CN117653686A

CN117653686A - Fennel oral preparation and preparation method and application thereof

Info

Publication number: CN117653686A
Application number: CN202311687466.9A
Authority: CN
Inventors: 买买提·艾力; 斯拉甫·艾白; 窦勤; 刘桂花; 巴合沙拉·马乃甫; 郭君婷; 李治建
Original assignee: HOSPITAL OF XINJIANG TRADITIONAL UYGHUR MEDICINE
Current assignee: HOSPITAL OF XINJIANG TRADITIONAL UYGHUR MEDICINE
Priority date: 2023-12-11
Filing date: 2023-12-11
Publication date: 2024-03-08

Abstract

The invention relates to an fennel oral preparation and a preparation method and application thereof. The oral preparation has effects in regulating abnormal mucilage, enhancing forceps and relieving cough and asthma. Oral preparation for treating bronchial asthma and chronic bronchitis caused by exogenous pathogenic factor and cough variant asthma.

Description

Fennel oral preparation and preparation method and application thereof

Technical Field

The invention relates to an fennel oral preparation, a preparation method and application thereof, wherein the oral preparation has the functions of regulating abnormal mucilage, enhancing the forceps force, relieving asthma and relieving asthma. Oral preparation for treating bronchial asthma and chronic bronchitis caused by exogenous pathogenic factor and cough variant asthma.

Background

Bronchial asthma is a common disease of the clinical respiratory system, especially well developed in childhood. According to epidemiology statistics, the prevalence rate of the childhood asthma is about 3.2%, and due to the increasingly serious environmental and food problems, the number of new-born asthmatics is continuously increased in recent years, the course of the asthma is long, the illness is easy to repeat, and the trend is gradually growing. Is easy to cause great harm to the body and mind of the children. The standard of diagnosis and prevention guidelines for childhood bronchial asthma states that asthma needs to be treated as early as possible, especially in the acute stage, and should be treated effectively in time to prevent disease from progressing to respiratory failure and threaten the life safety of the child. Currently, the Western medicine is mostly used for treating asthma by adopting a stepped treatment scheme in the global asthma diagnosis and management guidelines, wherein the atomized inhaled glucocorticoid is the first medicament for treating and controlling asthma. However, most of the family members of the infant have concerns about adverse reactions caused by long-term administration of glucocorticoids, which results in reduced compliance in treatment and recurrent attacks of asthma in the infant. One of the public health and medical care problems that has become a need to be solved. The principle of treating the symptoms according to the urgent rules and treating the root causes according to the slow rules has unique advantages in the aspect of preventing and treating asthma. A large number of researches show that the traditional Chinese medicine achieves the effects of resisting inflammation, improving immunity, resisting airway inflammation, reducing airway hyperresponsiveness and the like through the regulation of an immune system, and has remarkable effect of treating acute attack of bronchial asthma and chronic bronchitis.

Cough variant asthma is a special type of bronchial asthma, cough is the only or major clinical manifestation thereof, without obvious symptoms or signs of wheezing, shortness of breath, etc., but with airway hyperreactivity. Traditional Chinese medicine generally belongs to cough, and is considered to be related to lung dysfunction and lung qi upward reversal caused by exogenous pathogenic factors invasion, viscera dysfunction and other factors. Cough variant asthma pathogenesis is basically the same as that of typical asthma, namely, the pathogenesis is combined by immune, genetic, environmental and other factors, and chronic airway inflammation and airway hyperresponsiveness which are participated by various inflammatory cells and inflammatory mediators, so that the treatment principle of the cough variant asthma is similar to that of the typical asthma. Initial treatment of cough variant asthma generally recommends the first choice of inhaled glucocorticoids in combination with bronchodilators, and the recommended application time of inhaled glucocorticoids should not be less than 8 weeks. However, the single western medicine treatment of cough variant asthma still faces various disputes or problems at present, such as various adverse reactions of western medicines such as glucocorticoid, beta 2 adrenergic receptor agonist and the like which are used for a long time, high afterburning rate of chronic inflammation after the anti-inflammatory medicines such as inhaled hormone or leukotriene receptor antagonist and the like stop taking medicines, stable cough symptoms caused by bronchodilators, recurrent symptoms after stopping taking medicines and the like.

The fennel oral preparation is a compound preparation which is prepared according to the theory of traditional Chinese medicine, is composed of 7 medicinal materials of astragalus membranaceus, fennel root bark, origanum vulgare, safflower seed, radix sileris, malva fruit and bighead atractylodes rhizome, has the effects of regulating abnormal mucilage, enhancing the forceps strength and relieving cough and asthma. Can be used for treating bronchial asthma and chronic bronchitis caused by exogenous pathogenic factor, and cough variant asthma.

Disclosure of Invention

The invention aims at developing an fennel oral preparation which is prepared from raw materials such as astragalus membranaceus, fennel root bark, oregano, safflower seeds, divaricate saposhnikovia root, malva fruit and bighead atractylodes rhizome, and is prepared into an oral preparation by crushing, extracting, removing impurities and concentrating. The oral preparation has effects in regulating abnormal mucilage, enhancing forceps and relieving cough and asthma. Oral preparation for treating bronchial asthma and chronic bronchitis caused by exogenous pathogenic factor and cough variant asthma.

