CN114306453B - Application of pharmaceutical composition in preparation of medicines for treating inflammatory bowel disease - Google Patents

Abstract

The invention belongs to the technical field of new medical application, and particularly discloses application of a pharmaceutical composition in preparation of a medicine for treating inflammatory bowel disease. The medicinal composition comprises the following raw material extracts in parts by weight: 130-190 parts of pericarpium Granati, 120-160 parts of radix scutellariae, 100-140 parts of garden burnet and 100-140 parts of cortex phellodendri; in addition, the invention further prepares the pharmaceutical composition for treating inflammatory bowel disease into an enteric granule preparation for more accurate administration. The pharmaceutical composition for treating inflammatory bowel disease is a pure traditional Chinese medicine preparation, has a good effect of treating inflammatory bowel disease, is small in dosage, convenient to take, wide in application range, definite in curative effect, safe and reliable, free of drug dependence and side effects, and can achieve the effect of treating both symptoms and root causes.

Description

Application of pharmaceutical composition in preparation of medicines for treating inflammatory bowel disease

Technical Field

The invention belongs to the technical field of new medical application, and in particular relates to application of a pharmaceutical composition in preparation of a medicine for treating inflammatory bowel disease.

Background

Inflammatory bowel disease (Inflamatory bowel diseases, IBD) is a gastrointestinal recurrent inflammatory disease characterized by pain, diarrhea, bloody stool, and weight loss, and includes two major types: crohn's Disease (CD) and ulcerative colitis (Ulcerative colitis, UC). The etiology and pathogenesis of IBD are generally thought to be primarily due to environmental, genetic, immune, and intestinal dysbacteriosis. IBD is one of the modern refractory diseases that has been listed by the world health organization because of its prolonged and difficult recovery, even at risk of cancer. At present, single-target medicaments such as 5-aminosalicylic acid, glucocorticoid, immunosuppressant and the like are often used for clinical treatment, but the defects of low cure rate, easy recrudescence and the like exist, and adverse reactions such as intolerance, anaphylactic reaction and the like can be generated after long-term use. Faecal fungus transplantation (FMT) is a recently emerging modality for treating IBD, but FMT time and dosage are difficult to determine and there is also a risk of exposure to harmful strains, infectious diseases, etc. For complex diseases such as IBD, a single-target drug research and development mode based on a single gene-disease-drug research thought is challenged, and the success rate of new drug research and development is low, so that the complex diseases can not be met. The traditional Chinese medicine and the national medicine have advantages in the aspect of treating difficult and complicated diseases, mainly show the overall dialectical treatment of complex diseases, exert the multi-component, multi-target, multi-path and overall regulation treatment advantages aiming at complex etiology, and have remarkable effects. Therefore, effective therapeutic drugs are required to be searched from traditional Chinese medicines for treating IBD (IBD) which is a refractory disease.

IBD belongs to the categories of "diarrhea", "dysentery", "abdominal pain" and "acute appendicitis" in traditional Chinese medicine, and its etiology is related to congenital conditions (spleen and kidney deficiency), diets, seven emotions, exogenous pathogens, etc., and its disease location is mainly in the intestines and is related to viscera such as liver, spleen, kidney, lung, etc.

From the above, IBD is developed based on the deficiency of healthy qi and the opportunistic pathogen of the toxin, so the traditional Chinese medicine for treating IBD should be based on the effects of resolving toxin, strengthening spleen and promoting diuresis, regulating qi and blood, regulating liver and spleen, and warming kidney to consolidate the constitution in the remission stage. The effective components of the traditional Chinese medicine can regulate and control Th17/Treg balance, regulate intestinal flora, maintain immune microenvironment steady state, inhibit the secretion of pro-inflammatory factors, protect intestinal mucosa and promote the cure of IBD.

In recent years, the inventor of the application has conducted related research on traditional Chinese medicine anti-inflammatory, and has obtained a certain research result. The preparation provided by the invention is prepared from extracts of radix scutellariae, sanguisorba officinalis, pericarpium granati and cortex phellodendri, has the effects of clearing heat and drying dampness, removing stasis and detoxifying, cooling blood and stopping bleeding, purging intense heat and killing parasites, and astringing sores and intestines, and has obvious treatment effects on bacillary dysentery and gastroenteritis. However, as a hospital preparation, it has disadvantages such as unclear drug effect substances, unclear mechanism of action, and extremely limited application range. Based on this, it is necessary to conduct a perfection study.

Meanwhile, in order to expand the indications of the formulation in the preparation, the inventor of the application recently uses the composition in the curative effect research of the experimental IBD of mice, and finds that the effect is remarkable, and the composition is a novel medicinal effect, so the application is an invention patent.

Disclosure of Invention

The invention aims to avoid the problems that the side effect of the existing medicine for treating IBD and the FMT time and dosage are difficult to determine, and the risk of contacting harmful strains, infecting infectious diseases and the like exists, and the like, and to screen out an alternative medicine composition for treating IBD from traditional Chinese medicines.

