A method for constructing a diarrhea model in experimental mice using a combined medicament

Abstract

A method for constructing a diarrhea model of experimental mice using a combined medicament, characterized in that: using mice as the modeling object, after adaptive feeding, the combined medicament is given by gavage once a day for 7 consecutive days. After the gavage each day, the mice are placed in a breeding cage lined with filter paper, and the diarrhea situation is observed 4 hours after gavage; wherein the combined medicament is prepared by extracting senna leaf and fibraurea recisa. A method for constructing a diarrhea model of experimental mice using a combined medicament has good repeatability in the construction method, is simple and convenient, has good compliance of mice during the modeling process, high compliance, good living state, no restlessness phenomenon, small weight loss, long duration after modeling, which can be up to more than 28 hours, and the loose stool rate of mice is relatively high, comparable to that of the conventional senna leaf modeling, but the mice have better tolerance compared with the traditional senna leaf modeling. During the modeling process, the weight loss of mice is relatively small, and the modeling success rate is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal model construction, and particularly relates to a method for constructing a diarrhea model of experimental mice by using a combined medicament. Background Art

[0002] Animal model construction uses animals as experimental research objects or carriers to simulate the occurrence, progression, prognosis, etc. of a certain disease, and is one of the important means for research such as drug efficacy evaluation, screening, and gene therapy. In aspects such as clinical pathophysiology and disease mechanisms, animal models also play an important role. In the aspects of disease treatment and drug screening, the construction of animal models is the key for pharmaceutical companies to conduct preliminary screening and tests on new drugs. Common model animals include mice, rats, guinea pigs, pigs, monkeys, etc. In medical and life science research, animal models are widely used, and they can provide physiological and behavioral characteristics similar to those of humans to help scientists understand the development and treatment of human diseases.

[0003] Constructing animal models that conform to clinical practice has important functions, mainly including the following aspects: 1. Exploring disease mechanisms: Animal models can provide better conditions to understand the development and mechanisms of diseases. For example, by observing gene changes in mice, scientists can better understand the development process of human cancer. This method can accelerate research results and also reduce dangerous experiments on humans. 2. Evaluating drug safety: In the process of drug development, animal models can help scientists evaluate the safety and effectiveness of drugs. This method can pre-determine whether a drug is likely to cause adverse reactions, conduct small-scale tests before clinical trials to save time and resources, and protect the safety of human subjects. 3. Developing new treatment methods: Animal models can help scientists research and develop new treatment methods. For example, in the study of heart diseases, scientists can use mouse models to test new treatment methods to determine their effectiveness and further optimize treatment plans. 4. Popularizing public health: Animal models can also be used to detect and prevent the spread of infectious diseases. For example, when studying malaria and influenza vaccines, scientists can use mouse models to test the effectiveness of vaccines and determine the vaccination time and dose.

[0004] Senna (Senna alexandrina Milll) is a small herbaceous shrub of the genus Cassia in the Leguminosae family. The leaves are ovate-lanceolate to linear-lanceolate, glabrous or almost glabrous. The petals are yellow, obovate, and the two petals below are larger. The fruit is rectangular, flat, with a small and inconspicuous apex, and hairy when young. The seed coat is brown-green, with a fine linear stalk and warty wrinkles. The flowering period is from September to December, and the fruiting period is in March of the following year. The name of senna represents both its origin and its efficacy. "Fan" is foreign, coming from Alexandria, Egypt, "Xie" means the efficacy of diarrhea, and "Ye" means the medicinal part. Senna is native to tropical coastal or island areas in East Africa, southern Arabia, northwestern and southern India, and Pakistan. It is widely planted in India and Pakistan. It has been introduced and cultivated in Guangxi and Yunnan, China. Senna prefers dryness to wetness, and should be cultivated in high, dry, well-drained, and loose sandy loam. The main method of reproduction is seed propagation. Senna has the effect of purging and removing stagnation, and is a commonly used laxative in clinical practice. The "New Reference to Medicinal Pieces" records that senna "relieves heat, benefits the intestines and bowels, and relieves constipation." Modern pharmacological studies have shown that senna also has antibacterial, hemostatic, muscle relaxing, and spasmodic effects. Senna was used as medicine by Arab doctors in the 9th century AD, and was introduced to China in modern times. It was recorded in the "Chinese Pharmaceutical Dictionary" and "New Reference to Medicinal Pieces" published in 1935.