The invention relates to an fennel oral preparation, which is prepared from the following raw materials of astragalus, fennel root bark, oregano, safflower seed, ledebouriella root, malva fruit and bighead atractylodes rhizome, wherein the content of each component in 1 part is as follows: 90-180 g of astragalus membranaceus, 30-90 g of fennel root bark, 60-130 g of origanum vulgare, 50-120 g of safflower seeds, 20-60 g of radix sileris, 50-100 g of malva fruit and 40-90 g of bighead atractylodes rhizome, and the specific operation is carried out according to the following steps:

a. pulverizing radix astragali 90-180 g, foeniculum vulgare root bark 30-90 g, oregano 60-130 g, safflower seed 50-120 g, radix Saposhnikoviae 20-60 g, abutilus fruit 50-100 g, atractylodis rhizoma 40-90 g 7 medicinal materials by conventional method, sieving with 40-120 mesh sieve to obtain mixture;

b. placing the mixture obtained in the step a into an extraction tank, adding 4-12 times of water, decocting for 3 times, each time for 30 minutes-2 hours, filtering, and combining the filtrates;

c. concentrating the filtrate obtained in the step b at 40-80 ℃ under reduced pressure of-0.01-0.10 MPa to obtain medicinal materials with the relative density of 0.2-1:2-8 of the medicinal liquid being 1.01-1.05 and the temperature of 40-70 ℃, filtering and sieving with 200-300 meshes, taking the supernatant, concentrating the supernatant at 40-80 ℃ under reduced pressure of-0.01-0.10 MPa to obtain clear paste with the relative density of 0.9-1.25 and the temperature of 40-60 ℃ for later use;

d. adding auxiliary materials lactose and starch into the clear paste obtained in the step c according to the weight ratio of 1:1, and preparing the fennel oral preparation according to the conventional pharmaceutical method.

A preparation method of an fennel oral preparation comprises the following steps:

a. according to the content of each 1 part of each component, crushing 90-180 g of astragalus, 30-90 g of fennel root bark, 60-130 g of origanum vulgare, 50-120 g of safflower seed, 20-60 g of divaricate saposhnikovia root, 50-100 g of malva fruit and 40-90 g of bighead atractylodes rhizome by a pulverizer according to a conventional method, and sieving by a 40-120 mesh sieve to obtain a mixture;

The use of the fennel oral preparation in preparing medicines for treating asthma and acute attacks of chronic bronchitis caused by exogenous pathogens.

The use of the fennel oral preparation in preparing a medicament for treating cough variant asthma.

Drawings

FIG. 1 is a graph showing the effect of an oral preparation of fennel of the present invention on weight change in allergic asthmatic mice;

FIG. 2 shows the effect of the fennel oral formulation of the present invention on inflammatory cells in peripheral blood of allergic asthma mice;

FIG. 3 is a graph showing the effect of the fennel oral formulation of the present invention on BALF inflammatory cells in allergic asthma mice;

FIG. 4 shows the effect of the oral preparation of fennel of the present invention on pathological changes (HE staining) of lung tissue in allergic asthmatic mice; wherein the left part of fig. 4 is he×200, and the right part of fig. 4 is he×400;

FIG. 5 shows the effect of the oral preparation of fennel of the present invention on pathological changes of lung tissue (Masson staining) in allergic asthmatic mice; wherein the left side of fig. 5 is masson×200 and the right side of fig. 5 is masson×400.

Detailed Description

Example 1 (content of each component per 1 part)

a. Crushing 7 medicinal materials of 90 g of astragalus, 30 g of fennel root bark, 60 g of origanum vulgare, 50 g of safflower seed, 20 g of divaricate saposhnikovia root, 50 g of malva fruit and 40 g of bighead atractylodes rhizome by a conventional method by using a crusher, and sieving by a 40-mesh sieve to obtain a mixture;

b. placing the mixture obtained in the step a into an extraction tank, adding 4 times of water, decocting for 3 times, each time for 30 minutes, filtering, and combining filtrates;

c. concentrating the filtrate obtained in the step b under reduced pressure of-0.01 MPa at 40deg.C to obtain fluid extract with relative density of 1.01 and temperature of 40deg.C, filtering and sieving with 200 mesh sieve, collecting supernatant, concentrating under reduced pressure of-0.01 MPa at 40deg.C to obtain fluid extract with relative density of 0.9 and temperature of 40deg.C;

Example 2 (content of each component per 1 part)

a. Pulverizing 7 medicinal materials including 120 g of astragalus, 60 g of fennel root bark, 80 g of origanum vulgare, 70 g of safflower seed, 40 g of divaricate saposhnikovia root, 60 g of malva fruit and 50 g of bighead atractylodes rhizome by a pulverizer according to a conventional method, and sieving with a 60-mesh sieve to obtain a mixture;

b. placing the mixture obtained in the step a into an extraction tank, adding 8 times of water, decocting for 3 times, each time for 1 hour, filtering, and combining filtrates;

c. concentrating the filtrate obtained in the step b under reduced pressure of-0.02 Mpa at 60deg.C to obtain fluid extract with relative density of 0.7:7 and 50 deg.C, filtering with 250 mesh sieve, collecting supernatant, concentrating under reduced pressure of-0.02 Mpa at 60deg.C to obtain fluid extract with relative density of 0.1 and 50 deg.C;

