CN114984056B - Natural product extract for treating Crohn's disease and application thereof - Google Patents

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 medicines for preventing and treating Crohn's disease or medicines for preventing and treating intestinal fibrosis diseases. The traditional Chinese medicine nidus vespae for treating the Crohn disease has the effects of dispelling wind, relieving pain, counteracting toxic substances and relieving swelling, provides an effective therapeutic drug for clinically treating the Crohn disease, is economical and practical, has wide clinical application prospect, and has simple, quick and efficient preparation method, easy operation, convenient carrying and convenient administration.

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 application of 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 cause that affects the entire digestive tract, and is distributed in segments or hops with the terminal ileum and adjacent colon. Clinically, abdominal pain and diarrhea are the main characteristics, and simultaneously, the external manifestations such as bloody stool, fever, anemia, malnutrition, dysplasia, joint, skin, eyes, oral mucosa, liver lesions and the like can be accompanied. As a chronic disabling disease, fistula formation, celiac abscess, intestinal lumen stenosis, ileus and perianal lesions can result, severely affecting the quality of life of the patient. Wherein intestinal fibrosis and intestinal stenosis seriously affect the physical and mental health of patients suffering from Crohn's disease.

Intestinal fibrosis is the most important cause of CD intestinal structural injury, is easy to cause obstruction and fistula formation, is also the most important factor affecting middle-and-long-term clinical outcome, and no specific anti-fibrosis treatment means exists at present. In recent years, the application of biological agents has brought about new dawn for CD treatment, but there are still many problems to be solved clinically. In clinical practice, for CD where multiple biological agent treatments are ineffective, two different inflammatory pathways are used in combination, but the combination of drugs may increase the risk of side effects, infections and tumorigenesis, and even if multiple biological agents are used for IBD treatment, only a portion of patients can achieve long-term clinical relief, with a high surgical resection risk.

The Chinese medicine treatment is taken as the treatment characteristic of China, and an optimized treatment means is provided for patients. In addition to the pathological factors of damp-heat, CD is considered by traditional Chinese medicine to be the characteristic pathological factor of the CD, and the disease is caused by the stagnation of damp-toxin, qi-blood stasis and malnutrition of intestinal collaterals. The diseased region is in the stomach and intestine and is related to the spleen, liver, kidney, lung and other viscera. Has the characteristics of wide disease location, full digestive tract involvement, deep lesions, full tissue involvement, qi and blood co-diseases and damaged intestinal collaterals, and belongs to the category of collateral diseases of traditional Chinese medicine. The deficiency and excess are mixed and cause and effect with each other; cold and heat are mixed and transformed mutually; the viscera are simultaneously affected, and the deficiency of qi and blood is cured, so that the syndrome becomes a cluster. The nidus vespae is nest of wasp or multiple kinds of closely related insects of wasp family Huang Xing, has sweet taste and flat nature, enters liver, stomach and kidney channels, has the effects of dispelling wind, counteracting toxic substances, killing parasites, relieving pain and resisting allergy, and is mainly used for dispelling wind, relieving pain, counteracting toxic substances, resolving hard mass, killing parasites and relieving itching, and no report for treating Crohn's disease exists at present.

Disclosure of Invention

The invention aims to solve the technical problem of providing nidus Vespae extract for treating the refractory disease of Crohn 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 application of the nidus Vespae extract in preparing the medicine for preventing and treating the Crohn disease is within the protection scope of the invention. The control includes prevention and/or treatment.

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

Specifically, nidus Vespae extract is used as active ingredient to prepare pharmaceutically acceptable dosage forms.

Specifically, the dosage form is tablet, capsule, pill, suppository, aerosol, oral liquid, granule, powder, injection, syrup, medicated wine, tincture, distillate or film.

Specifically, the administration route of the dosage form is any one or a combination of a plurality of oral medicines, injection medicines, implantation medicines, external medicines, spray and inhalation medicines.

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

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

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

Specifically, nidus Vespae extract is used as active ingredient to prepare pharmaceutically acceptable dosage forms.

