dilution buffer were prepared according to the instructions and added to 96-well plates. The absorbance of each well was measured using a microplate reader at 562 nm. The protein concentration of each sample was calculated using the formula for the standard curve. 5g loading buffer, pre-mixed protein solution, and RIPA were added according to the specified ratio to prepare a 4g / µl loading protein solution for all samples. The protein was denatured by heating at 100℃ for 10 min.

[0078] (3) Gel preparation and sample loading: Use a 15-well comb to prepare the gel according to the instructions. After the gel solidifies, place it in pure water and store at 4°C. When loading the sample, add the required amount of protein according to each indicator. Add 20-30ug of protein to each lane.

[0079] (4) Electrophoresis, transfer, and blocking: Electrophoresis was performed using a constant voltage of 80V. The process was stopped when the sample reached the bottom of the gel. The transfer buffer was pre-cooled. The 0.22µm PVDF membrane was activated in methanol for 1 min and equilibrated in the transfer buffer for 3 min. After cutting the gel, the transfer clamp was placed with the black side down, and the membrane was covered in the following order: sponge pad-filter paper-gel-PVDF membrane-filter paper-sponge pad. Air bubbles between the gel and the PVDF membrane were removed. The transfer clamp was then closed. The transfer time was adjusted according to the molecular weight of the indicator protein, and the transfer was performed at a constant voltage of 100V. Electrophoresis buffer preparation: One packet of electrophoresis powder was dissolved in 1L of pure water and vortexed until no powder precipitate remained. Transfer buffer preparation: One packet of transfer powder was dissolved in 800mL of pure water, and 200mL of methanol was added. The membrane was vortexed until no powder precipitate remained. Blocking: The PVDF membrane was removed and blocked at room temperature for 30 min using a rapid blocking buffer.

[0080] (5) Incubation with primary antibody: After blocking, wash the membrane three times with TBST, 5 min each time. Cut the membrane to the appropriate size according to the marker markings and incubate overnight at 4°C with the corresponding antibody. TBST solution: Dissolve one packet of TBS powder in 2L of pure water, add 2mL of Tween-20, and vortex.

[0081] (6) Incubation of secondary antibody: Recover the primary antibody, wash the PVDF membrane with TBST, 5 min / time * 6 times. Select the appropriate secondary antibody according to the primary antibody, incubate at room temperature on a shaker for 1 h. After incubation, recover the secondary antibody, wash the PVDF membrane with TBST, 5 min / time * 6 times, and immerse the membrane in TBST to keep it moist.

[0082] (7) Development: Prepare Immobilon Western Chemiluminescent HRP Substrate at a 1:1 ratio according to the instructions, and expose and develop it on a gel imaging system. Use GraphPad Prism 8.02 for plotting and ImageJ for protein grayscale analysis.

[0083] VI. Statistical Methods The data in this section were statistically analyzed using SPSS 25.0, and graphs were created using GraphPad Prism 9.5.0 software. Count data were analyzed using (…). Data are expressed as x±s; continuous data are expressed as M(P25,P75). For multiple groups of data, if the data are normally distributed and have homogeneous variance, one-way ANOVA is used; if pairwise comparisons between multiple groups are required and the sample sizes of each group are equal, the Bonferroni correction method is used; if the sample sizes within groups are unequal, the Scheffe method is used for post-hoc comparisons between groups; if the data are normally distributed but have unequal variances, the Dunnett T3 method is used for pairwise comparisons between groups. For data that do not conform to a normal distribution, the Mann-Whitney U rank-sum test is used for comparisons between two groups, and the Kruskal-Wallis H test is used for comparisons between multiple groups.

[0084] VII. Experimental Results 1. Traditional Chinese medicine composition restores body weight gain in PI-IBS rats The results of the comparison of the body weight of rats in each group are as follows: Figure 3 As shown in the figure, A: Comparison of rat body weight among groups at different time points; B: Statistical chart of rat body weight among groups at week 6; Compared with the Control group, ***P<0.001; Compared with the PI-IBS group, ##P<0.01, ###P<0.001.

[0085] Table 1 shows the comparison of body weight of rats in each group at different time points.