Example 3 (content of each component per 1 part)

a. Crushing 7 medicinal materials of 150 g of astragalus, 80 g of fennel root bark, 100 g of origanum vulgare, 90 g of safflower seed, 50 g of divaricate saposhnikovia root, 60 g of malva fruit and 70 g of bighead atractylodes rhizome by a conventional method by using a crusher, and sieving by a 80-mesh sieve to obtain a mixture;

b. placing the mixture obtained in the step a into an extraction tank, adding 10 times of water, decocting for 3 times, each time for 1.5 hours, filtering, and combining the filtrates;

c. concentrating the filtrate obtained in the step b under reduced pressure of-0.05 Mpa at 70deg.C to obtain fluid extract with relative density of medicinal material to 0.8:6 of 1.05 and temperature of 60deg.C, filtering with 280 mesh sieve, collecting supernatant, concentrating under reduced pressure of-0.06 Mpa at 70deg.C to obtain fluid extract with relative density of 1.0 and temperature of 55deg.C;

Example 4 (content of each component per 1 part)

a. Crushing 130 g of astragalus, 70 g of fennel root bark, 110 g of origanum vulgare, 80 g of safflower seed, 30 g of divaricate saposhnikovia root, 40 g of malva fruit and 60 g of bighead atractylodes rhizome by a conventional method by using a crusher, and sieving by a 80-mesh sieve to obtain a mixture;

b. placing the mixture obtained in the step a into an extraction tank, adding 7 times of water, decocting for 3 times, each time for 50 minutes, filtering, and combining filtrates;

c. concentrating the filtrate obtained in the step b under reduced pressure of-0.08 Mpa at 50deg.C to obtain fluid extract with relative density of 0.9:6 of medicinal liquid of 1.04 and temperature of 45deg.C, filtering through 140 mesh sieve, collecting supernatant, concentrating under reduced pressure of-0.03 Mpa at 60deg.C to obtain fluid extract with relative density of 1.1 and temperature of 45deg.C;

d. and c, adding auxiliary materials into the clear paste obtained in the step c according to the weight ratio of 1:1 lactose to starch, and preparing the fennel oral preparation according to the conventional pharmaceutical method.

Example 5 (content of each component per 1 part)

a. Pulverizing 7 medicinal materials of 100 g of astragalus, 50 g of fennel root bark, 80 g of origanum vulgare, 100 g of safflower seed, 30 g of divaricate saposhnikovia root, 90 g of malva fruit and 70 g of bighead atractylodes rhizome by a pulverizer according to a conventional method, and sieving with a 40-120 mesh sieve to obtain a mixture;

b. placing the mixture obtained in the step a into an extraction tank, adding 11 times of water, decocting for 3 times, each time for 1 hour and 20 minutes, filtering, and combining the filtrates;

c. concentrating the filtrate obtained in the step b under reduced pressure of-0.15 Mpa at 65deg.C to obtain fluid extract with relative density of 0.6:3 and temperature of 65deg.C, filtering with 230 mesh sieve, collecting supernatant, concentrating under reduced pressure of-0.07 Mpa at 65deg.C to obtain fluid extract with relative density of 1.15 and temperature of 60deg.C;

Example 6 (content of each component per 1 part)

a. Crushing 160 g of astragalus, 85 g of fennel root bark, 100 g of origanum vulgare, 90 g of safflower seed, 50 g of divaricate saposhnikovia root, 90 g of malva fruit and 80 g of bighead atractylodes rhizome by a conventional method by using a crusher, and sieving by a 110-mesh sieve to obtain a mixture;

b. placing the mixture obtained in the step a into an extraction tank, adding 6 times of water, decocting for 3 times, each time for 40 minutes, filtering, and combining filtrates;

c. concentrating the filtrate obtained in the step b under reduced pressure of-0.03 Mpa at 65deg.C to obtain fluid extract with relative density of 1:5 and temperature of 65deg.C, filtering with 220 mesh sieve, collecting supernatant, concentrating under reduced pressure of 0.5Mpa at 75deg.C to obtain fluid extract with relative density of 1.15 and temperature of 58 deg.C;

Example 7 (content of each component per 1 part)

a. Crushing 180 g of astragalus, 90 g of fennel root bark, 130 g of origanum vulgare, 120 g of safflower seed, 20 g of divaricate saposhnikovia root, 100 g of malva fruit and 90 g of bighead atractylodes rhizome by a conventional method by using a crusher, and sieving by a 120-mesh sieve to obtain a mixture;

b. placing the mixture obtained in the step a into an extraction tank, adding 12 times of water, decocting for 3 times, each time for 2 hours, filtering, and combining filtrates;

c. concentrating the filtrate obtained in the step b at 80 ℃ under reduced pressure of 0.10Mpa until the relative density of medicinal liquid is 1.08 at 70 ℃ and the relative density of medicinal liquid is 0.6:8, filtering the filtrate through a sieve of 300 meshes, collecting supernatant, concentrating the supernatant at 80 ℃ under reduced pressure of 0.10Mpa until the relative density is 1.25 and the relative density of medicinal liquid is 60 ℃ for later use;

Example 8

The fennel oral preparation provided by the invention is proved to have the treatment effects and the mechanism of treating bronchial asthma, acute episode of chronic bronchitis and cough variant asthma by experimental researches on anti-inflammatory, phlegm eliminating, cough relieving, immunoregulation, treatment effects on allergic asthma and the like through the fennel oral preparation;

1. effect of fennel oral formulation on ear swelling in mice due to xylene:

1.1 test materials:

1.1.1 drugs and reagents:

the invention provides an fennel oral preparation, which comprises dimethylbenzene (North Union Fine chemical development Co., tianjin, lot number: 20210810), normal saline (Xinjiang pharmaceutical Co., ltd., lot number: 2302077), dexamethasone acetate tablets (Xinxiang Chang Zhu le pharmaceutical Co., lot number: D2207061, chinese medicine standard: H41020216), guilong Kechuanning capsules (Guilong pharmaceutical industry (Anhui) Limited company, lot number: K221203, chinese medicine standard: Z20053135;

