A method for constructing an experimental mouse diarrhea model

Abstract

A method for constructing an experimental mouse diarrhea model comprises: using mice as modeling subjects, adaptively feeding, and then administering a drug by gavage once daily for seven consecutive days. The mice are placed in a cage padded with filter paper after each gavage, and the diarrhea condition is observed four hours after gavage. The present invention provides a method for constructing an experimental mouse diarrhea model. The constructed model can effectively simulate diarrhea and evaluate the therapeutic effect of diarrhea drugs. The model has good repeatability and convenience, and has strong practical application value. During the modeling process of the present invention, the mice have good compliance and good living conditions. The mice also lose less weight during the modeling process, and the modeling duration is long, up to 23 hours or more.

Description

Technical Field

[0001] The invention belongs to the technical field of animal model construction, and particularly relates to a method for constructing an experimental mouse diarrhea model. Background Art

[0002] Senna leaves, a Chinese medicinal material, are the leaflets of the legume plants narrow-leaved senna or acuminata. ① Narrow-leaved senna: Pick the leaves before flowering, dry them in the shade, grade them according to leaf size and quality, and pack them with a hydraulic press. ② Acuminata: When the fruits are ripe, cut the branches, pick the leaves, dry them in the sun, and pack them separately as whole leaves and broken leaves. Functions and indications: Clearing heat and relieving stagnation. Treats constipation caused by heat, and abdominal distension caused by stagnation. ① "New Reference to Medicinal Pieces": Clears heat, benefits the intestines, and promotes bowel movements. ② "Modern Practical Chinese Medicine": Treats food stagnation, chest and abdominal distension, and constipation. Senna leaves are the leaflets of the legume plants narrow-leaved senna or acuminata.

[0003] Berberine hydrochloride is an organic compound with the chemical formula C 20 H 18 ClNO4 is a yellow crystalline powder, which is easily soluble in boiling water, slightly soluble in cold water, and almost insoluble in cold ethanol, chloroform and ether. It is mainly used as an antibacterial drug, and has inhibitory effects on Shigella dysenteriae, Escherichia coli, Pneumococcus, Staphylococcus aureus, Streptococcus, Salmonella typhi and ameba. It is mainly used clinically for intestinal infections and bacillary dysentery, etc. It has also been found to have anti-arrhythmic effects.

[0004] Diarrhea is one of the most common veterinary diseases, characterized by loose stools, increased bowel movements, dehydration, and weight loss, resulting in significant economic losses for the livestock industry. While studies have examined the clinical manifestations and other superficial indicators of senna-induced diarrhea, the duration of senna-induced diarrhea is short and spontaneous, leading to a critical challenge. To better understand the pathogenesis and treatment of diarrhea, establishing a mouse diarrhea model is crucial. This article describes the application of senna in establishing and evaluating a mouse diarrhea model. Summary of the Invention

[0005] The present invention aims to provide a method for constructing an experimental mouse diarrhea model.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for constructing an experimental mouse diarrhea model is characterized by: using mice as modeling objects, after adaptive feeding, administering a drug by gavage once a day for 7 consecutive days, placing the mice in a breeding cage padded with filter paper after each day's gavage, and observing the diarrhea condition 4 hours after gavage.

[0008] Furthermore, the dosage of the oral administration agent should be based on a ratio of mouse body weight to agent volume of 10:0.2, with the unit being g:ml.

[0009] Furthermore, the mice were 3 weeks old, of Kunming breed, and weighed 18 to 22 g.

[0010] Furthermore, the agent is composed of senna leaf extract and berberine hydrochloride in a mass ratio of 2 to 5:0.3 to 0.5. If the type and dosage of the agent are not properly selected, the mice will become hyperactive, have more diarrhea and stool, and lose more weight during the modeling process, which is not conducive to diarrhea modeling.

[0011] Furthermore, the senna leaf extract is prepared according to the following steps:

[0012] (1) Take senna leaves, crush them through a 12-mesh sieve to obtain senna leaf coarse powder;

[0013] (2) adding 50% to 70% by volume ethanol solution to the senna leaf coarse powder, refluxing and extracting for 2 to 3 hours, filtering the extract to obtain a filtrate;

[0014] (3) Take the above filtrate and place it in a rotary evaporator, set the temperature to 50-60 ° C, and the vacuum degree to

[0015] -0.05~-0.08MPa, recover ethanol and concentrate to a clear paste with a relative density of 1.18~1.22 at 60℃, and set aside;

[0016] (4) Take the above clear paste, place it in a vacuum drying oven with a temperature of 50-60°C and a vacuum degree of -0.05-0.08 MPa, and dry it under reduced pressure for 24-30 hours. Take it out to obtain a dry paste for later use;

[0017] (5) Take the dry paste, place it in a universal grinder, and grind it through a 100-mesh sieve to obtain senna leaf extract.