For this reason, the present invention firstly provides an application of a pharmaceutical composition in preparing a medicament for preventing and/or treating inflammatory bowel disease.

Further, the invention also provides a method for preparing the pharmaceutical composition into a pharmaceutical preparation for preventing and/or treating inflammatory bowel disease, and further the preparation is enteric granules.

The medicine composition is an extract of pericarpium Granati, radix Scutellariae, radix Sanguisorbae and cortex Phellodendri;

the raw material medicine for preparing the pharmaceutical preparation for treating inflammatory bowel disease comprises the following raw materials in parts by weight:

130-190 parts of pericarpium Granati, 120-160 parts of radix scutellariae, 100-140 parts of garden burnet and 100-140 parts of cortex phellodendri;

preferably, it is:

150-170 parts of pericarpium Granati, 130-150 parts of radix scutellariae, 110-130 parts of garden burnet and 110-130 parts of cortex phellodendri;

the best is:

160 parts of pericarpium Granati, 140 parts of radix scutellariae, 120 parts of garden burnet and 120 parts of cortex phellodendri.

The preparation method of the pharmaceutical preparation (enteric granule) for treating inflammatory bowel disease comprises the following steps:

(1) Weighing pericarpium Granati, pulverizing into coarse powder, adding 60v/v% ethanol with weight 6 times of that of pericarpium Granati coarse powder for the first time, soaking for 0.5h, heating and reflux-extracting for 15min, filtering to obtain filtrate, adding 60v/v% ethanol with weight 4 times of that of pericarpium Granati coarse powder into filter residue, heating and reflux-extracting for 10min, filtering to obtain filtrate, and mixing the two filtrates for use.

(2) Weighing radix Scutellariae, radix Sanguisorbae and cortex Phellodendri, pulverizing into mixed coarse powder of three materials, adding 60v/v% ethanol with the weight 6 times of that of the mixed coarse powder for the first time, soaking for 0.5h, heating and reflux extracting for 1h, and filtering to obtain filtrate for use; adding 60v/v% ethanol with the weight 4 times of that of the mixed coarse powder into the filter residue, heating and reflux extracting for 0.5h, filtering to obtain filtrate, and combining the two filtrates for standby.

(3) And (3) combining the filtrates in the steps (1) and (2), recovering ethanol, concentrating to a relative density of 1.25, and spray-drying to obtain dry extract powder for later use.

(4) And (3) forming a preparation: taking dry extract powder, preparing soft materials by 60v/v% ethanol solution, granulating by a 16-mesh sieve, drying at 50 ℃, finishing the granules by the 16-mesh sieve, and then placing in a coating pot for rounding for standby. Eudragit L30D-55 is used as enteric coating material, PEG6000 is used as plasticizer, talcum powder is used as anti-adhesion agent, a proper amount of water is added to prepare coating liquid, the above-mentioned granules are coated, and dried at 40 deg.C to obtain the enteric granules (the dry extract content in the described enteric granules is preferably 0.3 mg/g).

After being coated by an enteric coating material, the pharmaceutical composition can well release medicines in intestinal parts and play the roles of resisting inflammation, repairing intestinal mucosa and regulating intestinal flora so as to achieve the efficacy of treating IBD, and specific experimental operation, result record and conclusion analysis are shown in specific embodiments.

Compared with the prior art, the invention has the beneficial effects that:

the medicine composition for treating IBD provided by the invention is a pure traditional Chinese medicine compound preparation, can obviously improve the pathological characteristics of IBD mice, inhibit the release of pro-inflammatory factors NO, TNF-alpha, IL-1 beta and IL-6, increase the secretion of anti-inflammatory factors IL-10, further inhibit the activation of NF-kappa B and JAK/STAT3 channels, simultaneously maintain intestinal tract mechanical barriers by promoting the expression of tight junction proteins ZO-1, occludin-1 and Claudin-1, regulate intestinal flora to play a role in treating IBD together, and embody the treatment characteristics of complex disease system regulation and multi-component multi-target synergistic effect of traditional Chinese medicines.

Because the medicine is enteric-coated granule, the medicine is taken conveniently, the part which exerts the medicine effect is positioned at the colon part, the medicine is concentrated at the target lesion part, the concentration is high, the bioavailability is also high, and the curative effect is definite, safe and reliable.

The invention takes the secondary development of the clinically effective Chinese patent medicine as an entry point, deeply discusses the cooperative integration mechanism of the multi-component multi-target multi-path aiming at key pathological links and action paths, and provides a reference basis for further creating a novel compound preparation with clear components, clear mechanism, controllable quality and safety and effectiveness. The research is from clinic, returns to clinic, is one of important ways for creating novel compound traditional Chinese medicine, accords with the development direction of the transformation medical concept and the combination basis of traditional Chinese medicine and western medicine, and has great theoretical value and application prospect.

The applicant will now make further details of the present invention with reference to the accompanying drawings.