[0005] Huangteng is the dried stem of Fibraurea recisa, a plant of the Menispermaceae family. It is also known as Tianxianteng, Tuhuanglian, Jinsuoshi, and Huanglian. Its main component, Huangtenglin, is yellow needle-shaped crystals (water), with a melting point of 198-201°C (decomposition), extremely bitter, easily soluble in hot water, slightly soluble in water, slightly soluble in ethanol and chloroform, and almost insoluble in ether. The melting point of iodide is 240°C (decomposition), and the nitrate is light yellow needle-shaped crystals (methanol), with a melting point of 240°C (decomposition). Huangtenglin has antibacterial activity, anti-inflammatory effects, immune enhancement effects, and acts on conjunctivitis, bacterial dysentery, and respiratory system infections.

[0006] Diarrhea is characterized by loose stools, increased bowel movements, dehydration, and weight loss, which brings huge problems to the breeding industry. At present, there are studies on the clinical manifestations and other apparent indicators of diarrhea caused by senna leaves, but the retention time of diarrhea caused by senna leaves is short and can recover on its own in a short period of time. How to prolong its diarrhea time is a key issue that needs to be solved urgently. In order to better study the pathogenesis and treatment of diarrhea, it is particularly necessary to establish a mouse diarrhea model. Summary of the invention

[0007] The present invention aims to provide a method for constructing a diarrhea model of experimental mice by using a combination of drugs.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A method for constructing a diarrhea model in experimental mice using a combined medicament, characterized in that: using mice as the modeling object, after adaptive feeding, the combined medicament is given by gavage once a day for 7 consecutive days. After each gavage, the mice are placed in a breeding cage lined with filter paper, and the diarrhea situation is observed 4 hours after gavage; wherein the combined medicament is prepared by extracting senna leaf and fibraurea recisa pierre.

[0010] Furthermore, the mixed extraction of senna leaf and fibraurea recisa pierre is prepared according to the following steps:

[0011] (1) Take senna leaf and fibraurea recisa pierre, place them in a universal grinder, crush them through a 18-mesh sieve to obtain a mixed powder of senna leaf and fibraurea recisa pierre, and set aside.

[0012] (2) Add an ethanol solution with a volume fraction of 30% - 40% to the mixed powder, reflux and extract for 1 - 2 hours. After the extraction is completed, filter the extract to obtain a filtrate.

[0013] (3) Take the above filtrate, place it in a rotary evaporator, set the temperature at 55 - 60 °C, and the vacuum degree at

[0014] -0.05 - -0.08 MPa, recover ethanol and concentrate it to a clear paste with a relative density of 1.23 - 1.25 at 60 °C, and set aside.

[0015] (4) Take the above clear paste, add purified water, stir and mix, then let it stand for 15 - 20 hours, and set aside.

[0016] (5) Take the supernatant after standing, place it in a spray drying granulator, set the inlet air temperature at 100 - 120 °C, the inlet air frequency at 30 - 35 Hz, the spray pressure at 0.3 - 0.5 MPa, and the liquid spraying rotation speed at 100 - 130 rpm for spray granulation. After completion, obtain a mixed extract powder.

[0017] Furthermore, the mass ratio of senna leaf to fibraurea recisa pierre is 5:2 - 3, the mass ratio of the mixed powder to the ethanol solution with a volume fraction of 30% - 40% is 1:20 - 30, and the mass ratio of the clear paste before standing to purified water is 1:10 - 15.

[0018] Furthermore, the dosage of the gavage medicament needs to be carried out according to the dose of the ratio of mouse body weight to medicament volume of 10:0.5, with the unit of g:mL.

[0019] Furthermore, the mice are 3 - week - old, of the Kunming mouse strain, and weigh 18 - 22 g.

[0020] Further, for the medicament, take the mixed extract powder, place it in a grinder, first grind for 20 - 30 minutes, and then, while continuing to grind, slowly add normal saline. After the addition of normal saline is completed, continue to grind for 15 - 20 minutes to obtain the medicament; the mass ratio of the mixed extract powder to normal saline is 1:30 - 50. The present invention has the following technical effects:

[0021] A method for constructing a diarrhea model in experimental mice using a combined medicament, the construction method has good repeatability, is simple and convenient, the compliance of mice during the modeling process is good, the compliance is high, the clinical state is good, there is no restlessness phenomenon, the weight loss is small, the duration after modeling is long, up to more than 28 hours, the loose stool rate of mice is relatively high, comparable to that of the conventional senna leaf modeling, but the tolerance of mice is better than that of the traditional senna leaf modeling. During the modeling process, the weight loss of mice is small, and the modeling success rate is improved. Description of the Drawings

[0022] Figure 1 : Diarrhea condition diagrams of mice during the modeling processes of Example 1, Control Experiment 1, and Control Experiment 2. Detailed Embodiments

[0023] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the protection scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention belongs to the scope of the present invention.