Specifically, the dosage form is tablet, capsule, pill, suppository, aerosol, oral liquid, granule, powder, injection, syrup, medicated wine, tincture, distillate or film.

The beneficial effects are that:

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

(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 worth widely popularizing.

Drawings

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

FIG. 1 is a graph showing the effect of nidus Vespae extract on body weight of Crohn's disease model mice;

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

FIG. 3 is the effect of nidus Vespae extract on colon tissue pathology in a mouse model of Crohn's disease;

FIG. 4 is the effect of nidus Vespae extract on colon fibrosis in Crohn's disease model mice;

FIG. 5 is the effect of nidus Vespae extract on the expression of fibrotic factors in mice model of Crohn's disease.

Detailed Description

The invention will be better understood from the following examples.

Example 1

30g of nidus vespae is decocted with water for 2 times, 10 times of water is added for 1 hour in the first time, the first time is decocted for 1 hour, filtering is carried out, 10 times of water is added for the second time, the second time is decocted for 1 hour, filtering is carried out, and the filtrate is decompressed and concentrated to have the relative density of about 1.20 (60 ℃), so as to obtain concentrated solution. And (3) placing the dextrin with equal mass into a fluidized bed, setting the air inlet temperature to be 85-100 ℃, starting to feed the concentrated solution when the temperature of the material is increased 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, ending the spraying, and continuously drying at 70 ℃ for 1 hour to obtain the granules.

Example 2

30g of nidus vespae is decocted with water for 2 times, 10 times of water is added for 1 hour in the first time, the first time is decocted for 1 hour, filtering is carried out, 10 times of water is added for the second time, the second time is decocted for 1 hour, filtering is carried out, and the filtrate is decompressed and concentrated to have the relative density of about 1.25 (60 ℃), so as to obtain extract. Mixing the extract with dextrin of equal mass, stirring, vacuum drying in a vacuum drying oven (vacuum degree is-0.09-0.08 MPa, temperature is 25-60deg.C), pulverizing the dry extract, and sieving with 80 mesh sieve. Granulating the extract powder with appropriate amount of ethanol, drying the wet granules in a hot air circulation oven (60 ℃) and finishing. And (3) taking the granules after finishing, adding a lubricant magnesium stearate when needed, uniformly mixing, calculating the weight of the tablet, and pressing the tablet to obtain the tablet.

Example 3

30g of nidus vespae is decocted with water for 2 times, 10 times of water is added for 1 hour in the first time, the first time is decocted for 1 hour, filtering is carried out, 10 times of water is added for the second time, the second time is decocted for 1 hour, filtering is carried out, and the filtrate is decompressed and concentrated to have the relative density of about 1.25 (60 ℃), so as to obtain extract. Mixing the extract with dextrin of equal mass, stirring, vacuum drying in a vacuum drying oven (vacuum degree is-0.09-0.08 MPa, temperature is 25-60deg.C), pulverizing the dry extract, and sieving with 80 mesh sieve. Granulating the extract powder with appropriate amount of ethanol, drying the wet granules in a hot air circulation oven (60 ℃) and finishing. And (5) taking the whole granules, and filling the granules into capsules to obtain the capsule.

Example 4

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

Example 5

The natural product extract for treating Crohn's disease has the following therapeutic effects on a mouse model of Crohn's disease:

the research method comprises the following steps:

1. model preparation

Crohn's Disease (CD) mouse model preparation: after 1 week of adaptive feeding of BALB/C mice, 1.5cm of back shearing 1.5cm at day 1, 1%2,4, 6-trinitrobenzenesulfonic acid (TNBS) pre-sensitization solution was applied; the enema was started on the 8 th morning, fasted for 12 hours in advance, and after anesthesia (isoflurane inhalation), 0.1mL of a 40% absolute ethanol solution prepared from TNBS and absolute ethanol mixture was administered for enema, and the enema was inserted from the anus, generally with a length of 4-6 cm, with the head facing downward, and inverted for 1min. Gradually increasing doses (0.75 mg, 1.5mg, 2.5 mg) 1 time a week for a total of 7 times. After the enema is finished, in order to avoid that the enema solution overflows out of the anus, the tail of the hand needs to be lifted in time, the anus is lifted for 3 minutes, the liquid medicine uniformly reaches the whole colon, and the molding is finished. For recovery of the mice, a heat lamp was placed 20cm from the cage to keep the animals warm until recovery was good.