[0086] Table 1 Comparison of body weight (g) of rats in each group at different time points; x±s)

[0087] Note: In the table, a Compared to the Control group P <0.05, aaa Compared to the Control group P <0.001, b Compared with the PI-IBS group P <0.05, bb Compared with the PI-IBS group P <0.01, bbb Compared with the PI-IBS group P <0.001.

[0088] from Figure 3 As shown in Table 1, compared with the Control group, the body weight of rats after PI-IBS modeling was significantly reduced; after the corresponding drug intervention in the 3rd and 4th weeks, the body weight of rats in the MTXYF-M group, MTXYF-H group and CGEC group was significantly higher than that of the PI-IBS group (P<0.05); by the 6th week, the body weight of rats in the PI-IBS group was still significantly lower than that of the Control group (P<0.001), and the body weight of rats in the MTXYF-M group, MTXYF-H group and CGEC group was significantly higher than that of the PI-IBS group (P<0.001 or P<0.01).

[0089] 2. Traditional Chinese medicine composition alleviates diarrhea phenotype in PI-IBS rats The results of the comparison of fecal water content of rats in each group at week 6 are as follows: Figure 4 As shown in the figure, compared with the Control group, ***P<0.001; compared with the PI-IBS group, ##P<0.01.

[0090] from Figure 4 As can be seen, by week 6, the fecal water content of rats in the PI-IBS group was still significantly higher than that in the Control group (P < 0.001), while the fecal water content of rats in the MTXYF-M group, MTXYF-H group and CGEC group was significantly lower than that in the PI-IBS group (P < 0.01), suggesting that the traditional Chinese medicine composition of the present invention improved the diarrhea phenotype of PI-IBS rats.

[0091] 3. Traditional Chinese medicine composition improves the depressive phenotype in PI-IBS rats Comparison of sucrose preference index among rats in each group at week 6: Figure 5 As shown in the figure, compared with the Control group, ***P<0.001; compared with the PI-IBS group, ##P<0.01.

[0092] from Figure 5 It can be seen that by week 6, the sucrose preference index of rats in the PI-IBS group was significantly lower than that in the Control group (P<0.001), indicating a depressive phenotype; while the sucrose preference index of rats in the MTXYF-M group and MTXYF-H group was significantly higher than that in the PI-IBS group (P<0.01), indicating that the traditional Chinese medicine composition of the present invention improved the depressive phenotype of PI-IBS rats.

[0093] 4. Traditional Chinese medicine composition improves activity level in PI-IBS rats Comparison of open field test results of rats in different groups at week 6: Figure 6 As shown, A: Route map of rats in each group; B: Statistical chart of total distance, average speed, number of grid crossings, and time spent in the central area; In the figure, compared with the Control group, **P<0.01.

[0094] from Figure 6 As can be seen, by week 6, open field route maps showed that the Control group rats were more inclined to walk along the walls, exhibiting high exploration activity. The total distance and average speed of the PI-IBS group rats were significantly lower than those of the Control group (P < 0.01), indicating that the activity level of the PI-IBS group rats decreased and their desire to explore the surrounding environment was low. In addition, the number of grid crossings and the time spent in the central area of ​​the PI-IBS group rats both showed a decreasing trend compared with the Control group. The total distance, average speed, and number of grid crossings of the various dosage groups of the traditional Chinese medicine composition and the CGEC group showed an increasing trend compared with the PI-IBS group. The above results suggest that the traditional Chinese medicine composition of the present invention improves the activity level of PI-IBS rats.

[0095] 5. Traditional Chinese medicine composition reduces visceral hypersensitivity in PI-IBS rats. The visceral motor responses of rats in each group at week 6 are as follows: Figure 7 As shown in the figure, compared with the Control group, *P<0.05, ***P<0.001; compared with the PI-IBS group, #P<0.05, ##P<0.01.

[0096] from Figure 7 It can be seen that by week 6, compared with the PI-IBS group, the fluctuation frequency of rats in the MTXYF-M group, MTXYF-H group and CGEC group during 80 mmHg colorectal dilation (CRD) was significantly reduced (P<0.05 or P<0.01), suggesting that the traditional Chinese medicine composition of the present invention can alleviate the visceral hypersensitivity reaction of PI-IBS rats.