1.1.2 main instruments:

electronic balance (Beijing 00000246, sadolisco instruments (Beijing)) TC3K-H electronic balance (Su 00000193, double Jie test instruments Co., ltd.);

1.1.3 animals:

72 SPF-grade healthy ICR mice (male) with the mass of (13.6+/-20.6) g are provided by animal experiment centers of Xinjiang medical university, the use license number SYXK (new) 2018-0002 of experimental animals is fed for 3 days in an environment adaptation mode before the experiment, drinking water is free to eat every day, the room temperature is kept at 23-26 ℃ for 12 hours every day, and the experiment is approved by the ethical committee of the Uygur medical institute of Xinjiang Uygur autonomous region, and the ethical number is nGLP2023-04;

1.2 test method:

after 72 ICR male mice are adaptively fed for 3d, the ICR male mice are randomly divided into a control group, a dexamethasone group, a Guilong Kechuanning group and 12 groups/group according to the weight, wherein the fennel oral preparation is in low, medium and high dose groups; each administration group was lavaged 1 time per day for 10 days in addition to the control group, which was given an equal volume of physiological saline; after 1h of gastric lavage on day 10, the two sides of the right auricle of the mouse are smeared with dimethylbenzene, 40 mu L of the mouse is inflamed, the two sides of the left auricle are smeared with equal volume of physiological saline, after 30min, cervical vertebra is removed, the ears are sheared along the auricle baseline;

1.2.1 groupings and doses:

ICR mice were randomly divided into a control group (pair), a dexamethasone acetate tablet group (cation 1), a Guilong Kechuanning capsule group (cation 2), a fennel oral preparation low dose (low), a fennel oral preparation medium dose group (medium), and a fennel oral preparation high dose group (high) according to body weight;

table 1 effects of fennel oral preparation on ear swelling in mice caused by xylene test group and dose

1.2.2 dosing and observations:

each administration group was lavaged 1 time a day for 10 days, and the control group was given an equal volume of physiological saline;

1.2.3 test observations and detection indicators:

taking round lugs at the same part of ears at two sides by using a puncher with the diameter of 0.6cm, weighing by using a precision electronic balance, and calculating the mass difference of the two auricles as swelling degree (mg) and the swelling degree inhibition rate;

1.2.4 statistical treatment:

the data obtained all use mean ± standard deviationStatistical treatment was performed using SPSS25.0 data analysis software, using GraphPad Prism 10.0 mapping, and each set of experimental indicators was performed using random design One-way ANOVA (One-way ANOVA) with P<0.05 indicates that the difference is statistically significant;

1.3 test results:

1.3.1 effects of fennel oral formulation on ear swelling in mice due to paraxylene:

TABLE 2 Effect of Foeniculum vulgare oral preparation on ear swelling in mice due to paraxylene

Note that: p <0.05, < P <0.01 compared to control;

compared with a control group, the three dosage groups of the fennel oral preparation and the positive control group can reduce the ear swelling degree of mice, wherein the positive control group and the middle and high dosage groups have statistically significant significance in the difference of statistical results (P is less than 0.01=; the swelling inhibition rate of the high dosage group is optimal in the three dosage groups of the fennel oral preparation, and the test results show that the fennel oral preparation has a certain anti-inflammatory effect;

2 effects of fennel oral formulation on the amount of tracheal phenol red excretion in mice:

2.1 test materials:

2.1.1 drugs and reagents:

the invention relates to an fennel oral preparation, phenol red (Beijing Soy Bao technology Co., ltd., batch No. 328Q 034), ambroxol hydrochloride dispersible tablets (Zhuhai Hospital Co., ltd., batch No. 20220602, national drug standard code: H20052313), guilong Kechuanning capsules (Guilong drug Co., ltd., batch No. K221203, national drug standard code: Z20053135, physiological saline (national drug group Xinjiang pharmaceutical Co., batch No. 2302077);

2.1.2 main instruments:

TC3K-H electronic balance (Su's 00000193, double Jie test instruments Co., ltd.), ultraviolet spectrophotometer;

2.1.3 animal and feeding conditions:

72 SPF-grade healthy ICR mice (male) with the body mass of (18+/-22) g are provided by animal experiment centers of Xinjiang medical university, the use license number SYXK (new) 2018-0002 of experimental animals is fed for 3 days in an environment-adaptive manner before the experiment, the experimental animals eat and drink water freely every day, illumination is continued for 12 hours every day, the room temperature is kept at 23-26 ℃, and the experiment is approved by the ethical committee of the Xinjiang Uygur autonomous region drug institute, and the approval document number is nGLP2023-05;

2.2 test method:

after 72 ICR male mice are adaptively fed for 3d, the mice are randomly divided into a control group, an ambroxol hydrochloride group, a Guilong cough and asthma relieving group and a fennel oral preparation low, medium and high dose group according to the weight, wherein the number of the fennel oral preparations is 12/group; each administration group was lavaged 1 time per day for 10 days in addition to the control group, which was given an equal volume of physiological saline; after 1h of stomach irrigation on day 10, the mice are injected with 20mL/kg of 5% phenol red physiological saline solution in the abdominal cavity, and after 30min, cervical vertebra is removed and killed, the trachea is taken down and put into 4mL of physiological saline, 0.2mL of 5% sodium bicarbonate solution is added, and the vibration is carried out for 30min to completely release the phenol red in the trachea;

