CN114984056A

CN114984056A - Natural product extract for treating Crohn's disease and application thereof

Info

Publication number: CN114984056A
Application number: CN202210806036.3A
Authority: CN
Inventors: 沈洪; 朱磊; 胡静怡
Original assignee: Jiangsu Provincial Hospital of Chinese Medicine
Current assignee: Jiangsu Provincial Hospital of Chinese Medicine
Priority date: 2022-07-08
Filing date: 2022-07-08
Publication date: 2022-09-02
Anticipated expiration: 2042-07-08
Also published as: CN114984056B

Abstract

The invention discloses a natural product extract for treating Crohn's disease and application thereof. The nidus vespae extract can be directly used for preparing a medicament for preventing and treating Crohn's disease or used for preparing a medicament for preventing and treating intestinal fibrosis diseases. The Chinese medicinal wasp's nest for treating the Crohn's disease has the effects of dispelling wind, relieving pain, attacking toxin and diminishing swelling, provides an effective treatment medicament for clinically treating the intractable disease of the Crohn's disease, is economical and practical, has wide clinical application prospect, and has the advantages of simple, quick and efficient preparation method, easy operation, convenient carrying and convenient taking.

Description

Natural product extract for treating Crohn's disease and application thereof

Technical Field

The invention belongs to the field of traditional Chinese medicines, and relates to an application of a nidus vespae extract in preparation of a medicine for preventing and treating Crohn's disease.

Background

Crohn's Disease (CD) is a chronic nonspecific inflammatory disease of unknown origin, with lesions affecting the whole digestive tract, frequently seen in the distal ileum and adjacent colon, in a segmental or jumping distribution. Clinically, it is mainly characterized by abdominal pain and diarrhea, and may be accompanied by bloody stool, fever, anemia, malnutrition, dysgenesis and other extra-intestinal manifestations such as joint, skin, eye, oral mucosa and liver pathological changes. As a chronic disabling disease, fistula formation, abdominal abscess, intestinal stenosis, intestinal obstruction and perianal lesion may be caused, seriously affecting the quality of life of the patient. Wherein intestinal fibrosis and intestinal stenosis seriously affect the physical and mental health of patients with Crohn's disease.

Intestinal fibrosis is the most main reason of CD intestinal structural injury, is easy to cause obstruction and fistula formation, is also the most important factor influencing middle-term and long-term clinical outcome, and no specific anti-fibrosis treatment means exists at present. In recent years, the application of biological agents brings new eosin treatment of CD, but many problems still need to be solved in clinic. In clinical practice, for CD which is not effective in multiple biological agent treatment, two biological agents with different inflammatory pathways are used in combination, but the drug combination may increase the risks of side effects, infection and tumor occurrence, and even if multiple biological agents are used for treating IBD, only part of patients can achieve long-term clinical remission, and the surgical resection risk is still high.

The traditional Chinese medicine treatment is taken as the treatment characteristic in China, and provides an optimizable treatment means for patients. According to the traditional Chinese medicine, except for the pathological factors of damp-heat, the toxic or stasis of the CD is a characteristic pathological factor, and the CD is caused by retention of damp-toxin, qi and blood stasis and malnutrition of intestinal collaterals. The diseased part of the disease is in the stomach and intestine, and is related to the spleen, liver, kidney, lung and other viscera. Has the characteristics of wide disease position, involvement of the whole digestive tract, deep lesion, involvement of the whole tissue layer, qi-blood sympathy and intestinal collateral damage, and belongs to the category of collateral diseases of the traditional Chinese medicine. The deficiency and excess are mixed and cause and effect mutually; the cold and heat are mixed and transformed with each other; all the zang-organs are diseased together, and the deficiency of qi and blood makes the disease difficult to cure and the transformation syndrome is complicated. The wasp nest is the nest of wasp family insect yellow star-foot wasp or a plurality of kindred insects, has sweet taste and even nature, enters liver, stomach and kidney channels, has the effects of dispelling wind, counteracting toxic substances, killing parasites, relieving pain and resisting allergy, is mainly used for dispelling wind, relieving pain, counteracting toxic substances, dissipating binds, killing parasites and relieving itching, and has no report for treating the Crohn's disease at present.

Disclosure of Invention

The invention aims to solve the technical problem of providing the nidus vespae extract for treating the intractable disease of Crohn's disease aiming at the defects of the prior art.

In order to solve the technical problems, the technical scheme of the invention is as follows:

the nidus vespae extract is applied to the preparation of the drugs for preventing and treating Crohn's disease within the protection scope of the invention. The prevention and treatment comprise prevention and/or treatment.

Wherein the nidus vespae extract is prepared by the following method: decocting nidus Vespae in water, filtering, and concentrating the filtrate under reduced pressure.