[0024] Example 1

[0025] A method for constructing a diarrhea model in experimental mice using a combined medicament, which comprises the following steps:

[0026] 1. Preparation of the mixed extract:

[0027] (1) Take senna leaf and fibraureae caulis, place them in a universal grinder, pulverize and sieve through a 18 - mesh sieve to obtain the mixed powder of senna leaf and fibraureae caulis for standby; the mass ratio of senna leaf to fibraureae caulis is 5:2;

[0028] (2) Add an ethanol solution with a volume fraction of 35% to the mixed powder, reflux and extract for 1.5 hours. After the extraction is completed, filter the extract to obtain a filtrate; the mass ratio of the mixed powder to the ethanol solution with a volume fraction of 30% - 40% is 1:25;

[0029] (3) Take the above filtrate, place it in a rotary evaporator, set the temperature to 58°C and the vacuum degree to -0.07 MPa, recover ethanol and concentrate it to a clear paste with a relative density of 1.24 at 60°C for standby;

[0030] (4) Take the above-mentioned clear extract, add purified water, stir and mix, then let it stand for 18 hours for standby; the mass ratio of the clear extract to purified water before standing is 1:13;

[0031] (5) Take the supernatant after standing, place it in a spray drying granulator, set the inlet air temperature at 110 °C, the inlet air frequency at 32 Hz, the spray pressure at 0.4 MPa, and the liquid spraying rotation speed at 120 rpm for spray granulation. After completion, obtain the mixed extract powder.

[0032] 2. Preparation of the combined medicament: Take the mixed extract powder, place it in a grinder, first grind for 25 minutes, and then, while continuing to grind, slowly add physiological saline. After the addition of physiological saline is completed, continue to grind for 18 minutes to obtain the medicament; the mass ratio of the mixed extract powder to physiological saline is 1:40.

[0033] 3. Modeling: Use 10 Kunming mice at 3 weeks old with a body weight of 18 - 22 g. After adaptive feeding, intragastrically administer the medicament to the Kunming mice at a dose of 0.5 mL per 10 g body weight once a day for 7 consecutive days. After each intragastric administration, place them in a breeding cage lined with filter paper, and observe the diarrhea situation 4 hours after intragastric administration.

[0034] Comparative experiment 1: (According to the preparation method of Example 1, the combined medicine does not contain Fibraurea recisa)

[0035] A method for constructing a diarrhea model of experimental mice using a combined medicament, which includes the following steps:

[0036] 1. Preparation of the mixed extract:

[0037] (1) Take Folium Sennae, place it in a universal crusher, crush it through an 18-mesh sieve to obtain Folium Sennae powder for standby;

[0038] (2) Add an ethanol solution with a volume fraction of 35% to the Folium Sennae powder, reflux and extract for 1.5 hours. After the extraction is completed, filter the extract to obtain a filtrate; the mass ratio of the mixed powder to the ethanol solution with a volume fraction of 30% - 40% is 1:25;

[0039] (3) Take the above-mentioned filtrate, place it in a rotary evaporator, set the temperature at 58 °C and the vacuum degree at -0.07 MPa, recover ethanol and concentrate it to a clear extract with a relative density of 1.24 at 60 °C for standby;

[0040] (4) Take the above-mentioned clear extract, add purified water, stir and mix, then let it stand for 18 hours for standby; the mass ratio of the clear extract to purified water before standing is 1:13;

[0041] (5) Take the supernatant after standing still, place it in a spray drying granulator, set the inlet air temperature at 110 °C, the inlet air frequency at 32 Hz, the spray pressure at 0.4 MPa, and the liquid spraying rotation speed at 120 rpm to conduct spray granulation. After completion, obtain the extract powder.

[0042] 2. Preparation of the combined medicament: Take the extract powder and place it in a grinder. First, grind for 25 minutes, and then, while continuing to grind, slowly add normal saline. After the addition of normal saline is completed, continue to grind for 18 minutes to obtain the medicament; the mass ratio of the extract powder to normal saline is 1:40.

[0043] 3. Modeling: Use 10 Kunming mice at 3 weeks old with a body weight of 18 - 22 g. According to the dosage of 0.5 mL of the medicament per 10 g of body weight, intragastrically administer the medicament to the Kunming mice once a day for 7 consecutive days. After the intragastric administration is completed each day, place them in a breeding cage lined with filter paper. Observe the diarrhea situation 4 hours after intragastric administration.

