Application of qi stagnation and stomachache preparation in preparation of medicine for treating irritable bowel syndrome

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to application of a qi-stagnation stomachache preparation in preparation of a medicine for treating irritable bowel syndrome. According to the application of the qi-stagnation stomachache preparation in preparation of the medicine for treating the irritable bowel syndrome, the diarrhea condition of the diarrhea type irritable bowel syndrome can be improved, meanwhile, anxiety and visceral hypersensitivity caused by the irritable bowel syndrome can be relieved, and therefore the preparation can be used for preparing the medicine for treating the qi-stagnation stomachache. The qi stagnation and stomachache preparation has a remarkable improvement effect on diarrhea type irritable bowel syndrome (IBS-D).

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine preparations, and in particular to application of a qi stagnation and stomachache preparation in the preparation of medicines for treating irritable bowel syndrome. Background Art

[0002] Irritable bowel syndrome (IBS) is a clinical functional bowel disorder characterized by abdominal discomfort, urgency during bowel movements, and changes in stool form. These symptoms, which also include constipation and bloating, can occur continuously or intermittently, and their severity can be exacerbated by work, school, and life stress. Furthermore, factors ranging from external environmental factors to internal factors can mediate the development of the disease, including dietary stimulation, emotional disturbances, gastrointestinal hypermotility, increased intestinal sensitivity, intestinal flora imbalance, brain-gut axis regulation disorders, immune activation, and family heredity. Combined with individual differences, IBS presents a diverse range of symptoms.

[0003] Currently, there is no specific treatment plan for IBS in the clinic. Most of the treatments are symptomatic treatments such as antispasmodics and antidiarrheal drugs based on the symptoms reported by IBS patients and observed by doctors. These can temporarily relieve symptoms but have disadvantages such as poor efficacy and high recurrence rate. In recent years, the prevalence of IBS has been increasing year by year. During the COVID-19 pandemic, it was found that the number of COVID-19 patients with IBS increased six times that of healthy people, which greatly affected people's physical and mental health. In addition, repeated medical treatment has seriously affected patients' work efficiency, increased people's economic burden, and also caused a large waste of medical resources. Studies have shown that IBS patients who are not adequately treated have complex clinical manifestations and even have a lower quality of life than patients with gastroesophageal reflux disease and diabetes. Four IBS subtypes were obtained based on clinical stool morphology as the main classification basis, among which diarrhea-type IBS (IBS-D) is the most common, accounting for up to 65% of IBS cases.

[0004] IBS is not specifically documented within the theoretical framework of Traditional Chinese Medicine (TCM), but TCM has extensive experience in studying the symptom patterns and dialectical treatment methods associated with IBS. Based on the clinical symptoms of IBS-D, such as watery stools, intermittent abdominal pain, loss of appetite, and emotional and psychological abnormalities, TCM classifies the condition under the categories of "diarrhea," "abdominal pain," "large intestinal diarrhea," and "intestinal depression." Specifically, it falls under the "chronic diarrhea" category within "diarrhea." Further classifications based on the primary symptoms associated with diarrhea include spleen deficiency and dampness obstruction, liver depression and spleen deficiency, and spleen and kidney yang deficiency. Currently, TCM considers diarrhea to be located in the spleen, stomach, and intestines, with the liver and spleen playing a leading role in the development of symptoms. Etiology primarily involves internal injury from the seven emotions, internal invasion of external pathogens, dietary indiscriminateness, deficient endowment, and postnatal deficiency. The pathogenesis is due to large intestinal deficiency and coldness, dysfunction of the liver and gallbladder, and spleen and stomach transport and transformation. Therefore, clinical treatment should focus on soothing the liver and strengthening the spleen, warming the kidneys and strengthening the intestines, and removing dampness and stopping diarrhea. Commonly used prescriptions for pain and diarrhea and Shenling Baizhu powder are used.

[0005] Qizhi Weitong preparations are widely used clinically to treat gastrointestinal disorders such as chronic gastritis and functional dyspepsia, with excellent therapeutic effects. Qizhi Weitong preparations are recommended as over-the-counter Chinese patent medicines for patients with stomachaches. They are based on the "Sini Decoction" formula, which soothes the liver and relieves depression, and incorporate Cyperus rotundus and Corydalis yanhusuo. Corydalis yanhusuo promotes blood circulation, relieves pain, and reduces gastrointestinal rigidity. Bupleurum, Cyperus rotundus, White Peony Root, and Licorice Root all have effects on regulating menstruation and promoting Qi circulation, as well as nourishing the stomach. Together, these herbs achieve the effects of soothing the liver, regulating Qi, and alleviating pain, consistent with Traditional Chinese Medicine's theory of syndrome differentiation and treatment. Modern basic research suggests that Qizhi Weitong granules may alleviate the clinical pathological manifestations of chronic gastritis and gastric ulcers by influencing secondary inflammatory processes and the release of endocrine hormones. Based on these benefits, Qizhi Weitong granules are considered by experts and scholars to have considerable medicinal and research value. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the defect of the prior art in the lack of effective medicaments for irritable bowel syndrome, thereby providing a use of a qi stagnation and stomach pain preparation in the preparation of a medicament for treating irritable bowel syndrome.