Drawings

FIG. 1 is a graph of the effect of drug composition particles on IBD mice in general, 3.1;

FIG. 2 is a graph of the effect of drug composition particles on the DAI index of IBD mice in 3.2;

FIG. 3 is a graph of the effect of drug composition particles in 3.3 on the immune organ index of IBD mice;

FIG. 4 is a graph of the effect of drug composition particles in 3.4 on colon length in IBD mice;

FIG. 5 is a graph of the effect of particles of a pharmaceutical composition on colon tissue pathology in IBD mice in 3.5;

FIG. 6 is a graph of the effect of drug composition particles in 3.6 on NO content in colon tissue of IBD mice;

FIG. 7 is a graph showing the effect of drug composition particles on MPO content in colon tissue of IBD mice at 3.7;

FIG. 8 is a graph of the effect of drug composition particles in 3.8 on the levels of inflammatory factors TNF- α, IL-6, IL-1β and IL-10 in colon tissue of IBD mice;

FIG. 9 is a graph of the effect of drug composition particles in 3.9 on expression of HSP70/NF- κB pathway and JAK/STAT3 pathway in colon tissue of IBD mice;

FIG. 10 is a graph of the effect of drug composition particles on expression of zonulin ZO-1, occludin-1 and Claudin-1 in colon tissue of IBD mice at 3.10;

FIG. 11 is a graph of the effect of drug composition particles on short chain fatty acids of the cecal content of mice at 3.11;

FIG. 12 is a graph of the effect of drug composition particles in 3.12 on intestinal flora in the colon content of mice;

in the figure: alpha diversity: a Chao1 index and a Shannon index; beta diversity: o: blank control group

Model group Δ: a group of particles of the pharmaceutical composition, each group based on a PCoA profile of the unweighted_uniarray distance; C. the relative abundance of different flora gates; D. the relative abundance of different flora; E. different species at each panel gate level; F. differential species at the genus level of each group. Data are expressed as +.>

(n＝5)，#P<Normal group 0.05vs P<0.05vs model group.

Fig. 13 is a schematic of the dosing regimen of the in vivo animal experiment of 2.2.

Detailed Description

In the following, the applicant will make a clear and complete description of the technical solutions according to the embodiments of the present invention, with reference to the accompanying drawings. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In the following examples, the parts by weight were 10g in one part, and this is explained in detail, whereby the reader can see the production scale of each example.

1 examples 1-8: preparation of pharmaceutical composition preparation for treating inflammatory bowel disease

Example 1:

the embodiment provides a pharmaceutical composition preparation for treating inflammatory bowel disease, which comprises the following raw material medicinal materials in parts by weight: 160 parts of pericarpium Granati, 140 parts of radix scutellariae, 120 parts of garden burnet and 120 parts of phellodendron.

The preparation method of the pharmaceutical composition preparation for treating inflammatory bowel disease of the embodiment comprises the following steps:

(1) 160 parts of pericarpium Granati are weighed and crushed into coarse powder, 60v/v% ethanol which is 6 times of the weight of the coarse powder of the pericarpium Granati is added for the first time, the mixture is soaked for 0.5 hour and then heated and refluxed for extraction for 15 minutes, the filtrate is obtained after filtration, 60v/v% ethanol which is 4 times of the weight of the coarse powder of the pericarpium Granati is added into filter residues for heating and refluxing for extraction for 10 minutes, the filtrate is obtained after filtration, and the two filtrates are combined for standby.

(2) Weighing 140 parts of radix scutellariae, 120 parts of garden burnet and 120 parts of amur corktree bark, crushing the mixture into mixed coarse powder of three medicinal materials, adding 60v/v% ethanol which is 6 times of the weight of the mixed coarse powder for the first time, soaking for 0.5h, heating, refluxing and extracting for 1h, and filtering the filtrate for later use; adding 60v/v% ethanol with the weight 4 times of that of the mixed coarse powder into the filter residue, heating and reflux extracting for 0.5h, filtering to obtain filtrate, and combining the two filtrates for standby.

(3) Mixing the filtrates obtained in the steps (1) and (2) to obtain mixed extract, recovering ethanol, and spray drying to obtain dry extract powder for later use;

concentrating the mixed extractive solution to relative density of 1.25, and spray drying.

(4) And (3) forming a preparation: taking dry extract powder, preparing soft materials by 60v/v% ethanol solution, granulating by a 16-mesh sieve, drying at 50 ℃, finishing the granules by the 16-mesh sieve, and then placing in a coating pot for rounding for standby. Eudragit L30D-55 is used as enteric coating material, PEG6000 is used as plasticizer, talcum powder is used as anti-adhesion agent, a proper amount of water is added to prepare coating liquid, the above-mentioned granules are coated, and dried at 40 deg.C to obtain enteric granules (in which the dry extract content is 0.3 mg/g) for subsequent medicine effect and pharmacological experiment.