[0044] Comparative experiment 2: (Blank group, without giving any drug, only giving normal saline)

[0045] Use 10 Kunming mice at 3 weeks old with a body weight of 18 - 22 g. After adaptive feeding, intragastrically administer normal saline at a dosage of 0.5 mL per 10 g of body weight once a day for 7 consecutive days. After the intragastric administration is completed each day, place them in a breeding cage lined with filter paper. Observe the diarrhea situation 4 hours after intragastric administration.

[0046] With Example 1, Comparative experiment 1, and Comparative experiment 2, observe after continuous intragastric administration for 7 days to obtain the following results:

[0047] 1. Mouse status during the modeling process:

[0048]

[0049] As can be seen from the above table, during the entire modeling period, the mouse status in Comparative experiment 1 is worse than that of the product in Example 1.

[0050] 2. Time when the mice start to have diarrhea and the duration of diarrhea during the modeling process:

[0051]

[0052] As can be seen from the above table, in the group of Example 1, after administration, the duration of diarrhea is longer than that in Comparative experiment 1, and it can better meet the model requirements.

[0053] 3. Change in mouse body weight during the modeling process:

[0054]

[0055] As can be seen from the above table, based on the body weights of the mice before and after modeling, the body weight change of the modeling method in Example 1 is smaller compared to that of the control experiment group 1. This indicates that the mice are in a better state under the modeling method of Example 1.

[0056] 4. The loose stool rate of mice during the modeling process:

[0057]

[0058] There was no loose stool in the test mice of the blank group during the test period. In Example 1 group and the control experiment group 1, after the mice took the corresponding medicaments, their loose stool rates increased significantly. There was no obvious difference in the loose stool rates between Example 1 group and the control experiment group 1.

[0059] Example 2

[0060] A method for constructing a diarrhea model of test mice using a combined medicament, which comprises the following steps:

[0061] 1. Preparation of the mixed extract:

[0062] (1) Take senna leaf and fibraurea recisa pierre, place them in a universal pulverizer, pulverize and sieve through a 18-mesh sieve to obtain a mixed powder of senna leaf and fibraurea recisa pierre, and set aside; the mass ratio of senna leaf to fibraurea recisa pierre is 5:2;

[0063] (2) Add an ethanol solution with a volume fraction of 30% to the mixed powder, reflux and extract for 1 hour. After the extraction is completed, filter the extract to obtain a filtrate; the mass ratio of the mixed powder to the ethanol solution with a volume fraction of 30% - 40% is 1:20;

[0064] (3) Take the above filtrate, place it in a rotary evaporator, set the temperature to 55°C and the vacuum degree to -0.05 MPa, recover ethanol and concentrate it to a clear paste with a relative density of 1.23 at 60°C, and set aside;

[0065] (4) Take the above clear paste, add purified water, stir and mix, and then let it stand for 15 hours, and set aside; the mass ratio of the clear paste to the purified water before standing is 1:10;

[0066] (5) Take the supernatant after standing, place it in a spray drying granulator, set the inlet air temperature to 100°C, the inlet air frequency to 30 Hz, the spray pressure to 0.3 MPa, and the liquid spraying rotation speed to 100 rpm for spray granulation. After completion, obtain a mixed extract powder.

[0067] 2. Preparation of the combined medicament: Take the mixed extract powder, place it in a grinder, first grind for 20 minutes, and then, while continuing to grind, slowly add normal saline. After the addition of normal saline is completed, continue to grind for 15 minutes to obtain the medicament; the mass ratio of the mixed extract powder to normal saline is 1:30.

[0068] 3. Modeling: Use 10 Kunming mice at 3 weeks old with a body weight of 18 - 22 g. Administer the medicament to the Kunming mice by gavage at a dose of 0.5 mL per 10 g body weight once a day for 7 consecutive days. After each gavage, place the mice in a breeding cage lined with filter paper, and observe the diarrhea situation 4 hours after gavage.

[0069] Example 3

[0070] A method for constructing a diarrhea model of experimental mice using a combined medicament, which includes the following steps:

[0071] 1. Preparation of mixed extract:

[0072] (1) Take senna leaf and fibraurea recisa pierre, place them in a universal grinder, crush them through a 18 - mesh sieve to obtain a mixed powder of senna leaf and fibraurea recisa pierre for standby; the mass ratio of senna leaf to fibraurea recisa pierre is 5:3.