2.2.1 groupings and doses:

the ICR mice are randomly divided into a control group (pair), an ambroxol hydrochloride dispersible tablet group (cation 1), a Guilong Kechuanning capsule group (cation 2), a fennel oral preparation low dose (low), a fennel oral preparation medium dose group (medium) and a fennel oral preparation high dose group (high) according to the weight;

table 3 effects of fennel oral formulations on the amount of tracheal phenol red excretion in mice test groups and dosages

2.2.2 dosing and observations:

each administration group was lavaged 1 time per day for 10 days in addition to the control group, which was given an equal volume of physiological saline;

2.2.3 test observations and detection indicators:

OD value is measured at 546nm, and the excretion amount of the phenol red is calculated according to a standard curve of the phenol red;

2.2.4 statistical methods:

2.3 test results:

2.3.1 effects of fennel oral formulations on the amount of tracheal phenol red excretion in mice are shown in Table 4;

TABLE 4 Effect of Fennel oral formulations on the amount of mouse tracheal phenol Red excretion

Note that: p <0.05, < P <0.01 compared to control;

compared with a control group, the three dosage groups and the positive control group of the fennel oral preparation can obviously increase the tracheal phenol red excretion of mice, wherein the statistical result difference of the positive 1, the positive 2, the middle and the high dosage groups has statistically significant significance (P is less than 0.01 or P is less than 0.05); among three doses of the fennel oral preparation, the high-dose group has the best phlegm eliminating index, and the fennel oral preparation can obviously increase the airway phenol red excretion amount of mice, and the test results prove that the fennel oral preparation has a certain phlegm eliminating effect;

3 effects of fennel oral formulation on cough latency and cough times in mice with concentrated ammonia:

3.1 test materials:

3.1.1 drugs and reagents:

the invention provides an fennel oral preparation, which comprises concentrated ammonia water (Tianjin Xin platinum specialty chemical Co., ltd., batch number: 20210802), citric acid pentovine tablet (Shanghai Yurui biotechnology pharmaceutical Co., ltd., batch number: 20220406, national medicine standard character number: H41024201), guilong Kechuanning capsule (Guilong pharmaceutical Co., ltd., batch number: K221203, national medicine standard character: Z20053135, physiological saline (national medicine group Xinjiang pharmaceutical Co., ltd., batch number: 2302077);

3.1.2 main instruments:

TC3K-H electronic balance (Su's 00000193, double Jie test instruments Co., ltd.) 500mL jar;

3.1.3 animal and feeding conditions:

72 SPF-class healthy ICR mice (males) were (18+ -22) g in body mass, supplied by the Xinjiang university animal laboratory center, and the laboratory animals used license number SYXK (New) 2018-0002. The experimental animals are adaptively fed for 3 days before the experiment, eat and drink water freely every day, illumination is continued for 12 hours every day, the room temperature is kept at 23-26 ℃, the experiment is approved by the ethical committee of the Xinjiang Uygur autonomous region drug institute, and the approval document is nGLP2023-06;

3.2 test method:

after the 72 ICR male mice are adaptively fed for 3d, the mice are randomly divided into a control group, a citric acid pentovine group, a Guilong cough and asthma relieving group and 12 groups according to the weight of the mice. Filling the stomach 1 time a day except for a control group, continuously filling the stomach for 10 days, wherein the control group is filled with an equal volume of normal saline, after filling the stomach for 1 hour on the 10 th day, reversely buckling a 500ml ore mouth bottle on an operation table, placing a group of cotton with 0.3ml of concentrated ammonia water in the ore mouth bottle, volatilizing for 20 seconds, rapidly placing the mice in the ore mouth bottle, and observing and recording the cough latency period and the cough times in 2 minutes of the mice;

3.2.1 groupings and doses:

ICR mice are randomly divided into a control group (pair), a citric acid pentovine tablet group (cation 1) and a Guilong Kechuanning capsule group (cation 2) according to the weight, wherein the low dose (low) of the fennel oral preparation, the medium dose group (medium) of the fennel oral preparation and the high dose group (high) of the fennel oral preparation are prepared according to the invention;

table 5 Effect test group and dose of Fennel oral preparation on cough latency and cough times of mice with concentrated Ammonia

3.2.2 dosing and observations:

3.2.3 test observations and detection indicators:

observing and recording the cough latency period of the mice and the cough times within 2 min;

3.2.4 statistical methods:

3.3 test results:

3.3.1 effects of fennel oral formulation on cough latency and cough times in mice with strong ammonia, see table 6;

table 6 effect of fennel oral formulation on cough latency and cough times in mice with concentrated ammonia

Note that: p <0.05, P <0.01 compared to control group

Compared with a control group, the three dosage groups and the positive control group of the fennel oral preparation can prolong the cough latency period of the mice and reduce the cough times within 2 minutes of the mice, wherein the statistical result difference of the positive 1, the positive 2 and the high dosage groups has statistically significant significance (P is less than 0.01 or P is less than 0.05); of the three doses of the fennel oral preparation, the high dose group has the best effect of prolonging the cough latency period of the mice and reducing the cough times of the mice within 2 min. The test results show that the fennel oral preparation has a certain cough relieving effect on mice cough caused by strong ammonia water;

4 effects of fennel oral formulation on immune function in immunocompromised mice:

4.1 test materials:

4.1.1 drugs and reagents:

fennel oral preparation, physiological saline (Xinjiang pharmaceutical Co., ltd., lot number 2302077). Cyclophosphamide for injection (Baxter Oncology Gmbh, lot number: 2D542A, national drug standard code: HJ 2060467), danxi Yupingfeng granule (Yunnan white drug powder group Co., ltd., lot number: ZHB2222, national drug standard code: Z5302556), levamisole hydrochloride tablet (Guinnan drug Co., ltd., lot number: ZX221103, national drug standard code H45020360). Mouse IgM, igG ELISA kit (KL 0342BT4923; KL064NT80553, batch number, respectively, available from Wohan Irett Biotechnology Co., ltd.);

4.1.2 main instruments:

TC3K-H electronic balance (Su 00000193, double Jie test instruments Co., ltd.), electronic balance (Beijing 00000246, sidoriko instrument Co., ltd.), bench-type high-speed refrigerated centrifuge (Shanghai Lu Xiangyi centrifuge instrument Co., ltd., model H1650 RB); multifunctional microplate reader (us Molecular Devices model SpectraMax M2); full-automatic blood cell analyzer for animals (Shenzhen Mairui biomedical electronics Co., ltd., model: BC-5000 Vet); ultralow temperature refrigerator: the Japanese Sanyang electric appliance, MDF-382E;

4.1.3 animal and feeding conditions:

84 SPF-class healthy ICR mice (male and female halves) were of mass (22+ -24) g, supplied by the Xinjiang university animal laboratory center, and used the license number SYXK (New) 2018-0002 for laboratory animals. The experimental animals are adaptively fed for 3 days before the experiment, eat and drink water freely every day, continuously illuminate for 12 hours every day, and keep the room temperature at 23-26 ℃;

4.2 test methods

After the 84 ICR mice are adaptively bred for 3 days, the mice are randomly divided into a control group, a model group, a levamisole hydrochloride group, a Yupingfeng granule group, a fennel oral preparation low, medium and high dose group and 12 animals/group according to the weight, each administration group is subjected to stomach infusion for 1 time per day except the control group and the model group, the continuous period is 14 days, and the control group and the model group are subjected to equal-volume physiological saline. Injecting cyclophosphamide 80mg/kg into abdominal cavity for 6,8, 10 and 12 days after administration to establish an immunosuppressed mouse model, fasted the mouse for 12 hours after administration for 14 days, taking 50 mu L of blood from the eye socket, injecting into an anticoagulation tube, fully and uniformly mixing, centrifugally separating the rest blood, freezing the serum at-80 ℃ for standby, killing by cervical dislocation, picking thymus and spleen under aseptic condition after opening the cavity, removing surrounding connective tissues and fat to weigh wet, and calculating thymus index and spleen index. The spleen is weighed and replayed and stored in a freezing tube at the temperature of minus 80 ℃;

4.2.1 groupings and doses:

ICR mice are randomly divided into a control group (pair), a levamisole hydrochloride tablet group (cation 1), a Danxi Yupingfeng granule group (cation 2), a fennel oral preparation low dose (low), a fennel oral preparation medium dose group (medium) and a fennel oral preparation high dose group (high) according to the weight;

table 7 effects of fennel oral formulations on immune function of immunocompromised mice test groups and dosages

4.2.2 test observations and detection indicators:

4.2.2.1 body weight and immune organ index:

the initial and final body weights of the experimental animals were weighed by an electronic balance. After taking blood from the eyeballs, killing the mice by neck breaking, peeling spleen and thymus, weighing by an electronic balance, and calculating spleen index and thymus index, wherein organ index=wet weight of organ (mg)/body weight (g);

4.2.2.2 peripheral blood white blood cell count:

taking a proper amount of whole blood from each animal into an ethylenediamine tetraacetic acid (ethylene diamine tetraacetic acid, EDTA) anticoagulation centrifuge tube, immediately reversing and uniformly mixing, and counting the number of white blood cells by using a full-automatic blood cell analyzer;

4.2.2.3 determination of immunoglobulin content in peripheral blood:

centrifuging the residual blood of each animal to separate serum, taking 200 mu L of serum, and detecting the mass concentration of immunoglobulin IgG and IgM in the serum by using an enzyme-linked immunosorbent assay (ELISA) kit;

4.2.3 statistical methods:

4.3 test results:

4.3.1 Foeniculum vulgare oral formulations for effect on the body mass of immunocompromised mice, see Table 8

Table 8 effects of fennel oral formulations on body mass of immunocompromised mice

Note that: compared with the control group, P<0.05，**P<0.01; in comparison with the set of models, ^# P<0.05，##P<0.01。

compared with the control group, the weight gain of the mice in the model group is obviously lower than that of the control group, and the difference of the statistical results has statistically significant significance (P < 0.05), which indicates that the experiment successfully establishes the mouse immunosuppression model. Compared with a model group, the weight gain of mice in each administration group has different degrees of rising trend, wherein the weight gain rate of the mice in the fennel oral preparation high-dose group is obviously higher than that of the mice in the model group, the difference of statistical results has statistically significant significance (P < 0.05), and the experimental results show that the fennel oral preparation has a certain recovery effect on the weight loss of immunosuppressive mice caused by cyclophosphamide;

4.3.2 effects of fennel oral formulation on spleen index and thymus index in immunocompromised mice:

table 9 effect of fennel oral formulation on spleen index and thymus index in immunocompromised mice

Note that: p <0.05, < P <0.01 compared to control; compared to the model group, #p <0.05, #p <0.01.