Examples 2 to 8:

the embodiments 2-8 respectively provide a pharmaceutical composition preparation for treating inflammatory bowel disease, the effective components of which are extracts of pericarpium Granati, radix Scutellariae, radix Sanguisorbae and cortex Phellodendri, and the formula of the raw materials in parts by weight of the pharmaceutical composition preparation for treating inflammatory bowel disease is shown in Table 1:

TABLE 1

Examples	Medicinal material raw material formula
		2	130 parts of pericarpium Granati, 120 parts of radix scutellariae, 100 parts of garden burnet and 100 parts of cortex phellodendri
3	140 parts of pericarpium Granati, 130 parts of radix scutellariae, 110 parts of garden burnet and 110 parts of cortex phellodendri
		4	150 parts of pericarpium Granati, 140 parts of radix scutellariae, 120 parts of garden burnet and 120 parts of amur corktree bark
5	160 parts of pericarpium Granati, 140 parts of radix scutellariae, 120 parts of garden burnet and 120 parts of amur corktree bark
		6	170 parts of pericarpium Granati, 140 parts of radix scutellariae, 120 parts of garden burnet and 120 parts of amur corktree bark
7	180 parts of pericarpium Granati, 150 parts of radix scutellariae, 130 parts of garden burnet and 130 parts of cortex phellodendri
		8	190 parts of pericarpium Granati, 160 parts of radix scutellariae, 140 parts of garden burnet and 140 parts of amur corktree bark

The steps of the preparation method of the pharmaceutical composition preparation for treating inflammatory bowel disease of examples 2-8 are the same as those of example 1, and finally enteric granules with dry extract content of 0.3mg/g are obtained.

In the following, enteric coated granules (hereinafter referred to as "pharmaceutical composition granules") prepared in example 1 are all taken as examples, and the efficacy and mechanism of action of the enteric coated granules for treating IBD by combined action of promoting intestinal mucosal membrane repair and regulating intestinal flora are studied by using an in vivo animal experiment model and an in vitro cell model experiment.

2 pharmaceutical efficacy and pharmacological experiment method

2.1 experimental groupings and dosages

C57BL/6 male mice, 6-8 weeks old, 22+ -2 g, were randomly divided into 6 groups of 5 animals each, and normal (Control) and Model (Model) groups were given physiological saline; the Positive drug group (Positive) was given with 0.45 g/kg/day enteric-coated tablet of sulfasalazine (SASP, shanghai Yi balance pharmaceutical Co., ltd., each tablet containing 0.25g of sulfasalazine); group of particles of pharmaceutical composition: low dose group (YQG-L) 0.312 g/kg/day; medium dose group (YQG-M) 0.624 g/kg/day; the high dose group (YQG-H) was 1.248 g/kg/day. The positive medicine and the medicine particles are dissolved or suspended by normal saline, and each group is administrated by the mode of stomach irrigation, and the volume is 0.10mL/10 g/day.

2.2 in vivo animal experiments

30C 57BL/6 male mice were randomly divided into 6 groups of 5 mice each. After 7 days of adaptive feeding, each group of mice was given a solution of dextran sodium sulfate (DSS, m.w40000, shanghai Aladdin Biochemical Technology co., ltd. Hereinafter) formulated with sterile water at a concentration of 3% (by mass volume), i.e., 3g/100 mL) for free drinking for one week, modeling day 8, and each group of mice was changed to sterile water for free drinking for 3 days, in addition to normal groups of mice given sterile water. Each group of mice was dosed 1 time a day at the same time, starting from the day of modeling, for 10 consecutive days. The dosing regimen is shown in figure 13.

2.2.1 evaluation of colon inflammation model

Daily changes in body weight, diarrhea and bloody stool were recorded for each mouse and scored for disease activity index (Disease activity index, DAI) according to the scoring criteria in table 1.

TABLE 1 disease Activity index (Disease activity index, DAI) scoring criteria

Feces were collected from each mouse every 2 days after the start of the experiment. After the end of the administration, the mice were not fasted and not water-inhibited. On day 18, mice were sacrificed after ether anesthesia, the entire colon was collected, the colon length was recorded and assessed, a 0.5cm section of the colon was taken 1cm away from the anus, fixed in 4% paraformaldehyde, and the remaining colon was divided into 4 portions and placed in a freezing tube, quick frozen with liquid nitrogen, and stored at 80 ℃ for subsequent experiments.

2.2.2 histopathological analysis

After 4% paraformaldehyde is used for fixing colon tissues, ethanol (70 v/v%, 80v/v%, 90v/v%, 95v/v%, 100 v/v%) with different concentrations is used for gradient dehydration, 4 mu m-thick colon tissue slices are prepared by paraffin embedding, and after roasting, xylene and ethanol gradient dewaxing, hematoxylin-eosin staining and neutral resin sealing are carried out on the colon tissue slices in the next day. The pathological morphology of the colon tissue was observed under an optical microscope, and images were collected and scored. The scoring rules are as follows in table 2:

TABLE 2 colo-pathology scoring criteria

Determination of 2.2.3MPO content

The tissue was weighed according to MPO kit (Nanjing institute of biological engineering, 48T) instructions and added with the prepared reagent II to prepare a 5% tissue homogenate in a ratio of 1:19, 270. Mu.L of the 5% tissue homogenate was taken, 30. Mu.L of reagent III was added, and after mixing, incubated in a 37℃water bath for 15min. Taking 80 mu L of the mixture, adding 100 mu L of reagent IV and 1.2mL of color reagent (each group of control tubes is replaced by double distilled water) according to the proportion, incubating for 30min at 37 ℃, adding 20 mu L of reagent seven, mixing uniformly, placing in a 60 ℃ constant temperature water bath for incubating for 10min, taking out, and rapidly reading OD value at 460nm wavelength. The calculation formula is as follows:

MPO (U/g) = (sample OD value-control OD value)/11.3×sample size (g)

2.2.4NO content detection

The procedure was performed according to the NO detection kit (bi yun tian, 500T) instructions: taking frozen colon tissue at-80 ℃, adding 9 times of phosphate buffer solution (PBS, 0.01mol/L, pH7.4, the same applies below) to prepare 10% colon tissue homogenate, centrifuging at 4 ℃ and 12000rpm for 10min, taking tissue homogenate supernatant, adding 50 mu L of Griess I and Griess II into each hole, detecting OD value at 540nm wavelength, and calculating the concentration of NO in each group of samples according to a standard curve.

2.2.5ELISA detection of the content of inflammatory factors TNF-alpha, IL-6, IL-1 beta, IL-10

Taking 10% colon tissue homogenate supernatant prepared by PBS in 2.2.4, and detecting the content of TNF-alpha, IL-6, IL-1 beta and IL-10 in the colon tissue homogenate of the mice by adopting an ELISA method according to the instruction steps of the mouse TNF-alpha ELISA kit (Xinbo, 96T), the mouse IL-6ELISA kit (Xinbo, 96T), the mouse IL-1 beta ELISA kit (Xinbo, 96T) and the mouse IL-10ELISA kit (Xinbo, 96T).

2.2.6Western Blot detection of expression of related proteins

Colon tissue was removed from the-80 ℃ refrigerator and weighed, and protease inhibitor cocktail (beyotime, P1045,2ml,50 x) was added to RIPA lysate (beyotime, P0013F,100 ml). 1mL of the prepared cell lysate is added to every 100mg of colon tissue, homogenized by a homogenizer, cracked on ice for 30min, the tissue homogenate is centrifuged at 12000rpm for 15min at 4 ℃, and the supernatant is sucked and transferred to a new EP tube. The BCA method detects the protein concentration so as to detect the protein expression conditions of NF-kappa B p, p-p65, p-Ikappa B, I kappa B, p-JAK, JAK, p-STAT3, ZO-1, occludin-1, beta-actin and the like.

20. Mu.g and 40. Mu.g of the protein were separated by electrophoresis on 8%, 10% and 13.5% SDS-PAGE gels, respectively, and the target protein was transferred onto a polyvinylidene fluoride (PVDF) membrane by electrotransfer. The PVDF membrane was then blocked for 2h at room temperature with a 5% solution of Bovine Serum Albumin (BSA) in TBST buffer (1 ml of Tween 20 in 1L of Tris-HCl buffer), and after TBST washing, the primary antibody was incubated overnight at 4 ℃. NF- κ B p65, p-p65, p-Iκ B, I κ B, p-JAK, JAK, p-STAT3, ZO-1 diluted 1:1000, occludin-1 diluted 1:2000, claudin-1 diluted 1:500 dilutions, beta-actin 1:50000 dilutions. After three TBST washes, PVDF membranes were incubated with secondary antibody diluted 1:5000 for 1.5h at room temperature.

Protein bands were detected by chemiluminescence (ECL) and all blots were run with beta-actin protein as reference and analyzed by Image J software.

2.2.7 content determination of short chain fatty acids of cecal content

Taking out the preserved cecum content from a refrigerator at-80 ℃, precisely weighing a proper amount of sample into a 2ml centrifuge tube, adding methanol (the ratio of the content to the methanol is 1g:1 mL), swirling for 30s, centrifuging the mixed solution in a centrifuge at 4 ℃ (the rotating speed is 12000 g) for 10min, taking a proper amount of supernatant into a 2ml centrifuge tube, adding metaphosphoric acid (the volume ratio of the supernatant is 25% metaphosphoric acid=5:1), uniformly mixing, and standing at 4 ℃ overnight. The next day, centrifuge at 4deg.C (rotational speed 12000 g) for 10min, take appropriate amount of supernatant in sample bottle, store in 4deg.C refrigerator for use. The content of short chain fatty acids in the supernatant was then determined by gas chromatography. Chromatographic conditions: the chromatographic column is an Agilent DB-WAX capillary column; the detector is a Flame Ionization Detector (FID) (230 ℃); the sample feeding amount is 1 mu L, no flow division is performed, the temperature of the sample feeding line and the temperature of the transmission line are 230 ℃, the hydrogen flow is 35ml/min, the air flow is 350ml/min, and the tail blowing air is 30ml/min; heating to 75deg.C at 6deg.C/min to 180deg.C, maintaining for 2min, and collecting carrier gas as high purity nitrogen.