[0007] To this end, the present invention provides an application of a qi stagnation and stomach pain preparation in preparing a medicine for treating irritable bowel syndrome.

[0008] In some embodiments, the irritable bowel syndrome is diarrhea-type.

[0009] In some embodiments, the dosage form of the qi stagnation and stomach pain preparation includes at least one of an extract, granules, capsules, tablets, powder or oral solution.

[0010] In some embodiments, the drug for treating irritable bowel syndrome includes at least one of an oral type and an injectable type.

[0011] In some embodiments, the gastric pain preparation for qi stagnation includes bupleurum root, vinegar-cured corydalis, fructus aurantii, vinegar-cured cyperus, angelica dahurica and prepared liquorice root.

[0012] In some embodiments, the qi stagnation and stomachache preparation comprises the following components in parts by weight: 300-400 parts of bupleurum; 350-450 parts of vinegar-soaked corydalis; 350-450 parts of fructus aurantii; 350-450 parts of vinegar-soaked cyperus; 420-520 parts of angelica dahurica; and 150-250 parts of roasted liquorice.

[0013] On the other hand, the present invention provides a method for detecting compounds in a Qizhiweitong preparation, comprising the following steps: preparing a Qizhiweitong preparation solution; and determining the compounds in the Qizhiweitong preparation using a UHPLC / MS / MS method.

[0014] In some embodiments, the method for preparing the Qizhiweitong preparation solution comprises mixing an alcohol solution and the Qizhiweitong preparation to form a 0.5-2 mg / mL solution, wherein the alcohol solution comprises 40-60 vol% methanol in water.

[0015] In some embodiments, the chromatographic conditions are as follows: T3 chromatographic column; mobile phase A is an aqueous solution containing 0.1 vol% formic acid, mobile phase B is an acetonitrile solution; the flow rate is 0.4 mL / min;

[0016] The elution conditions were as follows: 0 min, the volume ratio of mobile phase A to mobile phase B was 9:1, 20 min, the volume ratio of mobile phase A to mobile phase B was 6:4, 25 min, and the volume ratio of mobile phase A to mobile phase B was 4:6, 30 min, mobile phase B.

[0017] In some embodiments, the mass spectrometry conditions are as follows: gas temperature 320°C, drying gas flow 8 L / min, nebulizer pressure 35 psig, sheath gas temperature 350°C, sheath gas flow 11 L / min, voltage 3.5 kV.

[0018] The technical solution of the present invention has the following advantages:

[0019] The use of the Qizhi Weitong preparation provided by the present invention in the preparation of a medicament for treating irritable bowel syndrome can improve the diarrhea of ​​diarrhea-predominant irritable bowel syndrome, and at the same time can relieve the anxiety and visceral hypersensitivity caused by irritable bowel syndrome. Therefore, the Qizhi Weitong preparation has a significant improvement effect on diarrhea-predominant irritable bowel syndrome (IBS-D). BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is the ion chromatogram of the Qizhi Weitong extract in the UHPLC / MS / MS positive ion mode in Example 1 of the present invention;

[0022] Figure 2 This is the ion chromatogram of the Qizhi Weitong extract in the UHPLC / MS / MS negative ion mode in Example 1 of the present invention;

[0023] Figure 3 This is a graph showing the weight growth rate of rats in Example 2 of the present invention;

[0024] Figure 4 This is a graph showing the food intake results of rats in Example 2 of the present invention;

[0025] Figure 5 This is a graph showing the diarrhea index results of rats on the 14th day of the experiment in Example 2 of the present invention;

[0026] Figure 6 This is a graph showing the diarrhea index results of rats on day 21 of the experiment in Example 2 of the present invention;

[0027] Figure 7 This is a diagram showing the fecal typing results of rats on the 14th day of the experiment in Example 2 of the present invention;

[0028] Figure 8 This is a diagram showing the fecal typing results of rats on the 21st day of the experiment in Example 2 of the present invention;

[0029] Figure 9 This is a graph showing the moisture content of rat feces on the 14th day of the experiment in Example 2 of the present invention;

[0030] Figure 10 This is a graph showing the moisture content of rat feces on day 21 of the experiment in Example 2 of the present invention;

[0031] Figure 11 This is a graph showing the number of grid penetrations in the open field in Example 2 of the present invention;

[0032] Figure 12 This is a graph showing the number of open-field standing times in Example 2 of the present invention;

[0033] Figure 13 This is a diagram showing the total distance traveled in an open field in Example 2 of the present invention;

[0034] Figure 14This is the result diagram of the central distance of the open field in Example 2 of the present invention;

[0035] Figure 15 This is a result diagram of the overhead arm opening dwell time in Example 2 of the present invention;

[0036] Figure 16 This is a result diagram of the number of overhead arm opening entries in Example 2 of the present invention;

[0037] Figure 17 This is a diagram showing the abdominal wall withdrawal reflex results in Example 2 of the present invention. DETAILED DESCRIPTION

[0038] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0039] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0040] Example 1

[0041] This example provides a method for detecting compounds of a Qi-stagnation and stomach-ache preparation, and the specific steps and parameters are as follows:

[0042] Qizhiweitong extract (provided by Benxi National Traditional Chinese Medicine Engineering Technology Research Center Co., Ltd.) was accurately weighed and prepared into a 1 mg / mL clear solution using a 50 vol% methanol-water solution. Compounds in the Qizhiweitong extract were detected and identified using UHPLC / MS / MS. The instrument used was an Agilent 6540 HPLC-MS system with a Waters ACQUITY UPLCHSS T3 column. Mobile phase A was water containing 0.1% formic acid, and mobile phase B was pure acetonitrile. The elution conditions are shown in Table 1.