Compared with the control group, the spleen index and thymus index of the mice in the model group are obviously lower than those in the control group, and the difference of the statistical results has statistically significant significance (P < 0.05), which indicates that the experiment successfully establishes the mouse immunosuppression model. The spleen index and thymus index of mice in each administration group had different levels of ascending trend compared to the model group. The test results show that the fennel oral preparation has a certain recovery effect on spleen index and thymus index reduction of the immunosuppressed mice caused by cyclophosphamide;

4.3.3 effects of fennel oral formulation on total number of peripheral blood leukocytes in immunocompromised mice:

table 10 effect of fennel oral formulation on total number of peripheral blood leukocytes in immunocompromised mice

Note that: compared with the control group, P<0.05，**P<0.01; in comparison with the set of models, ^# P<0.05， ^## P<0.01。

compared with a control group, the WBC of the mice in the model group is obviously reduced, and the difference of statistical results has statistically significant significance (P < 0.05), which indicates that cyclophosphamide can obviously reduce the immune defenses of the mice, and indicates that the experiment successfully establishes an immunosuppression model of the mice. Compared with the model group, the WBC of the mice in each administration group has different degrees of rising trend, wherein the WBC of the mice in the fennel oral preparation high dose group is significantly raised (P < 0.05), and the change of the WBC level is an important reference index for observing and diagnosing hypoimmunity diseases and acute infectious diseases. The test results show that the fennel oral preparation has a certain recovery effect on WBC reduction of the immunosuppressed mice caused by cyclophosphamide;

effects of 4.3.4 fennel oral formulation on changes in immunoglobulin IgM, igG content in serum of immunocompromised mice:

table 11 effect of fennel oral formulation on the variation of immunoglobulin IgM, igG content in serum of immunocompromised mice

compared with the control group, the serum IgG and IgM contents of the mice in the model group are obviously lower than those in the control group, and the difference of the statistical results has statistical significance (P < 0.01), which indicates that the experiment successfully establishes a mouse immunosuppression model. Compared with the model group, the serum IgG and IgM content of the mice in each administration group has different degrees of rising trend, wherein the serum IgG and IgM content of the mice in the positive 2 and low dose groups is obviously increased, and the difference of the statistical results has statistically significant significance (P < 0.05). The test results show that the fennel oral preparation has a certain recovery effect on the content reduction of immune suppression mouse serum IgG and IgM caused by cyclophosphamide, and can improve the body immune activity;

5 research on mechanism of fennel oral preparation for inhibiting inflammatory factor secretion and relieving allergic asthma:

5.1 test materials:

5.1.1 drugs and reagents:

the fennel oral preparation of the invention is normal saline (Xinjiang pharmaceutical Co., ltd., batch number: 2302077) ovalbumin OVA (Sigma Co., V grade, batch number: SLCR 2806); physiological saline diluent of aluminum hydroxide gel (Harrow pharmaceutical group biological vaccine Co.); dexamethasone acetate tablet (lot number: D2207061, national drug standard: H41020216), guilong Kechuanning capsule (Guilong drug industry (Anhui) Limited liability company, lot number: K221203, national drug standard: Z20053135), PBS buffer (Beijing blue Jie Ke Zhi Limited, lot number: 23113279), mouse IFN-gamma, IL-4, IL-17A, TGF-beta 1, igE ELISA kit (Wuhan Yillere Biotechnology Co., ltd., lot number: KL096TD88528; KL102 RN; KL07H600321; KL080 25888; PA064DT 63001), DEPC, respectively)

Amerco company No. D60029-500 ml), agarose (Shanghai chemical company No.: a610013-0250), M5Hiclear DL2000 DNA marker (Polymer America, cat. MF 025) TRIzol ^TM Reagent (ambion company, cat# 15596026), 5 Xall-In-One RT Master mix (abm company, cat# G492), evaGreen Express 2 XqPCR Master mix-Low Rox (abm company, cat# G891);

5.1.2 main instruments:

TC3K-H electronic balance (Su 00000193, double Jie test instruments Co., ltd.), electronic balance (Beijing 00000246, sidoriko instrument Co., ltd.), bench-type high-speed refrigerated centrifuge (Shanghai Lu Xiangyi centrifuge instrument Co., ltd., model H1650 RB); multifunctional microplate reader (us Molecular Devices model SpectraMax M2); full-automatic blood cell analyzer for animals (Shenzhen Mairui biomedical electronics Co., ltd., model: BC-5000 Vet); ultralow temperature refrigerator: the Japanese Sanyang electric appliance, MDF-382E; PCR instrument (Bio-Rad, USA MyCycler Thermal Cycler) Real Time PCR instrument (ABI 7500 Fast);

5.1.3 animal and feeding conditions:

84 SPF-class healthy BABL/c mice (females) had a body mass of (18.+ -.20) g. The experimental animal is provided by Beijing Fukang Biotechnology Co., ltd, and the use license number SCXK (Beijing) 2019-0008 is used for the experimental animal, the experimental animal is adaptively fed for 3 days before the experiment, the experimental animal is free to eat and drink water every day, the experimental animal is continuously illuminated for 12 hours every day, and the room temperature is kept at 23-26 ℃;

5.2 test method:

after the 84 BABL/c mice are adaptively fed for 3d, the mice are randomly divided into a control group, a model group, a dexamethasone group, a Guilong cough and asthma relieving group and 12 fennel oral preparations in low, medium and high dose groups according to the weight;

1. sensitization phase: except for the control group, the other groups were injected intraperitoneally with 0.2ml of an OVA solution (0.5g.L-1 OVA, physiological saline dilution of aluminum hydroxide gel) on both the first and eighth days of the experiment;