2.2.8 16sDNA sequencing method for determining changes in intestinal flora in colon contents

DNA was extracted from each set of colon content samples using DNA extraction kit (MN Nucleospin 96 Soi), and specific primers were synthesized according to the V4 sequencing region: V4:515F-806R2 (515F: 5'-GTGYCAGCMGC CGCGGTAA-3',806R2:5 '-GGACTACNVGGGTWTCTAAT-3') performing PCR amplification, constructing a library by using the PCR product as a template, normalizing the constructed library, mixing samples, sequencing by using Illumina Novaseq 6000, and analyzing the result.

Splicing the original data by using FLASH (version 1.2.11), and performing quality filtering by using trimmatic (version 0.33) software; double-end sequence splicing is carried out by using Usearch (version 10) software, and length filtering is carried out on spliced data according to the length ranges of different areas; the chimaera sequences were identified and removed using UCHIME v4.2 software to yield final Effective Reads. And performing operation classification unit cluster analysis by taking SILVA as a reference database, and performing deep analysis on species community diversity, species classification and abundance by using QIIME.

2.2.7 statistical analysis

All experimental data in this document are expressed as mean ± standard error (x±sem). Statistical analysis uses t-test and one-factor analysis of variance. * P <0.05 and P <0.01 indicate that the inter-group differences are statistically significant.

3. Drug efficacy and pharmacological results and analysis

3.1 particles of pharmaceutical composition for alleviating clinical symptoms of DSS-induced IBD in mice

Compared with the normal group, the model group mice have diarrhea, bloody stool, weight reduction and other conditions. The pharmaceutical composition particles significantly improved the symptoms described above compared to the model group: mice had reduced bloody stool, gradually recovered stool morphology, increased intake of water, gradually active mental state, increased coat gloss, significantly reduced body weight loss compared to the model group, and exhibited significant dose dependence (×p)<0.05 or P<0.01). The medicinal composition particles have better curative effect on IBD. See fig. 1, A, B, C, D, E for specific results: A. observing the anus Zhou Rouyan of the mice; B. observing the shape of the mouse feces; C. food intake of mice; D. water intake of mice; E. mice body weight change. The data is expressed as

(n＝5)，#P<0.05、##P<Normal group 0.01vs P<0.05、**P<0.01vs model group.

3.2 Effect of the particles of the pharmaceutical composition on the DAI index of IBD mice

The DAI index is an index for comprehensively evaluating the disease condition by the body weight change, the stool dryness and the stool condition of the mice. The result of the study shows thatThe pharmaceutical composition particles were able to significantly reduce DAI index score (×p) compared to the model group<0.05 Indicating that it has a dose-dependent effect of alleviating clinical symptoms of IBD in mice. The DAI index scores for each group of mice are shown in fig. 2, where: the data is expressed as

(n＝5)，#P<0.05、##P<Normal group 0.01vs P<0.05、**P<0.01vs model group.

3.3 Effect of the particles of the pharmaceutical composition on the organ index of IBD mice

The results are shown in fig. 3, where: A. organ index of mouse heart; B. organ index of mouse liver; C. organ index of mouse spleen; D. organ index of lung; E. organ index of left kidney; F. organ index of right kidney; G. thymus index of mice. The data is expressed as

(n＝5)，#P<0.05、##P<Normal group 0.01vs P<0.05、**P<0.01vs model group.

DSS molding has no obvious effect on organs such as heart, lung, kidney and the like of the C57BL/6 mice, and each dose of the drug composition particles and SASP administration have no obvious effect on the organs of the mice after molding. DSS modeling has different degrees of effect on mice thymus and spleen, compared to normal mice, model mice have splenomegaly, thymus shrinkage (< 0.01) and pharmaceutical composition particles can alleviate symptoms of splenomegaly and thymus shrinkage (< 0.01) in mice.

3.4 Effect of the particles of the pharmaceutical composition on the colon Length of IBD mice

The colon of each experimental group was taken, its length was measured and photographed. From the figure, it can be seen that the colon of the mice in the model group is significantly shortened (P)<0.01 Blood stool in the intestinal lumen can be seen by naked eyes. Compared with the model group, the medicinal composition particles can reduce colon shortening of mice, and the colon length is obviously increased (P)<0.01 And is dose dependent. The results are shown in FIG. 4, where: A. a mouse colon; B. colon length statistics. The data is expressed as

(n＝5)，#P<0.05、##P<Normal group 0.01vs P<0.05、**P<0.01vs model group.

3.5 Effect of the particles of the pharmaceutical composition on the pathological conditions of colon tissue in IBD mice

Colon tissue section H for 6 groups of mice&E staining found that colon tissue mucosal structure disappeared, goblet cells were lost significantly, mucosal and submucosal inflammatory infiltrates severely, edema increased (×P) in model group mice compared to normal group mice<0.01). The particles of the pharmaceutical composition have reduced goblet cell loss, complete colonic mucosa structure recovery, reduced inflammatory infiltration, reduced edema level with increasing dose, and the most significant effect in high dose group (P)<0.01). Scoring each group of colon tissue found: model group score significantly increased (×p)<0.01 A) is provided; the pharmaceutical composition particles reduced the colon score of mice to a different extent compared to the model group and were dose dependent (×p)<0.05). The results are shown in FIG. 5, where: A. mouse colon tissue HE stained sections (100×); B. pathology scoring. The data is expressed as

(n＝5)，#P<0.05、##P<Normal group 0.01vs P<0.05、**P<0.01vs model group.