Specifically, the nidus vespae extract is used as an active ingredient to prepare a pharmaceutically acceptable dosage form.

Specifically, the dosage form is tablets, capsules, pills, suppositories, aerosols, oral liquid preparations, granules, powder, injections, syrups, medicated liquors, tinctures, lotions or films.

Specifically, the administration route of the preparation is any one or the combination of more of oral drugs, injection drugs, implant drugs, external drugs, sprays and inhalation drugs.

Specifically, the Crohn's disease, the major intestinal structural damage is intestinal fibrosis.

The application of the nidus vespae extract in preparing the medicine for preventing and treating intestinal fibrosis diseases is also within the protection scope of the invention.

Specifically, the nidus vespae extract is prepared by the following method: decocting nidus Vespae in water, filtering, and concentrating the filtrate under reduced pressure.

Has the advantages that:

(1) the invention discovers for the first time that the nidus vespae extract can effectively inhibit intestinal fibrosis;

(2) the nidus vespae used in the invention is a traditional Chinese medicinal material, has low price and small toxic and side effects, can obviously inhibit the development of intestinal fibrosis, and is worthy of wide popularization.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1 is a graph of the effect of wasp's nest extract on body weight in mice of a Crohn's disease model;

FIG. 2 is a graph of the effect of wasp's nest extract on the ratio of colon weight to colon length and the ratio of colon weight to mouse body weight in a Crohn's disease model mouse;

FIG. 3 is a graph of the effect of wasp's nest extract on colon histopathology in a Crohn's disease model mouse;

FIG. 4 is a graph of the effect of wasp's nest extract on colonic fibrosis in a Crohn's disease model mouse;

FIG. 5 is a graph of the effect of wasp's nest extract on the expression of fibrotic factor in mice that are models of Crohn's disease.

Detailed Description

The invention will be better understood from the following examples.

Example 1

Nidus Vespae 30g, decocting in water for 2 times, adding 10 times of water for the first time, soaking for 1h, decocting for 1h, filtering, adding 10 times of water for the second time, decocting for 1h, filtering, concentrating the filtrate under reduced pressure to relative density of about 1.20(60 deg.C), and concentrating to obtain concentrated solution. Putting equal mass dextrin into a fluidized bed, setting the air inlet temperature to be 85-100 ℃, starting to feed concentrated solution when the material temperature is raised to 70 ℃, controlling the liquid inlet speed to be 80-150 rpm/min, controlling the atomization pressure to be 0.2MPa outside and 0.15MPa inside, finishing spraying, and continuously drying for 1 hour at 70 ℃ to obtain the granules.

Example 2

Nidus Vespae 30g, decocting in water for 2 times, adding 10 times of water for the first time, soaking for 1h, decocting for 1h, filtering, adding 10 times of water for the second time, decocting for 1h, filtering, and concentrating the filtrate under reduced pressure to relative density of about 1.25(60 deg.C) to obtain extract. Mixing the extract with dextrin with equal mass, stirring uniformly, vacuum drying in a vacuum drying oven (vacuum degree of-0.09 to-0.08 MPa, temperature of 25-60 ℃), pulverizing the dry extract, and sieving with a 80-mesh sieve. And (3) taking the extract powder, adding a proper amount of ethanol for granulation, drying the wet granules in a hot air circulation oven (at 60 ℃), and finishing granules. And (3) taking the granules after finishing, adding a lubricant magnesium stearate if necessary, uniformly mixing, calculating the weight of the tablets, and pressing the tablets into the tablets to obtain the tablets.

Example 3

Nidus Vespae 30g, decocting in water for 2 times, adding 10 times of water for the first time, soaking for 1h, decocting for 1h, filtering, adding 10 times of water for the second time, decocting for 1h, filtering, and concentrating the filtrate under reduced pressure to relative density of about 1.25(60 deg.C) to obtain extract. Mixing the extract with dextrin with equal mass, stirring uniformly, vacuum drying in a vacuum drying oven (vacuum degree of-0.09 to-0.08 MPa, temperature of 25-60 ℃), pulverizing the dry extract, and sieving with a 80-mesh sieve. Adding appropriate amount of ethanol into the extract powder, granulating, drying the wet granules in a hot air circulation oven (60 deg.C), and grading. And (4) taking the granules after finishing, and filling the granules into capsules to obtain the capsules.

Example 4

Adding 30g of nidus vespae, decocting for 2 times by adding water, adding 10 times of water for the first time, soaking for 1h, decocting for 1h, filtering, adding 10 times of water for the second time, decocting for 1h, filtering, concentrating the filtrate under reduced pressure (the vacuum degree is-0.08 to-0.04 MPa, the temperature is 70-80 ℃) to 900mL, centrifuging at high speed (the rotating speed is 16000r/min, the flow is 4-6L/min), adding water to adjust to 1000mL, stirring uniformly, subpackaging, and sterilizing at 100 ℃ for 30min to obtain a liquid preparation.