2. atomization excitation stage: starting on day 15, mice were placed in a home-made nebulizing box and challenged with 2% ova solution for 30min each for a total of 3 weeks, resulting in a mouse asthma model. The control group uses the same amount of physiological saline control in the sensitization and the excitation phases; the dosing group was gavaged 1 hour prior to each challenge from day 15 of the experiment for 3 weeks; in the excitation process, mice with shortness of breath, severe cases of cyanosis of lips, slow response, slow respiration or irregular rhythm, slow movement and the like are taken as excitation success marks;

5.2.1 groupings and doses:

randomly dividing BABL/c mice into a control group (pair), a dexamethasone acetate tablet group (cation 1), a Guilong Kechuanning tablet group (cation 2), a fennel oral preparation low dose (low), a fennel oral preparation medium dose group (medium), and a fennel oral preparation high dose group (high) according to body weight;

table 12 Foeniculum vulgare oral preparation mechanism study test group for inhibiting inflammatory factor secretion and relieving allergic asthma and dosage

5.2.2 test observations and detection indicators:

effects of 5.2.2.1 fennel oral formulation on allergic asthma mice weight change:

weighing the body weight of the experimental animal in the non-fed state by an electronic balance every three days;

5.2.2.2 peripheral blood inflammatory cell counts:

taking 50 mu L of whole blood from each animal in an ethylenediamine tetraacetic acid (ethylene diamine tetraacetic acid, EDTA) anticoagulation centrifuge tube, immediately reversing and mixing uniformly, and counting the number of white blood cells and the number of various white blood cell subsets (including eosinophils, neutrophils, lymphocytes and monocytes) by using a full-automatic blood cell analyzer;

5.2.2.3 alveolar lavage fluid (BALF) inflammatory cell count:

taking BALF heavy suspension of each group of mice, and detecting the number of white blood cells in the BALF and the number of various white blood cell subclasses (including eosinophils, neutrophils, lymphocytes and monocytes) by using a fully automatic animal blood cell analyzer (Mindray BC-5000) provided with a five-class blood cell kit;

5.2.2.4 observation of pathological changes of lung tissue:

taking out right lung lobe tissues of each group of mice, fixing the right lung lobe tissues with 4% paraformaldehyde, embedding the right lung lobe tissues with conventional paraffin, slicing, performing HE staining on lung slices, performing Masson staining, performing microscopic examination, and performing image acquisition analysis;

5.2.3 statistical methods

5.3 test results

5.3.1 effects of fennel oral formulations on allergic asthma mice mass-change:

the results are shown in figure 1, where the body mass of each group of mice tended to increase gradually prior to OVA nebulization challenge (W2); in the atomization excitation process, other groups of mice except the control group have the phenomena of dysphoria, slow reaction, hypokinesia, hair erection, abdominal-style dyspnea and the like with different degrees. The above-described phenomenon model group mice were most evident compared to the control group. The above phenomena were alleviated to different degrees after mice in each dosing group compared to the model group, with the high dose group having the best degree of remission.

5.3.2 effects of fennel oral formulation on inflammatory cells in peripheral blood of allergic asthma mice and BALF:

as shown in fig. 2 and 3, the total White Blood Cell (WBC), neutrophil (Neu), lymphocyte (Lym), monocyte (Mon) and eosinophil (Eos) numbers were significantly increased in the peripheral blood of mice in the model group, and BALF, compared to the control group, and the differences in the statistical results were statistically significant (P <0.01 or P < 0.05). After the drug administration intervention, the total white blood cell number, the neutrophil number, the lymphocyte number and the eosinophil number in the peripheral blood and the BALF of each drug administration group are obviously reduced, and the difference of the statistical results has statistically significant significance (P <0.01 or P < 0.05); the test results show that the fennel oral preparation has a certain recovery effect on the increase of relevant inflammatory factors in the peripheral blood and BALF of mice with OVA induced allergic asthma;

5.3.3 effects of fennel oral formulation on pathological changes in lung tissue in allergic asthma mice:

as shown in fig. 4, HE staining results show that the control group has clear alveolar structure, no obvious inflammatory cell infiltration around bronchi, and compared with the control group, a large amount of inflammatory cell infiltration around bronchi of lung tissue of the model group mice can be seen, and mucus secretion is increased; compared with the model group, the lung tissue airway inflammation of the mice in each administration group is obviously reduced, and mucus secretion is reduced;

the results are shown in fig. 5, and the Masson staining results show that the control group has clear alveolar structure, the alveolar walls are not thickened, and collagen deposition is not found; compared with the control group, the lung tissue collagen deposition of the mice in the model group is obvious; the lung tissue collagen deposition was significantly reduced in mice from each of the dosing groups compared to the model group.

Claims

1. An fennel oral preparation is characterized by being prepared from the following raw materials of astragalus mongholicus, fennel root bark, origanum vulgare, safflower seeds, radix sileris, malva fruit and bighead atractylodes rhizome, wherein the content of each component in each 1 part is as follows: 90-180 g of astragalus membranaceus, 30-90 g of fennel root bark, 60-130 g of origanum vulgare, 50-120 g of safflower seeds, 20-60 g of radix sileris, 50-100 g of malva fruit and 40-90 g of bighead atractylodes rhizome, and the specific operation is carried out according to the following steps:

2. The preparation method of the fennel oral preparation is characterized by comprising the following steps:

3. Use of an oral preparation of fennel according to claim 1 or 2 for the preparation of a medicament for the treatment of asthma caused by exogenous pathogenic factors and acute episodes of chronic bronchitis.

4. Use of an oral preparation of fennel according to claim 1 or 2 for the preparation of a medicament for the treatment of cough variant asthma.