[0018] Furthermore, the medicament is prepared by grinding senna leaf extract and berberine hydrochloride for 10 to 15 minutes to obtain a mixed powder; while continuing to grind the mixed powder, slowly adding physiological saline, and after the addition of the physiological saline is completed, continuing to grind for 20 to 30 minutes to obtain the medicament; the mass ratio of the mixed powder to the physiological saline is 1:30 to 50.

[0019] The present invention has the following technical effects:

[0020] The present invention provides a method for constructing an experimental mouse diarrhea model. The constructed model can effectively simulate diarrhea, can evaluate the therapeutic effect of diarrhea drugs, has good repeatability and convenience, and has strong practical application value. During the modeling process of the present invention, the mice have good compliance and good living conditions. The mice also lose less weight during the modeling process, and the modeling duration is long, which can reach more than 23 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Example 1, control experiment 1, control experiment 2, loose stools in mice during the modeling process. DETAILED DESCRIPTION

[0022] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0023] Example 1

[0024] A method for constructing an experimental mouse diarrhea model comprises the following steps:

[0025] 1. Preparation of modeling agents:

[0026] (1) Take senna leaves, crush them through a 12-mesh sieve to obtain senna leaf coarse powder;

[0027] (2) adding a 70% by volume ethanol solution to the senna leaf coarse powder, and performing reflux extraction for 3 hours. After the extraction is completed, the extract is filtered to obtain a filtrate;

[0028] (3) Take the above filtrate, place it in a rotary evaporator, set the temperature to 60°C, and the vacuum degree to -0.08 MPa, recover the ethanol and concentrate it to a clear paste with a relative density of 1.22 at 60°C, and set aside;

[0029] (4) Take the above clear paste, place it in a vacuum drying oven with a temperature of 60°C and a vacuum degree of -0.08 MPa, and dry it under reduced pressure for 24 hours. Take it out to obtain a dry paste for later use;

[0030] (5) Take the dry paste, place it in a universal grinder, and grind it through a 100-mesh sieve to obtain senna leaf extract.

[0031] (6) Senna leaf extract and berberine hydrochloride were placed in a grinder at a mass ratio of 5:0.3, and ground for 15 minutes to obtain a mixed powder; while continuing to grind the mixed powder, physiological saline was slowly added, and after the addition of the physiological saline was completed, the mixture was ground for 30 minutes to obtain the medicine; the mass ratio of the mixed powder to the physiological saline was 1:30.

[0032] 2. Preparation of modeling agent: Ten 3-week-old Kunming mice weighing 18-22 g were gavaged with 0.2 mL of the agent per 10 g of body weight, once daily for 7 consecutive days. After each day's gavage, the mice were placed in a cage lined with filter paper and observed for diarrhea 4 hours after gavage.

[0033] Comparative Experiment 1: (Prepared according to the method of Example 1, berberine hydrochloride was not added to the preparation)

[0034] A method for constructing an experimental mouse diarrhea model comprises the following steps:

[0035] 1. Preparation of modeling agents:

[0036] (1) Take senna leaves, crush them through a 12-mesh sieve to obtain senna leaf coarse powder;

[0037] (2) adding a 70% by volume ethanol solution to the senna leaf coarse powder, and performing reflux extraction for 3 hours. After the extraction is completed, the extract is filtered to obtain a filtrate;

[0038] (3) Take the above filtrate, place it in a rotary evaporator, set the temperature to 60°C, and the vacuum degree to -0.08 MPa, recover the ethanol and concentrate it to a clear paste with a relative density of 1.22 at 60°C, and set aside;

[0039] (4) Take the above clear paste, place it in a vacuum drying oven with a temperature of 60°C and a vacuum degree of -0.08 MPa, and dry it under reduced pressure for 24 hours. Take it out to obtain a dry paste for later use;

[0040] (5) Take the dry paste, place it in a universal grinder, and grind it through a 100-mesh sieve to obtain senna leaf extract.