Example 5

The therapeutic effect of the natural product extract for treating the Crohn disease on a mouse model with the Crohn disease is as follows:

the research method comprises the following steps:

1. preparation of the model

Preparation of a mouse model for Crohn's Disease (CD): after the BALB/C mice are adaptively fed for 1 week, the back of the BALB/C mice is sheared by 1.5cm on the 1 st morning, and 1% of 2,4, 6-trinitrobenzenesulfonic acid (TNBS) pre-sensitization liquid is coated on the back of the BALB/C mice; beginning enema on the 8 th morning, fasting for 12h in advance, anesthetizing (isoflurane inhalation), preparing a 40% absolute ethanol solution 0.1mL with a mixture of TNBS and absolute ethanol, administering the enema, inserting the enema from the anus, generally entering the enema with the length of 4-6 cm, turning the head down, and inverting for 1 min. The dose (0.75mg, 1.5mg, 2.5mg) was gradually increased 1 time per week for a total of 7 times. After the enema is finished, in order to prevent the enema solution from overflowing out of the anus immediately, the rat tail needs to be lifted in time, the anus is lifted for 3min, the liquid medicine uniformly reaches the whole colon, and the molding is finished. For mouse recovery, a heating lamp was placed 20cm from the cage to keep the animals warm until recovery was good.

2. Experimental groups and drug intervention

The mice were randomly divided into 4 groups, a normal group (Ctrl group) 5, a model group (TNBS), a nidus Vespae Low dose group (TNBS + NVL, 1.34975g/ml) and a nidus Vespae high dose group (TNBS + NVH group, 2.6995 g/ml). Normal control group: feeding the chicken regularly, and freely drinking water to eat the chicken; model group: after the model is made, equal volume of normal saline is given every day for intragastric administration; nidus Vespae extract is prepared by the same method as example 4, mixing filtrates, concentrating to the above high and low dosage concentration, and performing intragastric administration for 1 time/day from the first enema molding. The gavage administration of each group lasted 49 days.

3. Specimen collection method

After 49 days of administration, the mice are killed by cervical dislocation after anesthesia, all colon tissues are taken out and washed by PBS liquid quickly, then the tissues at the proximal end of the colon, which is about 1cm away from the anus, are cut off, fixed in 10% formalin, fixed at room temperature, embedded by conventional paraffin, the section thickness is 4-5 mm, the pathological histopathology and immunohistochemistry examination are carried out sequentially by a conventional hematoxylin-eosin staining method (HE staining) and Masson trichrome staining method, the mouse colon is observed and fibrosis is evaluated under a microscope, and the rest tissues are immediately placed in a refrigerator at-80 ℃ for the detection of fibrosis related cytokine indexes.

4. Observation items

(1) General conditions of the mice: the stool character, hair color, activity and mental state of the mice were observed daily, weighed and recorded daily.

(2) Pathology H & E detection and Masson staining of mouse colon tissue: after model making administration, mice were sacrificed on day 49, and colon tissues of the mice were left to be fixed in 10% formalin, embedded in paraffin by a conventional method, sliced to a thickness of 4-5 mm, subjected to conventional HE staining and Masson trichrome staining, and observed and fibrosis of the colon of the mice was evaluated under a microscope. The colon was generally observed, and the entire colon was weighed, length measured and a colon general score was performed, the specific scores are given in table 1 below.

TABLE 1 gross score criteria for fibrotic colon morphology in mice

And (4) observing under a mirror: the fibrosis characteristics of histopathology under the specimen section are observed, the severity of the fibrosis is evaluated, and relevant indexes under the microscope are scored according to the scoring standards shown in the following table 2.

TABLE 2 mouse fibrosis colonoscopy scoring criteria

(3) Colon fibrosis factor expression: the expression of mRNA of fibrosis factors Fibronetin, alpha-SMA, N-Cadherin, E-Cadherin, Col1 and TIMP1 in colon tissues of each group of mice was detected by a reverse transcription quantitative PCR (qRT-PCR) method.

5. Statistical processing

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The data processing is carried out by adopting Graphpad 8.0.1 statistical software, the result is expressed in the form of mean number plus or minus standard deviation (x plus or minus s), the comparison among a plurality of groups adopts one-factor variance analysis, and the difference with p less than 0.05 has statistical significance.

6. The research results are as follows:

(1) weight condition: the mice were induced to model intestinal fibrosis by rectal administration of TNBS 1 time per week in a volume of 0.1mL and weekly in concentrations of 0.75% (g/mL), 1.5% (g/mL), 2.5% (g/mL). The nidus vespae low-dose group and the nidus vespae high-dose group are respectively and simultaneously administered with different concentrations of medicine intervention, and the normal group and the model group are administered with distilled water with the same volume. 4 th to 7 th circulation: the body weight of the model group is obviously reduced compared with that of the normal group, and the body weight of the nidus vespae drug group is recovered (figure 1).