3.6 Effect of the particles of the pharmaceutical composition on NO content in colon tissue of IBD mice

As shown in fig. 6, in the drawing: the data is expressed as

(n＝3)，#P<0.05、##P<Normal group 0.01vs P<0.05、**P<0.01vs model group.

The level of NO in the mice of the model group was significantly increased compared to the level in the mice of the normal group (< P < 0.01), and the high dose in the particles of the pharmaceutical composition significantly reduced the level of NO (< P <0.05, < P < 0.01) compared to the high level of NO in the model group. The above results demonstrate that the particles of the pharmaceutical composition are capable of reducing the NO content in DSS-induced colon tissue and inhibiting inflammatory responses.

3.7 Effect of the particles of the pharmaceutical composition on MPO content in colon tissue of IBD mice

Myeloperoxidase (MPO) is secreted mainly by neutrophils, and its level in colon tissue is detected by colorimetry, which can indirectly reflect the degree of inflammation at the colon site. The results of this experiment show that the MPO content in colon tissue of mice in the model group is significantly increased compared to the normal group (<0.01). Whereas the particles of the pharmaceutical composition are capable of significantly reducing the MPO content (P)<0.01 A dose-dependent aspect). It is demonstrated that DSS increases the number of neutrophils migrating to the colon site, while particles of the pharmaceutical composition can down-regulate MPO levels, reducing inflammatory responses to some extent. The results are shown in FIG. 7, where: the data is expressed as

(n＝3)，#P<0.05、##P<Normal group 0.01vs P<0.05、**P<0.01vs model group.

3.8 effects of the particles of pharmaceutical composition on TNF- α, IL-6, IL-1β and IL-10 levels in colon tissue of IBD mice are shown in FIG. 8, which shows: TNF-alpha; IL-6; IL-1 beta; IL-10. The data is expressed as

(n＝3)，#P<0.05、##P<Normal group 0.01vs P<0.05、**P<0.01vs model group.

TNF- α, IL-6, IL-1β levels were significantly increased (P < 0.01) and IL-10 levels were significantly decreased (P < 0.01) in model mice compared to normal mice; the pharmaceutical composition particles can significantly down-regulate the levels of TNF-alpha, IL-6 and IL-1 beta (P < 0.01), and increase the content of anti-inflammatory factor IL-10 (P < 0.01). The results demonstrate that the pharmaceutical composition particles are capable of reducing expression of a colon site-associated pro-inflammatory factor induced by DSS induction, up-regulating expression of an anti-inflammatory factor, to inhibit inflammatory response.

3.9 Effect of the particles of the pharmaceutical composition on expression of the HSP70/NF- κB pathway and the JAK/STAT3 pathway in the colon tissue of IBD mice

As shown in fig. 9, in the drawing: NF- κB pathway protein expression; B.HSP70/beta-acA tin; C.p-P65/P65; D.p-IκB/IκB. The data is expressed as

(n＝3)，#P<0.05、##P<Normal group 0.01vs P<0.05、**P<0.01vs model group.

Compared with the normal group, the DSS model group has obviously reduced expression of HSP70 and up-regulated expression of NF- κB channel protein (P < 0.01). The ratio of P-ikb/ikb and P-P65/P65 and P-JAK/JAK and P-STAT3/STAT3 also significantly increases (< 0.01) with significantly up-regulated ratios (< 0.01) indicating that the NF- κb pathway is activated resulting in increased colonic inflammation. The pharmaceutical composition particles can up-regulate the expression of HSP70, effectively reduce the phosphorylation of IκB and P65 proteins (P < 0.05) and reduce inflammation. The results of this experiment demonstrate that the pharmaceutical composition particles are able to treat IBD by inducing up-regulation of HSP70 protein while inhibiting activation of NF- κB pathway to inhibit inflammatory response.

3.10 Effect of the particles of the pharmaceutical composition on the expression of the Tight-connector protein in the colon tissue of IBD mice

As shown in fig. 10, in the drawing: A. tight junction protein expression; ZO-1/beta-actin; occludin-1/beta-actin; claudin-1/beta-actin. The data is expressed as

(n＝3)，#P<0.05、##P<Normal group 0.01vs P<0.05、**P<0.01vs model group.

The expression of the model group of zon-1, occludin-1 and Claudin-1 was significantly reduced (< 0.01, < 0.05) after DSS induction compared to the normal group, and the high dose group of the pharmaceutical composition particles was able to significantly up-regulate the expression of ZO-1, occludin-1 and Claudin-1 proteins (< 0.01). The above results demonstrate that the pharmaceutical composition particles may restore colonic mucosal permeability by up-regulating the expression of the claudin.