[0097] 6. Traditional Chinese medicine composition improves colonic pathological damage in PI-IBS rats HE staining results of rats in each group at week 6 are as follows: Figure 8As shown, 20×, the black scale bar represents 200μm. From Figure 8 It can be seen that, compared with the PI-IBS group, the colonic mucosal epithelium of rats in the MTXYF-M group, MTXYF-H group and CGEC group recovered continuously without obvious inflammatory infiltration, suggesting that the traditional Chinese medicine composition of the present invention can improve the pathological damage of the colon in PI-IBS rats.

[0098] Ultrastructural observation (transmission electron microscopy) of colonic epithelial cells and intercellular connections in rats at week 6 in each group is as follows: Figure 9 As shown in the figure, A: 10000x; B: 20000x.

[0099] from Figure 9 It can be seen that the colonic epithelial cells of rats in the Control group were tightly connected and the desmosome structure was clear; the colonic epithelial cells of rats in the PI-IBS group had a loose structure and the desmosome structure disappeared; the colonic epithelial cell connection structure and desmosome structure of rats in the MTXYF-M group, MTXYF-H group and CGEC group were restored to varying degrees.

[0100] 7. Traditional Chinese medicine composition regulates the Th1 / Th2 immune imbalance in the blood of PI-IBS rats. Changes and ratios of Th1 and Th2 cells in the blood PBMCs of rats in each group at week 6 are as follows: Figure 10 As shown in the figure, A: CD4+INF-γ+ Th1 cells; B: CD4+IL-4+ Th2 cells; Q2 quadrant represents the target cell population; compared with the Control group, **P<0.01; compared with the PI-IBS group, #P<0.05, ##P<0.01.

[0101] from Figure 10 It can be seen that the Th1 / Th2 ratio of peripheral blood PBMCs in the PI-IBS group was significantly higher than that in the Control group (P < 0.01); compared with the PI-IBS group, the Th1 / Th2 ratios of rats in the MTXYF-M group, MTXYF-H group and CGEC group were significantly lower (P < 0.01 or P < 0.05), suggesting that the traditional Chinese medicine composition of the present invention can restore the immune imbalance of Th1 and Th2 in the blood of PI-IBS rats.

[0102] 8. Traditional Chinese medicine composition relieves intestinal mucosal inflammation in PI-IBS rats The results of TNF-α and IL-10 content detection in colon tissue of rats in each group at week 6 are as follows: Figure 11 As shown in the figure, A: TNF-α content; B: IL-10 content; compared with the Control group, ***P<0.001; compared with the PI-IBS group, ##P<0.01.

[0103] from Figure 11 As can be seen, compared with the Control group, the TNF-α content in the colonic tissue of rats in the PI-IBS group was significantly increased (P < 0.001), and the IL-10 content showed an increasing trend but no statistical difference; compared with the PI-IBS group, the TNF-α content in the colonic tissue of rats in the MTXYF-M group, MTXYF-H group, and CGEC group was significantly decreased (P < 0.01), and the IL-10 content showed a decreasing trend but no statistical difference. This suggests that the traditional Chinese medicine composition of the present invention can alleviate intestinal mucosal inflammation in PI-IBS rats.

[0104] 9. Traditional Chinese medicine composition reduces intestinal permeability in PI-IBS rats The serum levels of LPS, DAO, and D-LA in each group of rats at week 6 were as follows: Figure 12 As shown in the figure, A: LPS content; B: DAO content; C: D-LA content; Compared with the Control group, *P<0.05, ***P<0.001; Compared with the PI-IBS group, #P<0.05, ###P<0.001.

[0105] from Figure 12 As can be seen, compared with the Control group, the serum LPS, DAO, and D-LA levels in the PI-IBS group rats were significantly increased (P < 0.001 or P < 0.05), indicating that the intestinal mucosal permeability of the PI-IBS group rats was significantly increased. Compared with the PI-IBS group, the LPS content in each dose group of the traditional Chinese medicine composition was significantly decreased (P < 0.001), the DAO content in the MTXYF-H group was significantly decreased (P < 0.05), and the D-LA content in the MTXYF-M and MTXYF-H groups was significantly decreased (P < 0.05), indicating that the traditional Chinese medicine composition of the present invention can significantly improve the intestinal mucosal permeability of PI-IBS rats.