2. Experimental grouping and pharmaceutical intervention

Mice were randomly divided into 4 groups, 5 normal (Ctrl) groups, model (TNBS), low nidus Vespae (tnbs+nvl, 1.34975 g/ml) and high nidus Vespae (tnbs+nvh, 2.6995 g/ml). Normal control group: conventional feeding, free drinking and eating; model group: after molding, the stomach is irrigated by an equal volume of physiological saline every day; the preparation method of nidus Vespae extract is the same as in example 4, and the filtrates are combined and concentrated to the above high and low dosage group concentrations, and each group is administered by lavage for 1 time/day from the first time of clysis molding. Each of the above groups was administered by intragastric administration for 49 days.

3. Specimen collection method

49 days after administration, the mice are killed after cervical dislocation, all colon tissues are taken out and quickly washed by PBS liquid, then the colon is cut off about 1cm from the proximal tissue of the anus, fixed in 10% formalin, fixed at room temperature, embedded in conventional paraffin, and the slice thickness is 4-5 mm, the pathological histopathological examination and the immunohistochemical examination are sequentially carried out by using a conventional hematoxylin-eosin staining method (HE staining) and Masson trichromatic staining, the colon of the mice is observed and fibrosis assessed under a microscope, and the rest tissues are immediately placed in a refrigerator at-80 ℃ for detection of relevant cytokine indexes of fibrosis.

4. Viewing items

(1) General status of mice: mice were observed daily for stool traits, hair color, activity, mental status, and weighed and recorded daily.

(2) Pathological H & E detection of mouse colon tissue and Masson staining: after the molding administration, mice were sacrificed on day 49, colon tissues of the mice were left to be fixed in 10% formalin, paraffin-embedded conventionally, and sections were 4-5 mm thick, and the colon of the mice was observed and fibrotic assessed under a microscope by conventional HE staining and Masson trichromatic staining. Colon gross observation, taking total colon weight, measuring length, and performing colon gross scoring, specific scores are given in table 1 below.

TABLE 1 general scoring criteria for mouse fibrotic colon morphology

And (5) observing under a mirror: the fibrosis characteristics of the histopathology under the specimen sections were observed, the severity thereof was evaluated, and the relevant index under the mirror was scored, and the scoring criteria are shown in table 2 below.

TABLE 2 criteria for scoring under a mouse fibrotic colonoscope

(3) Colon fibrosis factor expression: the fibrosis factors fibroetin, alpha-SMA, N-Cadherin, E-Cadherin, col1 and TIMP1mRNA expression in colon tissue of each group of mice were detected by reverse transcription quantitative PCR (qRT-PCR) method.

5. Statistical treatment

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Data processing is carried out by adopting Graphpad 8.0.1 statistical software, the result is expressed in the form of mean ± standard deviation (x ± s), the comparison among multiple groups adopts single factor analysis of variance, and p < 0.05 is taken as the difference to have statistical significance.

6. Study results:

(1) Weight conditions: the mice were induced into intestinal fibrosis models by rectal administration of TNBS 1 time per week at a volume of 0.1mL and at a weekly concentration of 0.75% (g/mL), 1.5% (g/mL), 2.5% (g/mL). The low dosage group and the high dosage group of nidus Vespae are simultaneously administered with different concentrations of drug for intervention, and the normal group and the model group are administered with distilled water with the same volume. Cycle 4-7: the weight of the model group was significantly reduced compared with the normal group, and the weight of the nidus Vespae drug group was recovered (figure 1).

(2) Colon length and colon weight: after the end of the experiment, mice were euthanized with carbon dioxide. The abdomen of the mice was dissected longitudinally, the colon of the mice was isolated, the colon length and the colon weight of the mice were measured and recorded, the colon weight to colon length ratio and the colon weight to mouse weight ratio of the model group were significantly increased compared to the normal group, and the colon weight to colon length ratio and the colon weight to mouse weight ratio of the mice were significantly decreased compared to the model group after administration of the nidus Vespae drug dry prognosis (fig. 2).