[0043] Table 1 Elution conditions

[0044] time A B flow rate 0min 90％ 10％ 0.400mL / min 20.00min 60％ 40％ 0.400mL / min 25.00min 40％ 60％ 0.400mL / min 30.00min 0％ 100％ 0.400mL / min

[0045] Mass spectrometry conditions:

[0046] The ion source parameters were as follows: gas temperature 320°C, drying gas flow 8 L / min, nebulizer pressure 35 psig, sheath gas temperature 350°C, sheath gas flow 11 L / min, and voltage 3.5 kV. Data processing was performed using Qua-Analysis software.

[0047] See the results Figure 1 and Figure 2 As shown in Tables 2 and 3, under the positive ion mode, the detection method provided in this embodiment can detect 34 compounds in the Qizhi Weitong extract, and under the negative ion mode, the detection method provided in this embodiment can detect 13 compounds in the Qizhi Weitong extract. In summary, by combining different ion modes, 40 compounds can be identified from the Qizhi Weitong extract.

[0048] Table 2 Compounds identified in Qizhiweitong extract using UHPLC / MS / MS positive ion mode

[0049]

[0050]

[0051] Table 3 Compounds identified in Qizhiweitong extract using UHPLC / MS / MS negative ion mode

[0052]

[0053]

[0054] Example 2

[0055] This example provides a pharmacodynamic study method for a Qi-stagnation and stomach-ache preparation, and the specific steps and parameters are as follows:

[0056] 2.1 Establishment of diarrhea-predominant irritable bowel syndrome model

[0057] 2.1.1 Experimental Materials

[0058] 2.1.1.1 Experimental Animals

[0059] SPF-grade SD rats (180 ± 20 g, 6-7 weeks), half male and half female, totaling 108, were purchased from Southern Medical University with the experimental animal use license number: SCXK (Guangdong) 2021-0041 and the animal ethics number: 20221002. They were kept in the International Institute of Translational Medicine of Guangzhou University of Chinese Medicine with the experimental animal use license number: SYXK (Guangdong) 2019-0144.

[0060] Before the experiment, the rats were adaptively raised and given free access to food and water. The indoor air circulation and natural day and night light were maintained. The indoor temperature was maintained at 22-25°C and the relative humidity was about 60%.

[0061] 2.1.1.2 Experimental instruments, reagents and drugs

[0062] The experimental instruments, reagents and drugs used in this example are shown in Tables 4-6.

[0063] Table 4 Main experimental instruments

[0064] name Production Analytical balance (accuracy of one hundred thousandth) Sartorius, Germany electronic scales Xiangheng Electronics Desktop Refrigerated Centrifuge TCL-16A Shanghai Lu Xiangyi Milli-Q Ultrapure Water System Milipore, USA Constant temperature water bath Esco Singapore 8F urinary catheter Zhejiang Utechel Medical Supplies Co., Ltd. Elevated plus maze Shanghai Xinruan Information Technology Co., Ltd. Conditioned Stimulus Controller Shanghai Xinruan Information Technology Co., Ltd. open field Shanghai Xinruan Information Technology Co., Ltd.

[0065] Table 5 Main reagents

[0066] name Manufacturer Sodium chloride Sangon Bioengineering (Shanghai) Co., Ltd. liquid paraffin Sinopharm Chemical Reagent Co., Ltd. Isoflurane Hebei Jindafu Pharmaceutical Co., Ltd.

[0067] Table 6 Main drugs

[0068] name batch number Manufacturer Qizhi Weitong Granules 20200122 Liaoning China Resources Benxi Sanyao Co., Ltd. Qizhiweitong granule extract powder Liaoning China Resources Benxi Sanyao Co., Ltd. Pinaverium Bromide Tablets 715257 MYLAN LABORATORIES LIMITED Senna 220102 Guangdong Huikangyuan Pharmaceutical Co., Ltd.

[0069] 2.2 Experimental Methods

[0070] 2.2.1 Animal Grouping

[0071] A total of 108 rats were randomly divided into blank group, model group, low-dose qi stagnation and stomach pain group, medium-dose qi stagnation and stomach pain group, high-dose qi stagnation and stomach pain group and pinaverium bromide group, with 9 males and 9 females in each group, for a total of 18 rats in each group.

[0072] 2.2.2. Establishment of animal model and medication

[0073] An animal model of diarrhea-predominant irritable bowel syndrome (IBS-D) was established using senna leaf catharsis and chronic unpredictable stress. Rats in each group received prophylactic medication for 7 days. Starting on the eighth day, rats were gavage-administered a 3g / kg senna leaf solution daily at 9:00 AM. After a 1-hour interval, the rats were randomly assigned to one of the following modeling methods for 14 consecutive days. After successful modeling, each group received medication for 21 consecutive days.