[0106] 10. Traditional Chinese medicine composition increases the expression of intestinal barrier-related proteins in PI-IBS rats. The expression of intestinal mucosal barrier-related proteins in each group of rats at week 6 is as follows: Figure 13 As shown in the figure, compared with the Control group, **P<0.01; compared with the PI-IBS group, #P<0.05, ##P<0.01.

[0107] from Figure 13As can be seen, compared with the Control group, the expression levels of intestinal tight junction proteins ZO-1, CLDN1, and E-Cadherin in the PI-IBS group rats were significantly decreased (P < 0.01), and B-Catenin protein showed a decreasing trend. Compared with the PI-IBS group, ZO-1 was significantly increased in the MTXYF-H group (P < 0.05), CLDN1 was significantly increased in both the MTXYF-M and MTXYF-H groups (P < 0.05), E-Cadherin was significantly increased in the MTXYF-M group (P < 0.05), and B-Catenin showed an increasing trend in both the MTXYF-M and MTXYF-H groups. This suggests that the traditional Chinese medicine composition of the present invention can improve intestinal barrier function in PI-IBS model rats.

[0108] In summary, the therapeutic effects of the traditional Chinese medicine composition of this invention on rats with PI-IBS liver stagnation and spleen deficiency syndrome are manifested in the following ways: improving the diarrhea phenotype and depressive symptoms in PI-IBS rats with liver stagnation and spleen deficiency syndrome, reducing visceral hypersensitivity, restoring the tight junction structure and desmosome structure of the colon, upregulating the expression of tight junction (TJ) proteins (ZO-1, CLDN1, and E-Cadherin, etc.), restoring the Th1 / Th2 immune imbalance in the blood, reducing the level of pro-inflammatory factors (TNF-α) in colonic tissue, and upregulating the levels of LPS, DAO, and D-LA in serum. This suggests that the traditional Chinese medicine composition of this invention can effectively treat liver stagnation and spleen deficiency syndrome in post-infectious irritable bowel syndrome.

[0109] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A traditional Chinese medicine composition for treating post-infectious irritable bowel syndrome, characterized in that, The raw materials, by weight, include: 10-15 parts of Atractylodes macrocephala, 10-20 parts of Paeonia lactiflora, 3-7 parts of Citrus reticulata peel, 5-15 parts of Saposhnikovia divaricata, 10-20 parts of Brussels sprout leaves, 10-20 parts of Plumeria rubra, 5-15 parts of Bombax ceiba flower, 25-35 parts of Smilax glabra, and 5-15 parts of Albizia julibrissin flower.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The raw materials, by weight, include: 12 parts of Atractylodes macrocephala, 15 parts of Paeonia lactiflora, 5 parts of Citrus reticulata peel, 10 parts of Saposhnikovia divaricata, 15 parts of Brussels sprout leaves, 15 parts of Plumeria rubra, 9 parts of Bombax ceiba, 30 parts of Smilax glabra, and 10 parts of Albizia julibrissin flowers.

3. The traditional Chinese medicine composition according to claim 1 or 2, characterized in that, The Atractylodes macrocephala mentioned is stir-fried Atractylodes macrocephala, and the Paeonia 4. The traditional Chinese medicine composition according to claim 1 or 2, characterized in that, Pharmaceutically acceptable excipients can also be added.

5. The traditional Chinese medicine composition according to claim 1 or 2, characterized in that, The dosage form of the traditional Chinese medicine composition is decoction, granules, powder, tablets, pills or capsules.

6. A method for preparing the traditional Chinese medicine composition for treating post-infectious irritable bowel syndrome according to any one of claims 1-5, characterized in that, Includes the following steps: Soak the raw materials in 4-8 times their weight of water for 20-40 minutes, then boil over high heat for 30-40 minutes, then simmer over low heat for 30-40 minutes. Filter the mixture and repeat the boiling process on the residue 1-2 times. Combine the filtrates to obtain the final product.

7. The use of the traditional Chinese medicine composition for treating post-infectious irritable bowel syndrome according to any one of claims 1-5 in the preparation of a medicament for treating post-infectious irritable bowel syndrome.

8. The use of the traditional Chinese medicine composition for treating post-infectious irritable bowel syndrome according to any one of claims 1-5 in the preparation of a medicine for treating liver stagnation and spleen deficiency syndrome of post-infectious irritable bowel syndrome.

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