[0041] (6) Place the senna leaf extract in a grinder and grind for 15 minutes to obtain a mixed powder; while continuing to grind the mixed powder, slowly add physiological saline. After the addition of the physiological saline is completed, continue grinding for 30 minutes to obtain the medicine; the mass ratio of the senna leaf extract to the physiological saline is 1:30.

[0042] 2. Preparation of modeling agent: Ten 3-week-old Kunming mice weighing 18-22 g were gavaged with 0.2 mL of the agent per 10 g of body weight, once daily for 7 consecutive days. After each day's gavage, the mice were placed in a cage lined with filter paper and observed for diarrhea 4 hours after gavage.

[0043] Comparative experiment 2: (Blank group, no drugs were given, only saline)

[0044] Ten 3-week-old Kunming mice weighing 18-22 g were fed adaptively and then gavaged with 0.2 mL of normal saline per 10 g of body weight once a day for 7 consecutive days. After each day's gavage, the mice were placed in a cage padded with filter paper and observed for diarrhea 4 hours after gavage.

[0045] In Example 1, Comparative Experiment 1 and Comparative Experiment 2, continuous gavage was performed for 7 days to observe the following results:

[0046] 1. Mouse status during modeling:

[0047]

[0048] As can be seen from the above table, during the entire modeling period, the condition of the mice in comparative experiment 1 was worse than that of the product in embodiment 1.

[0049] 2. The time when mice start to have diarrhea and the duration of diarrhea during the modeling process:

[0050]

[0051] As can be seen from the above table, in the Example 1 group, after administration, the duration of diarrhea was longer than that in the comparative experiment 1, which better met the model requirements.

[0052] 3. Changes in mouse weight during modeling:

[0053] Group Before the test After 7 days of testing weight gain Example 1 group 24.32±0.67 24.02±1.36Bb -0.30±1.01Bb Comparative experiment group 1 24.45±1.05 21.48±1.64Bb -2.97±1.54Bb Comparative experiment group 2 24.60±0.65 31.33±2.53Aa 6.53±3.14Aa

[0054] As can be seen from the table above, based on the weight of mice before and after modeling, the weight change of the modeling method of Example 1 is smaller than that of the control experiment group 1, indicating that the modeling method of Example 1 is in better condition.

[0055] 4. The rate of loose stools in mice during the modeling process:

[0056]

[0057] The mice in the blank group had no loose stools during the test. In Example 1 combined with comparative experiment group 1, the loose stool rate of the mice increased significantly after taking the corresponding drugs. There was no significant difference in the loose stool rate between Example 1 group and comparative experiment group 1.

[0058] Example 2

[0059] A method for constructing an experimental mouse diarrhea model comprises the following steps:

[0060] 1. Preparation of modeling agents:

[0061] (1) Take senna leaves, crush them through a 12-mesh sieve to obtain senna leaf coarse powder;

[0062] (2) adding a 60% by volume ethanol solution to the senna leaf coarse powder, and performing reflux extraction for 2.5 hours. After the extraction is completed, the extract is filtered to obtain a filtrate;

[0063] (3) Take the above filtrate, place it in a rotary evaporator, set the temperature to 55°C, and the vacuum degree to -0.06 MPa, recover the ethanol and concentrate it to a clear paste with a relative density of 1.20 at 60°C, and set aside;

[0064] (4) Take the above clear paste, place it in a vacuum drying oven with a temperature of 55°C and a vacuum degree of -0.06 MPa, and dry it under reduced pressure for 28 hours. Take it out to obtain a dry paste for later use;

[0065] (5) Take the dry paste, place it in a universal grinder, and grind it through a 100-mesh sieve to obtain senna leaf extract.

[0066] (6) Senna leaf extract and berberine hydrochloride were placed in a grinder at a mass ratio of 3:0.4, and ground for 12 minutes to obtain a mixed powder; while continuing to grind the mixed powder, physiological saline was slowly added, and after the addition of the physiological saline was completed, the mixture was ground for 25 minutes to obtain the medicine; the mass ratio of the mixed powder to the physiological saline was 1:40.

[0067] 2. Preparation of modeling agent: Ten 3-week-old Kunming mice weighing 18-22 g were fed adaptively and then gavaged once daily for 7 consecutive days at a dose of 0.2 mL per 10 g of body weight. After each day's gavage, the mice were placed in a cage lined with filter paper and observed for diarrhea 4 hours after gavage.