(2) Colon length and colon weight: after the experiment was completed, the mice were euthanized using carbon dioxide. The colon length and the colon weight of the mouse are measured and recorded, the ratio of the colon weight to the colon length and the ratio of the colon weight to the mouse body weight of the model group are obviously increased compared with the normal group, and the ratio of the colon weight to the colon length and the ratio of the colon weight to the mouse body weight of the mouse are obviously reduced compared with the model group after the nidus vespae medicament is administered for drying (figure 2).

(3) Colon histopathology: pathological colon of each group of mice is observed by H & E staining, the colon tissue structure of normal mice is clear, and inflammatory cells are not infiltrated; the colon tissue structure of the mouse in the model group is damaged, inflammatory cell infiltration and colonic edema are achieved, the pathological score of the mouse is higher than that of the mouse in the normal group, the nidus vespae drug is given for prognosis, the colon epithelial cell is normal in shape, inflammatory cells are slightly infiltrated, and the pathological score is reduced compared with that of the mouse in the model group (figure 3).

(4) Masson staining: the colon fibrosis degree of each group of mice is inspected through Masson staining, the colon tissue structure of a normal mouse is clear, and the fibrosis layer of a muscle layer is thinner; the colon tissue structure of the model group mice is damaged, the fibrosis degree is heavier, and the colon epithelial cell morphology is normal and the fibrosis degree is reduced after the nidus vespae drug is given (figure 4).

(5) Fibrotic factor expression in colon tissue: the expression of the mRNA of the fibrosis factors Fibronetin, alpha-SMA, N-Cadherin, E-Cadherin, Col1 and TIMP1 in colon tissues of each group of mice was detected by a qRT-PCR method. The expression of mRNA of fibrosis factors fibrionetin, alpha-SMA, N-Cadherin, Col1 and TIMP1 in the colon of the mouse in the model group is obviously increased compared with that in the Ctrl group; the expression of the fibrosis factors fibrionetin, alpha-SMA, N-Cadherin, Col1 and TIMP1mRNA in the colon tissue of mice was significantly reduced with statistical differences after the intervention of nidus vespae drug administration (FIG. 5). The mRNA expression of Cadherin E-Cadherin in the colon of the model group mouse is obviously reduced compared with that of the Ctrl group; E-Cadherin expression was significantly elevated in colon tissue of mice following nidus vespae drug intervention with statistical differences (FIG. 5).

In conclusion, the wasp's nest extract is applied to the preparation of the medicine for preventing and treating the Crohn's disease, increases the weight of a mouse, reduces the ratio of the colon weight to the colon length and the ratio of the colon weight to the weight of the mouse, improves the pathological structure and the fibrosis score of the colon of the mouse, changes the expression of the fibrosis gene of colon tissues, can be used as a potential medicine for preventing and treating the Crohn's disease, and provides a new medicine selection for clinic.

The present invention provides a natural product extract for treating crohn's disease and a method for applying the same, and a plurality of methods and ways for implementing the technical scheme, and the above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, a plurality of improvements and modifications can be made without departing from the principle of the present invention, and the improvements and modifications should be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.

Claims

1. Application of nidus Vespae extract in preparing medicine for preventing and treating Crohn's disease is provided.

2. The use as claimed in claim 1, wherein the nidus Vespae extract is prepared by the following method: decocting nidus Vespae in water, filtering, and concentrating the filtrate under reduced pressure.

3. The use as claimed in claim 1, wherein nidus Vespae extract is used as active ingredient for preparing pharmaceutically acceptable dosage forms.

4. The use of claim 3, wherein the dosage form is a tablet, capsule, pill, suppository, aerosol, oral liquid, granule, powder, injection, syrup, medicated wine, tincture, lotion, or film.

5. The use of claim 3, wherein the administration route of the dosage form is any one or a combination of oral drug, injection drug, implant drug, external drug, spray and inhalation drug.

6. The use of claim 1, wherein the Crohn's disease, the major intestinal structural damage is intestinal fibrosis.

7. Application of nidus Vespae extract in preparing medicine for preventing and treating intestinal fibrosis diseases is provided.

8. The use as claimed in claim 7, wherein the nidus Vespae extract is prepared by the following method: decocting nidus Vespae in water, filtering, and concentrating the filtrate under reduced pressure.

9. The use as claimed in claim 7, wherein nidus Vespae extract is used as active ingredient for preparing pharmaceutically acceptable dosage forms.

10. The use of claim 9, wherein the dosage form is a tablet, capsule, pill, suppository, aerosol, oral liquid, granule, powder, injection, syrup, medicated wine, tincture, lotion, or film.