[0073] (2) Add an ethanol solution with a volume fraction of 40% to the mixed powder, reflux and extract for 2 hours. After the extraction is completed, filter the extract to obtain a filtrate; the mass ratio of the mixed powder to the ethanol solution with a volume fraction of 30% - 40% is 1:30.

[0074] (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 ethanol and concentrate it to a clear paste with a relative density of 1.25 at 60 °C for standby.

[0075] (4) Take the above clear paste, add purified water, stir and mix, then let it stand for 20 hours for standby; the mass ratio of the clear paste to purified water before standing is 1:15.

[0076] (5) Take the supernatant after standing, place it in a spray drying granulator, set the inlet air temperature to 120 °C, the inlet air frequency to 35 Hz, the spray pressure to 0.5 MPa, and the liquid spraying rotation speed to 130 rpm for spray granulation. After completion, obtain a mixed extract powder.

[0077] 2. Preparation of the combined medicament: Take the mixed extract powder, place it in a grinder, first grind for 30 minutes, and then, while continuing to grind, slowly add physiological saline. After the addition of physiological saline is completed, continue to grind for 20 minutes to obtain the medicament; the mass ratio of the mixed extract powder to physiological saline is 1:50.

[0078] 3. Modeling: Use 10 Kunming mice at 3 weeks old with a body weight of 18 - 22 g. Administer the medicament to the Kunming mice by gavage at a dose of 0.5 mL per 10 g body weight once a day for 7 consecutive days. After each gavage, place the mice in a breeding cage lined with filter paper, and observe the diarrhea situation 4 hours after gavage.

Claims

1. A method for constructing a diarrhea model in experimental mice using a combined medicament, characterized in that: Using mice as the modeling object, after adaptive feeding, the combined medicament was administered by gavage once a day for 7 consecutive days. After each gavage, the mice were placed in a breeding cage lined with filter paper. Four hours after gavage, the diarrhea situation was observed. The combined medicament was prepared by extracting senna leaf and fibraurea recisa. Specifically, senna leaf and fibraurea recisa were crushed into a mixed powder, and a 30% - 40% ethanol solution was added for reflux extraction. The filtrate obtained by filtering the extract was rotary evaporated into a clear paste with a relative density of 1.23 - 1.25 at 60°C. The clear paste was mixed with purified water according to a mass ratio of 1:10 - 15 and allowed to stand. The supernatant was taken for spray granulation to obtain a mixed extract powder. The mass ratio of senna leaf to fibraurea recisa was 5:2 - 3, and the mass ratio of the mixed powder to the ethanol solution was 1:20 - 30.

2. The method for constructing a diarrhea model of experimental mice using a combined medicament as described in claim 1, wherein, The mixed extraction of senna leaf and fibraurea recisa was prepared according to the following steps: (1) Take senna leaf and fibraurea recisa, place them in a universal grinder, crush them through a 18-mesh sieve to obtain a mixed powder of senna leaf and fibraurea recisa, and set aside. (2) Add an ethanol solution with a volume fraction of 30% - 40% to the mixed powder, reflux extract for 1 - 2 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 at 55 - 60°C, and the vacuum degree at -0.05 - -0.08 MPa, recover ethanol and concentrate it to a clear paste with a relative density of 1.23 - 1.25 at 60°C, and set aside. (4) Take the above clear paste, add purified water, stir and mix, and then let it stand for 15 - 20 hours, and set aside. (5) Take the supernatant after standing, place it in a spray drying granulator, set the inlet air temperature at 100 - 120°C, the inlet air frequency at 30 - 35 Hz, the spray pressure at 0.3 - 0.5 MPa, and the spray liquid rotation speed at 100 - 130 rpm for spray granulation. After completion, obtain a mixed extract powder.

3. The method for constructing a diarrhea model of experimental mice using a combined medicament according to claim 2, characterized in that, The dosage of the combined medicament needs to be carried out according to the dose with a volume ratio of mouse body weight to the combined medicament of 10:0.5, with the unit of g:mL.

4. The method for constructing a diarrhea model of experimental mice using a combined medicament according to claim 3, characterized in that, The mice were 3-week-old Kunming mice with a body weight of 18 - 22 g.

5. The method for constructing a diarrhea model of experimental mice using a combined agent as described in claim 4, characterized in that, For the combined medicament, take the mixed extract powder, place it in a grinder, grind it for 20 - 30 minutes first, and then, while continuing to grind, slowly add physiological saline. After the addition of physiological saline is completed, continue to grind for 15 - 20 minutes to obtain the combined medicament. The mass ratio of the mixed extract powder to physiological saline is 1:30 - 50.

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