[0074] Stress methods included: ① Restraint (rats were placed in disposable plastic bottles with multiple holes to allow for ventilation and restrict movement, for 120 minutes); ② Foot shock (current intensity 1.5 mA, three 30-second shocks within 2 minutes, with 10-second intervals); ③ Rocking-crowding (rats from the same group were placed in a mouse cage, covered, and secured with tape, for 60 minutes); ④ Cold water swimming (10 minutes, immediately removed if unable to swim, motionless, or sinking, indicating drowning); ⑤ Tail clamping (30 seconds, once in the morning and afternoon); ⑥ Solitary housing for 24 hours (rats were placed in mouse cages and given chow); and ⑦ Crowded housing for 24 hours (nine rats per cage). Each group received drug administration from the afternoon of the first day of the experiment until the completion of the behavioral experiments.

[0075] Among them, preventive administration refers to gavage of normal saline, feeding of low-dose gas stagnation and stomach pain drugs, medium-dose gas stagnation and stomach pain drugs, high-dose gas stagnation and stomach pain drugs and pinaverium bromide to each group of mice according to the grouping. Among them, the mice in the blank group and the model group were gavaged with normal saline, the low-dose gas stagnation and stomach pain group was fed with a low-dose gas stagnation and stomach pain drug, the medium-dose gas stagnation and stomach pain group was fed with a medium-dose gas stagnation and stomach pain drug, the high-dose gas stagnation and stomach pain group was fed with a high-dose gas stagnation and stomach pain drug, and the pinaverium bromide group was fed with pinaverium bromide.

[0076] Continuous administration refers to gavage of normal saline, low-dose of gastric stagnation and stomachache drug, medium-dose of gastric stagnation and stomachache drug, high-dose of gastric stagnation and stomachache drug and pinaverium bromide to mice in each group. Among them, mice in the blank group and model group were gavaged with normal saline, mice in the low-dose gastric stagnation and stomachache group were fed with low-dose of gastric stagnation and stomachache drug, mice in the medium-dose gastric stagnation and stomachache group were fed with medium-dose of gastric stagnation and stomachache drug, mice in the high-dose gastric stagnation and stomachache group were fed with high-dose of gastric stagnation and stomachache drug, and mice in the pinaverium bromide group were fed with pinaverium bromide.

[0077] Among them, the dosage for preventive administration and continuous administration was based on the body weight of the rats, and the dosage was the same. The low-dose group for qi stagnation and stomach pain was fed with a low-dose of qi stagnation and stomach pain drug at a dosage of 0.512 g / kg, the medium-dose group for qi stagnation and stomach pain was fed with a medium-dose of qi stagnation and stomach pain drug at a dosage of 1.024 g / kg, the high-dose group for qi stagnation and stomach pain was fed with a high-dose of qi stagnation and stomach pain drug at a dosage of 2.048 g / kg, and the pinaverium bromide group was fed with a pinaverium bromide dosage of 30 mg / kg.

[0078] 2.2.3 Drug preparation

[0079] Pinaverium bromide (30 mg / kg): Grind pinaverium bromide tablets in a mortar until powdered. Weigh 30 mg of pinaverium bromide powder and dissolve it in 10 mL of normal saline to a final concentration of 3 mg / mL.

[0080] Low dose for Qizhiweitong (0.512 g / kg): Weigh 2.048 g of Qizhiweitong extract and dissolve it in 40 mL of normal saline to obtain a concentration of 0.0512 g / mL.

[0081] Medium dose for Qizhi Weitong (1.024 g / kg): Weigh 4.096 g of Qizhi Weitong granule extract and dissolve it in 40 mL of normal saline to a concentration of 0.1024 g / mL.

[0082] High dose of Qizhiweitong (2.048g / kg): Weigh 8.912g of Qizhiweitong extract and dissolve it in 40mL of normal saline and mix well. The concentration after preparation is 0.2048g / mL.

[0083] Senna solution: Soak 100g of the drug in 5 times the amount of ultrapure water for 30 minutes, boil it on an induction cooker for 10 minutes, filter it, concentrate the decoction, make up to a certain volume, store it in a refrigerator at 4℃, and return it to room temperature before use. The dosage concentration of senna solution is 3g / kg.

[0084] 2.2.4. Evaluation methods of animal models

[0085] 2.2.4.1 General Observations

[0086] All animals had their body weight recorded daily during the experiment. Observations also included mental state, behavioral activity, feces, and diet and water intake.

[0087] 2.2.4.2. Test of rat weight growth rate

[0088] The rats were weighed on the first day of the experiment and every two days during the experiment, and the weight growth rate of the rats was calculated.

[0089] 2.2.4.3. Food intake test of rats

[0090] The rats' food intake was recorded on two days during the experiment. The feed given and the number of rats in each cage were recorded on the first day. The remaining feed weight was recorded at the same time on the second day. The difference in feed reduction divided by the number of rats in each cage was the average daily food intake of each rat in the cage.

[0091] 2.2.4.4. Diarrhea Index in Rat

[0092] On the 14th and 21st days of the experiment, clean filter paper was placed in the cages. The rats were observed between 8:00 AM and 12:00 PM. The total number of fecal pellets and the number of loose fecal pellets in the four-hour period were recorded. The loose stool rate was calculated by dividing the number of loose fecal pellets by the total number of pellets. The looseness grade of the feces was determined by the diameter of the fecal stain left on the filter paper. The diarrhea index of the rats was calculated by multiplying the loose stool grade by the loose stool rate.