[0068] During the modeling process, the mice were in high spirits and had normal urine on the first day, in high spirits and with yellowish urine on the second day, and in high spirits and with normal urine after the third day. By the seventh day, the duration of diarrhea in the mice was 23 hours, the weight gain was -0.32±1.63, and the loose stool rate was 60.21±5.36.

[0069] Example 3

[0070] A method for constructing an experimental mouse diarrhea model comprises the following steps:

[0071] 1. Preparation of modeling agents:

[0072] (1) Take senna leaves, crush them through a 12-mesh sieve to obtain senna leaf coarse powder;

[0073] (2) adding a 50% by volume ethanol solution to the senna leaf coarse powder, and performing reflux extraction for 2 hours. After the extraction is completed, the extract is filtered to obtain a filtrate;

[0074] (3) Take the above filtrate, place it in a rotary evaporator, set the temperature to 50°C, and the vacuum degree to -0.05 MPa, recover the ethanol and concentrate it to a clear paste with a relative density of 1.18 at 60°C, and set aside;

[0075] (4) Take the above clear paste, place it in a vacuum drying oven with a temperature of 50°C and a vacuum degree of -0.05 MPa, and dry it under reduced pressure for 30 hours. Take it out to obtain a dry paste for later use;

[0076] (5) Take the dry paste, place it in a universal grinder, and grind it through a 100-mesh sieve to obtain senna leaf extract.

[0077] (6) Senna leaf extract and berberine hydrochloride were placed in a grinder at a mass ratio of 2:0.5, and ground for 10 minutes to obtain a mixed powder; while continuing to grind the mixed powder, physiological saline was slowly added, and after the addition of the physiological saline was completed, the mixture was ground for 20 minutes to obtain the medicine; the mass ratio of the mixed powder to the physiological saline was 1:50.

[0078] 2. Preparation of modeling agent: Ten 3-week-old Kunming mice weighing 18-22 g were fed adaptively and then gavaged once daily for 7 consecutive days at a dose of 0.2 mL per 10 g of body weight. After each day's gavage, the mice were placed in a cage lined with filter paper and observed for diarrhea 4 hours after gavage.

[0079] During the modeling process, the mice were in high spirits and had normal urine on the first day, in high spirits and with yellowish urine on the third day, and in high spirits and with normal urine after the fourth day. By the seventh day, the duration of diarrhea in the mice was 26 hours, the weight gain was -0.32±1.63, and the loose stool rate was 58.30±7.11.

Claims

1. A method for constructing an experimental mouse diarrhea model, characterized by: Mice were used as modeling subjects. After adaptive feeding, the drug was administered by gavage once a day for 7 consecutive days. The mice were placed in a cage padded with filter paper after the daily gavage. The diarrhea was observed 4 hours after gavage. The dosage of the drug for gavage was based on a ratio of mouse body weight to drug volume of 10:0.2, with the unit of g:mL. The drug was composed of senna leaf extract and berberine hydrochloride, with a mass ratio of preferably 2-5:0.3-0.

5. The senna leaf extract was prepared according to the following steps: (1) Take senna leaves, crush them through a 12-mesh sieve to obtain coarse senna powder; (2) Add 50% to 70% ethanol solution by volume to the senna leaf coarse powder, reflux and extract for 2 to 3 hours. After the extraction is completed, filter the extract to obtain a filtrate; (3) Take the above filtrate, place it in a rotary evaporator, set the temperature to 50~60℃, and the vacuum degree to -0.05~-0.08MPa, recover the ethanol and concentrate it to a clear paste with a relative density of 1.18~1.22 at 60℃, and set aside; (4) Take the above clear paste, place it in a vacuum drying oven with a temperature of 50-60°C and a vacuum degree of -0.05-0.08 MPa, and dry it under reduced pressure for 24-30 hours. Take it out to obtain a dry paste for later use; (5) Take the dry paste, place it in a universal grinder, and grind it through a 100-mesh sieve to obtain senna leaf extract.

2. The method for constructing an experimental mouse diarrhea model according to claim 1, wherein: The mice were 3 weeks old, of the Kunming variety, and weighed 18-22 g.

3. The method for constructing an experimental mouse diarrhea model according to claim 2, wherein: The medicament is prepared by grinding senna leaf extract and berberine hydrochloride for 10 to 15 minutes to obtain a mixed powder; while continuing to grind the mixed powder, slowly adding physiological saline, and after the addition of the physiological saline is completed, continuing to grind for 20 to 30 minutes to obtain the medicament; the mass ratio of the mixed powder to the physiological saline is 1:30 to 50.

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