(3) Colon tissue pathology: observing colon pathology of each group of mice through H & E staining, wherein the colon tissue structure of normal mice is clear, and inflammatory cell infiltration is avoided; the model group mice had damaged colon tissue structure, infiltrated inflammatory cells, colonic edema, with a higher pathological score than the normal group, after administration of nidus Vespae drug, the colonic epithelial cells were morphologically normal, and slightly infiltrated inflammatory cells, with a lower pathological score than the model group (fig. 3).

(4) Masson staining: investigation on colon fibrosis degree of each group of mice is carried out through Masson staining, the colon tissue structure of normal mice is clear, and the myometrium fibrosis layer is thinner; the model group mice had a damaged colon tissue structure and a heavy degree of fibrosis, and after administration of nidus Vespae drug, the colonic epithelial cells were morphologically normal and the degree of fibrosis was reduced (fig. 4).

(5) Fibrosis factor expression in colon tissue: fibrosis factors fibroetin, alpha-SMA, N-Cadherin, E-Cadherin, col1 and TIMP1mRNA expression in colon tissue of each group of mice were examined by qRT-PCR. The mRNA expression of the fibrosis factors of fibrins, alpha-SMA, N-Cadherin, col1 and TIMP1 in the colon of the mice in the model group is obviously increased compared with that in the Ctrl group; the expression of fibrotic factors fibroetin, α -SMA, N-cadherein, col1 and TIMP1mRNA was significantly reduced in colon tissue of mice following administration of nidus Vespae drug dry, with statistical differences (fig. 5). The mRNA expression of Cadherin E-Cadherin in the colon of the mice in the model group is obviously reduced compared with that in the Ctrl group; E-Cadherin expression was significantly elevated in colon tissues of mice following administration of nidus Vespae drug dry, with statistical differences (FIG. 5).

In summary, the invention provides the application of the nidus Vespae extract in preparing the medicine for preventing and treating the Crohn's disease, the nidus Vespae extract increases the weight of mice, reduces the ratio of the weight of the colon to the length of the colon and the ratio of the weight of the colon to the weight of the mice, improves the pathological structure and fibrosis score of the colon of the mice, changes the expression of fibrotic genes of the colon tissue, 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 method and a way for realizing the technical scheme are numerous, and the above description is merely a preferred embodiment of the present invention, and it should be noted that, for a person skilled in the art, several improvements and modifications can be made, and these improvements and modifications should also be considered as the protection scope of the present invention, without departing from the principle of the present invention. The components not explicitly described in this embodiment can be implemented by using the prior art.

Claims (9)

1. The application of the nidus Vespae extract in preparing the medicine for preventing and treating the Crohn disease is characterized in that the nidus Vespae extract is prepared according to the following method: decocting nidus Vespae in water, filtering, and concentrating the filtrate under reduced pressure;

the main intestinal structural injury of the Crohn disease is intestinal fibrosis.

2. The use according to claim 1, wherein the nidus Vespae extract is used as active ingredient to prepare pharmaceutically acceptable dosage forms.

3. The use according to claim 2, wherein the dosage form is a tablet, capsule, pill, oral liquid or granule.

4. The use according to claim 3, wherein the oral liquid formulation is a syrup, a wine or a lotion.

5. The use according to claim 2, wherein the dosage form is administered orally.

6. The application of the nidus Vespae extract in preparing the medicine for preventing and treating the intestinal fibrosis disease is characterized in that the nidus Vespae extract is prepared according to the following method: decocting nidus Vespae in water, filtering, and concentrating the filtrate under reduced pressure.

7. The use according to claim 6, wherein the nidus Vespae extract is used as active ingredient to prepare pharmaceutically acceptable dosage forms.

8. The use according to claim 7, wherein the dosage form is a tablet, capsule, pill, oral liquid or granule.

9. The use according to claim 8, wherein the oral liquid formulation is a syrup, a wine or a lotion.

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