[0093] Diarrhea index = loose stool rate × loose stool grade;

[0094] Loose stool rate = number of loose stool particles of rats / total number of stool particles (loose stool is determined by whether there are stains on the filter paper). The loose stool grade can be divided into 4 levels according to the size of the stain on the filter paper;

[0095] Level 1, stain diameter <1 cm;

[0096] Level 2, stain diameter 1-1.9 cm;

[0097] Level 3, stain diameter 2-3 cm;

[0098] Level 4, stain diameter > 3 cm

[0099] 2.2.4.5. Feces moisture content

[0100] On the 14th and 21st days of the experiment, each group of rats was placed in a small cage, one rat per cage, and clean filter paper was placed in the cage. The rats had free access to food and water. The rat feces were collected from 8:00 to 12:00, within 4 hours, and the weight of the feces was weighed on an electronic analytical balance as the wet weight. The feces were dried in an oven (75℃, 24 hours) and then weighed on an electronic analytical balance as the dry weight. The difference between the wet weight of feces and the dry weight of feces divided by the wet weight of feces was the feces moisture content. The feces moisture content of each group of rats was calculated separately.

[0101] Calculation formula: feces moisture content = (wet weight - dry weight) / wet weight × 100%.

[0102] 2.2.4.6 Fecal Bristol typing

[0103] On the 14th and 21st days of the experiment, clean filter paper was placed in the cages, and the number of fecal spots excreted by the rats was observed from 8:00 to 12:00 in the morning, that is, within 4 hours. The rat feces were divided into 7 types according to the stool characteristics. The Bristol grading standard is shown in Table 7. Types 1 and 2 indicate constipation; type 3 is the ideal stool shape; type 4 is the shape that is easiest to defecate; types 5-7 indicate diarrhea.

[0104] Table 7 Bristol stool characteristics classification and scoring

[0105] Classification Stool characteristics score 1 loose, dry stools, like nuts, that are difficult to pass 1 point 2 Sausage-shaped, lumpy 2 points 3 Sausage-like, with cracks on the surface 3 points 4 Sausage-like or snake-like, smooth and soft 4 points 5 Soft mass with clear edges (easy to discharge) 5 points 6 Soft, flaky, or mushy stools 6 points 7 Watery stools with no solids 7 points

[0106] 2.2.4.7 Open field test

[0107] The open-field test chamber measured 80 cm × 80 cm × 50 cm and was divided into 16 equal-sized squares. A camera was placed above the center square. The experimental environment was quiet, and the indoor brightness matched the nighttime brightness of the animal room. All experiments began at 9:00 AM to minimize the influence of circadian rhythms. Rats were placed in the test chamber for 10 minutes to acclimate. Afterwards, the rats were gently placed in the center of the chamber, and their activity was recorded for 10 minutes.

[0108] The total score for the open field test is the sum of the horizontal score and the vertical score. The horizontal score is the number of squares crossed by the rat (with all four paws), and the vertical score is the number of times the rat stood upright. The total score for the open field test reflects the rat's level of arousal.

[0109] 2.2.4.8. Elevated plus maze test (EPM test)

[0110] The rats' movements within the EPM maze (purchased from Shanghai Xinruan Information Technology Co., Ltd.) were recorded using video and tracking equipment. Finally, the ratio of time spent in the open arms (OT value) was calculated for each rat: the sum of time spent in the two open arms / (total time spent in the maze minus time spent in the center area). The ratio of the model group rats was compared with that of the control group. A decrease in the ratio indicated that the model group rats were more anxious than the control group rats.

[0111] 2.2.4.9 Abdominal Withdrawal Reflex Test (AWR Test)

[0112] Rats were fasted for 24 hours before the experiment. Their anus was gently palpated to induce complete defecation. Under light anesthesia, an uninflated paraffin-oil-coated balloon catheter was slowly inserted through the anus. The balloon tip was approximately 1 cm from the anus and secured to the base of the rat's tail 1 cm outside the anus. Each rat was inflated three times with volumes of 1.0, 1.5, and 2.0 mL, respectively. Each inflated balloon lasted 3-5 minutes, with 30-second intervals. The AWR scoring criteria are shown in Table 8.

[0113] Table 8 AWR scoring criteria

[0114] Rats showed symptoms after colorectal stimulation score Emotionally stable 0 points Becoming unsteady and occasionally twisting the head 1 point The abdominal and back muscles are slightly contracted without lifting the abdomen off the ground 2 points The abdominal and back muscles contract strongly and lift the abdomen off the ground 3 points The abdominal muscles contract strongly, the abdomen arches, and the abdomen and perineum are lifted off the ground. 4 points

[0115] 2.2.4.10 Statistical Processing

[0116] SPSS 19.0 was used to analyze the data of each group. One-way analysis of variance was used to compare the groups for homogeneity of variance, and the LSD method was used for pairwise comparisons. P < 0.05 indicated statistically significant differences.

[0117] 2.3 Experimental Results

[0118] 2.3.1. Observation of general condition of rats

[0119] According to observation, all animals were weighed after balanced feeding as the initial body weight, and the body weight was measured before drug administration every day.

[0120] The rats in the blank group gained weight day by day, their fur became shiny, their mobility was good, their movements were agile, their reactions were quick, there was no fighting between the rats, no arching of the back and raising of the abdomen, their anus was clean, and the stool characteristics did not change.

[0121] The rats in the model group showed a decreased weight gain rate, no loose stools, dull, sparse fur, and no diarrhea. Their abdominal muscles contracted, their backs arched, and their abdomens lifted. They were prone to fighting. Their bowel movements increased only during stress, with increased fecal weight. Perianal fecal stains were more noticeable when restrained, and loose stools occurred during restraint, but not after release. The rats were also anxious and irritable.

[0122] Compared with the model group, the general condition of the rats in the Qizhiweitong group was significantly improved, with shiny fur, good mobility, less fighting between rats, no arched back and raised abdomen, cleaner anal area, no obvious odor on the bedding, and less loose stools when restrained.

[0123] 2.3.2 Effect of Qizhi Weitong Preparation on Body Weight Growth Rate in IBS-D Model Rats

[0124] At the beginning of model construction, there was no statistical difference in the weight growth rate of rats in each group. The weight growth rate of rats in each group during the experiment was shown in the table below. Figure 3 As shown in Table 9, on the 14th day of the experiment, compared with the blank group, the weight growth rate of the rats in the other groups decreased significantly; on the 21st day, compared with the blank group, the weight growth rate of the rats in the other groups was relatively slow, and the weight growth rate of the rats in the model group decreased significantly, with statistical differences; compared with the model group, the weight growth rate of each Qizhi Weitong drug-treated group increased, among which the weight growth rate of the Qizhi Weitong medium-dose group increased significantly, with statistical differences.

[0125] Table 9 Effect of Qi stagnation and stomachache on the body weight growth rate of IBS-D rats (X±s)

[0126] Group n Day 7 Day 14 Day 21 Blank group 18 21.1±6.9 39.0±14.9 47.8±18.7 Model Group 18 18.9±7.4 22.7±9.9 16.9±9.8** Qi stagnation stomach pain low dose group 18 21.2±9.6 25.4±8.5 23.8±13.3 Qi stagnation stomach pain medium dose group 18 20.2±7.4 26.3±6.7 24.8±11.7# Qi stagnation stomach pain high dose group 18 18.6±9.1 23.4±8.4 24.4±13.7 Pinaverium bromide group 18 20.8±4.6 24.9±5.1 26.2±10.8*

[0127] Note: **P<0.01, compared with the blank group, *P<0.05, compared with the blank group, # P<0.05, compared with the model group.

[0128] 2.3.3 Effect of Qi Stagnation and Stomach Pain on Food Intake in IBS-D Model Rats

[0129] The average food intake of rats in each group was Figure 4 As shown in Table 10, there is no significant difference in the food intake of rats among the treatment groups provided in the embodiments of the present invention.

[0130] Table 10 Effect of Qi stagnation and stomachache on food intake in IBS-D rats (X±s)

[0131] Group n Average food intake (g) Blank group 18 16.7±3.4 Model Group 18 16.2±3.6 Qi stagnation stomach pain low dose group 18 16.8±3.1 Qi stagnation stomach pain medium dose group 18 16.8±3.9 Qi stagnation stomach pain high dose group 18 16.7±3.0 Pinaverium bromide group 18 17.0±3.5

[0132] 2.3.4 Effect of Qi Stagnation and Stomach Pain on Diarrhea Index in IBS-D Model Rats

[0133] The diarrhea index is obtained by multiplying the loose stool rate and loose stool grade, which can reflect the diarrhea condition of rats. Before the experiment, the diarrhea index of rats in each group was 0.

[0134] During the experiment, the diarrhea of ​​rats was observed on the 14th and 21st days of administration. The results are shown in Table 11 and Figure 5-Figure 6It can be seen that compared with the blank group, the diarrhea index of the model group rats on the 14th and 21st days was significantly increased; compared with the model group, the diarrhea index of each Qizhi Weitong drug-treated group on the 14th and 21st days was significantly decreased. It can be seen that Qizhi Weitong granules can significantly improve the diarrhea of ​​IBS-D rats.

[0135] Table 11 Effects of Qi stagnation and stomachache on diarrhea index in IBS-D model rats

[0136] Group n Day 14 Day 21 Blank group 18 0.2±0.5 0.2±0.2 Model Group 18 2.8±1.0** 3.1±0.7** Qi stagnation stomach pain low dose group 18 <![CDATA[1.1±0.5 ## ]]> <![CDATA[1.0±0.7 ## ]]> Qi stagnation stomach pain medium dose group 18 <![CDATA[1.2±0.6 ## ]]> <![CDATA[1.0±0.7 ## ]]> Qi stagnation stomach pain high dose group 18 <![CDATA[0.8±0.4 ## ]]> <![CDATA[0.7±0.6 ## ]]> Pinaverium bromide group 18 <![CDATA[0.7±0.3 ## ]]> <![CDATA[0.6±0.4 ## ]]>

[0137] Note: ** P<0.01, compared with the blank group, ## P<0.01, compared with the model group.

[0138] 2.3.5 Effects of Qi Stagnation and Stomach Pain on Fecal Observation and Characteristic Scoring in IBS-D Model Rats

[0139] The anus of the rats in the blank group was clean and the feces were formed. The feces of the rats in the model group became soft or unformed, mostly watery, and there was feces adhering to the anus. In terms of the Bristol score of rats, the feces of the rats on the 14th and 21st days of administration were observed during the experiment. The results are shown in the figure. Figure 7-Figure 8 As shown in Table 12, compared with the blank group, the fecal typing scores of the model group rats on days 14 and 21 were significantly higher, and the difference was statistically significant. In contrast, compared with the model group, the Bristol fecal typing scores of the rats in the Qizhi Weitong groups at all doses were lower on days 14 and 21, and the fecal shape was improved. This suggests that Qizhi Weitong can improve diarrhea in IBS-D rats.

[0140] Table 12 Effects of Qi stagnation and stomachache on the trait classification scores of IBS-D model rats

[0141] Group n Day 14 Day 21 Blank group 18 2.4±0.7 2.6±0.8 Model Group 18 6.7±0.5** 6.7±0.5** Qi stagnation stomach pain low dose group 18 <![CDATA[5.3±0.7 ## ]]> <![CDATA[5.0±0.8 ## ]]> Qi stagnation stomach pain medium dose group 18 <![CDATA[5.0±0.8 ## ]]> <![CDATA[4.7±0.7 ## ]]> Qi stagnation stomach pain high dose group 18 <![CDATA[4.8±0.8 ## ]]> <![CDATA[4.7±0.7 ## ]]> Pinaverium bromide group 18 <![CDATA[4.9±0.7 ## ]]> <![CDATA[4.8±0.4 ## ]]>

[0142] Note: ** P<0.01, compared with the blank group, ## P<0.01, compared with the model group.

[0143] 2.3.6 Effect of Qi Stagnation and Stomach Pain on Fecal Water Content in IBS-D Model Rats

[0144] The feces moisture content of rats reflects the diarrhea condition of rats. During the experiment, feces of rats in each group were collected on the 14th and 21st days of administration to measure the feces moisture content. The results are shown in Figure 9-10and Table 13. Compared with the blank group, the fecal moisture content of the model group rats on days 14 and 21 was significantly increased; whereas, compared with the model group, the fecal moisture content of the Qizhi Weitong treatment groups on days 14 and 21 was significantly decreased, with statistically significant differences. This suggests that Qizhi Weitong has an ameliorative effect on diarrhea in IBS-D rats.

[0145] Table 13 Effect of Qi stagnation and stomachache on fecal water content in IBS-D model rats

[0146] Group n Day 14 (%) Day 21 (%) Blank group 18 3.7±2.4 2.9±0.6 Model Group 18 14.3±7.1** 6.0±2.2** Qi stagnation stomach pain low dose group 18 <![CDATA[8.4±7.1 ## ]]> <![CDATA[4.5±0.7 ## ]]> Qi stagnation stomach pain medium dose group 18 <![CDATA[5.5±4.2 ## ]]> <![CDATA[4.6±0.6 ## ]]> Qi stagnation stomach pain high dose group 18 <![CDATA[6.9±5.5 ## ]]> <![CDATA[4.5±0.7 ## ]]> Pinaverium bromide group 18 <![CDATA[10.2±3.6 # ]]> <![CDATA[4.5±1.0 ## ]]>

[0147] Note: ** P<0.01, compared with the blank group, # P<0.05, ## P<0.01, compared with the model group.

[0148] 2.3.7 Effects of Qi Stagnation and Stomach Pain on the Open Field Test in IBS-D Model Rats

[0149] The open field test explores the activity and excitement of rats by observing the autonomous behavior of animals in a new environment. Figure 11-14 .

[0150] In terms of horizontal movement grid counts, compared with the blank group, the model group showed a slight decrease in horizontal movement grid counts, but no significant difference. Compared with the model group, the low-dose Qizhiweitong group showed a significant increase in horizontal movement grid counts, indicating increased animal activity. The medium- and high-dose Qizhiweitong groups showed an increase in horizontal movement grid counts, but no significant difference was observed compared with the model group.

[0151] In terms of the number of vertical standing movements: compared with the blank group, the number of vertical standing movements of rats in the model group was significantly decreased, which was manifested as a decrease in the activity level of the animals; compared with the model group, the number of vertical standing movements of rats in each dose group of Qizhiweitong was significantly increased, which was manifested as an increase in the activity level of the animals.

[0152] In terms of total distance: compared with the blank group, the total distance traveled in the open field by the rats in the model group was slightly decreased; compared with the model group, the total distance traveled in the open field by the rats in each drug-treated group with qi stagnation and stomach pain was slightly increased, but there was no significant difference.

[0153] In terms of central distance: there was no significant difference in the central distance of the open field among the rats in each group.

[0154] The above results suggest that Qi-stagnation stomachache has a slight improvement effect on the anxiety-like behavior of IBS-D rats, but the effect is not significant.

[0155] 2.3.8 Effects of Qi Stagnation and Stomach Pain on the Cross-Elevated Maze Test in IBS-D Model Rats

[0156] The ratio of time spent in open arms (OT value) = the sum of time spent in the two open arms / (total time spent in the maze - time spent in the central area). These indicators can reflect the anxiety level of rats.

[0157] After the experiment, the time spent in the open arms and the number of times the rats entered the open arms were compared. The results are shown in Tables 15 and Figure 15-16 ,It can be seen that there was no significant difference among the groups in the ,open arm residence time and the number of open arm entries.

[0158] 2.3.9 Effects of Qi Stagnation and Stomach Pain on the Abdominal Withdrawal Reflex Test in IBS-D Model Rats

[0159] The abdominal wall reflex withdrawal test was conducted by expanding the rat intestine to evaluate the visceral sensitivity of the animals. After the experiment, the comparison of the pain levels of the rats in each group to different balloon expansion volumes in the abdominal wall withdrawal reflex test is shown in Table 16 and Figure 17 .

[0160] When the balloon was inflated to 1mL, 1.5mL, or 2mL, the pain intensity in the model group rats was significantly increased compared with the blank group, indicating increased visceral sensitivity. Compared with the model group, the pain intensity in the abdominal wall reflex test in each Qizhiweitong treatment group was significantly reduced, indicating decreased visceral sensitivity. This suggests that Qizhiweitong has an ameliorative effect on visceral hypersensitivity in IBS-D rats.

[0161] Table 16 Effects of Qi stagnation and stomachache on abdominal withdrawal reflex test in IBS-D model rats

[0162]

[0163] ** P<0.01, compared with the blank group, # P<0.05, ## P<0.01, compared with the model group.

[0164] In summary, the present invention established an IBS-D rat model induced by oral administration of senna leaves combined with chronic unpredictable mild stress CUMS modeling, and evaluated its efficacy by observing the effects of Qizhiweitong on the general state, diarrhea, anxiety-like behavior, depression-like behavior, and visceral hypersensitivity of the rats after administration. It was found that the Qizhiweitong preparation had no significant effect on the food intake and body weight of the rats in each group; by evaluating the water content of the feces of the rats in each group, typing and scoring the feces of the rats in each group, and recording the diarrhea index of the rats in each group, it was found that Qizhiweitong could improve The results showed that Qizhi Weitong could improve the diarrhea of ​​IBS-D rats. By evaluating the number of grid crossings, standing times, total distance and central distance of each group of rats in the open field test, as well as the ratio of open arm stay time and the number of open arm entries in the cross-elevated test, it was found that Qizhi Weitong could partially alleviate the anxiety-like behavior of IBS-D rats. By evaluating the pain degree of different balloon expansion volumes in the abdominal wall withdrawal reflex test, it was found that Qizhi Weitong could reduce the visceral hypersensitivity of IBS-D rats. In general, Qizhi Weitong has the pharmacological effect of improving IBS-D.

[0165] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of a qi stagnation and stomachache preparation in the preparation of a medicine for treating irritable bowel syndrome.

2. The use according to claim 1, characterized in that The irritable bowel syndrome is of diarrhea type.

3. The use according to claim 1, characterized in that The dosage form of the qi stagnation and stomachache preparation includes at least one of extract, granules, capsules, tablets, powders or oral liquids.

4. The use according to claim 1, characterized in that The medicine for treating irritable bowel syndrome includes at least one of an oral type and an injectable type.

5. The use according to claim 1, characterized in that The gastric pain preparation for qi stagnation comprises bupleurum root, vinegar-cured corydalis, fructus aurantii, vinegar-cured cyperus, angelica dahurica and roasted liquorice.

6. The use according to claim 5, characterized in that The Qi stagnation and stomachache preparation comprises the following components in parts by weight: 300-400 parts of Bupleurum chinense; 350-450 parts of vinegared Corydalis yanhusuo; 350-450 parts of Citrus aurantium; 350-450 parts of vinegar-soaked cyperus rotundus; 420-520 parts of Angelica dahurica; 150-250 parts of roasted licorice root.

7. A method for detecting compounds in a Qi-stagnation and stomach-ache preparation, characterized in that: The following steps are included: Prepare the Qi stagnation and stomachache preparation solution; The compounds in the Qizhiweitong preparation were determined by UHPLC / MS / MS.

8. The method for detecting compounds in the Qizhiweitong preparation according to claim 7, characterized in that: The method for preparing the Qizhiweitong preparation solution comprises mixing an alcohol solution and the Qizhiweitong preparation to form a 0.5-2 mg / mL solution, wherein the alcohol solution comprises a 40-60 vol% methanol aqueous solution.

9. The method for detecting compounds in the Qizhiweitong preparation according to claim 7, characterized in that: The chromatographic conditions are as follows: T3 chromatographic column; mobile phase A is an aqueous solution containing 0.1 vol% formic acid, and mobile phase B is an acetonitrile solution; the flow rate is 0.4 mL / min; The elution conditions are: 0min, the volume ratio of mobile phase A to mobile phase B was 9:1, 20min, the volume ratio of mobile phase A to mobile phase B is 6:4, 25min, the volume ratio of mobile phase A to mobile phase B is 4:6, 30 min, mobile phase B.

10. The method for detecting compounds in the Qizhiweitong preparation according to claim 7, characterized in that: Mass spectrometry conditions are as follows: Gas temperature 320°C, drying gas flow 8 L / min, nebulizer pressure 35 psig, sheath gas temperature 350°C, sheath gas flow 11 L / min, voltage 3.5 kV.

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