3.11 pharmaceutical composition particles promote the production of short chain fatty acids

Detection of Short Chain Fatty Acids (SCFAs) content in the cecum content of mice by gas chromatographyFig. 11 shows: A. short chain fatty acid mixed standard solution and sample chromatogram. a. Mixing a standard solution b, sample 1, acetic acid 2, propionic acid 3, isobutyric acid 4, butyric acid 5, isovaleric acid 6, valeric acid; B. total short chain fatty acids; C. acetic acid; D. propionic acid; E. isobutyric acid; F. butyric acid; G. isovaleric acid; H. valeric acid. The data is expressed as

(n＝3)，#P<0.05、##P<Normal group 0.01vs P<0.05、**P<0.01vs model group.

The total SCFAs content of the model group was significantly reduced (P < 0.05) compared to the placebo group, with significant reductions in acetic acid (P < 0.05), isobutyric acid (P < 0.01), isovaleric acid (P < 0.05). Compared with the model group, the content of acetic acid (P < 0.05) and butyric acid (P < 0.05) in the drug composition particle group is obviously increased, and the content of total SCFAs in the drug composition particle group is obviously increased (P < 0.01).

The experimental results show that: it was found that the pharmaceutical composition particles were able to improve the clinical symptoms of IBD mice by increasing the content of acetic acid, propionic acid, butyric acid.

3.12. Intestinal microbiota analysis

The effect of the drug composition particles on DSS-induced IBD mice colon content intestinal flora was investigated using 16sRNA gene sequencing. The Alpha diversity (Alpha diversity) analysis results show that the species Coverage (Good Coverage's) >99.9%, the flora richness (Chao) index and Shannon (Shannon) index of the model group are reduced, and the Chao index is increased after the particle treatment of the pharmaceutical composition. Beta diversity analysis showed that the species populations of the control, model and drug composition particle groups were all different. As shown in FIG. 12C, at the phylum level, the dominant bacterial groups of each group were Bacteroides, firmides, verrucomicrobia, proteus Proteobacteria, epsilonbacteraeota; analysis of the relative abundance of the intestinal flora in mice revealed that the relative abundance of Verrucomicrobia was reduced in the model group compared to the placebo group (fig. 12E), and that the relative abundance increased significantly after treatment with the pharmaceutical composition particles (P < 0.05); the relative abundance of Firmicutes was significantly increased (P < 0.05) in the model group, whereas the relative abundance was significantly decreased (P < 0.05) after particle treatment of the pharmaceutical composition.

At the genus level (FIG. 12D), the dominant bacterial population of each group was uncultured_bacteria_f_Muribacueae, achroman muciniphila Akkermansia. The ruminococcaceae_ucg-013 of the model group was significantly decreased (P < 0.05), defloccultaceae_ucg-011 was significantly increased (P < 0.05), the relative abundance of coriobacteriaceae_ucg-002, ncultrued_bacteria_f_atobiaceae was significantly decreased (P < 0.05), the relative abundance of mucin ackermannia, bifidobacterium Bifidobacterium, defluviitaleaceae _ucg-011, ruminococcaceae_ucg-013 was significantly increased (P < 0.05) following particle treatment of the pharmaceutical composition (fig. 12F).

The experimental results show that: the drug combination particles can alleviate the clinical symptoms of IBD mice by increasing the relative abundance of the beneficial bacteria mucin Ackermansia (Akkermansia), bifidobacterium (Bifidobacterium), oenococcus (Ruminococcaceae_UCG-013).

Summarizing: in recent decades, the incidence rate of IBD in China is continuously rising, and because the etiology and pathogenesis of IBD are not completely elucidated, the IBD still lacks curative effect and low toxic and side effect therapeutic drugs clinically. At present, main therapeutic drugs for treating IBD by western medicine are aminosalicylic acid, immunosuppressant, adrenoglucocorticoid and the like, but the long-term use of the traditional Chinese medicine has the problems of large side effect, easy recurrence after stopping the drug and the like. For complex diseases such as IBD, a single-target substance research and development mode based on a single gene-disease-drug research thought is challenged, the complex disease prevention and treatment requirements cannot be met, and the success rate of new drug research and development is obviously reduced. The characteristics of the traditional Chinese medicine are presented in the overall dialectical treatment of the traditional Chinese medicine compound for complex diseases, meanwhile, the compatibility formula of the prescription focuses on the principle of monarch, minister, assistant and guide, and the regulation of the unbalanced and disordered state of the organism is realized through the coordination and coordination of multiple traditional Chinese medicines, and the ideas fully embody the ideas of multi-component, multi-target and systematic regulation.

Cortex Phellodendri has effects of clearing heat, eliminating dampness, purging pathogenic fire, removing steam, removing toxic substances, and treating sore, and can be used for treating damp-heat dysentery, jaundice, dark urine, pyocutaneous disease, and eczema, and wet sore, and is used as monarch drug; the baikal skullcap root has the effects of clearing heat and drying dampness, purging fire and removing toxin, cooling blood and stopping bleeding, and is mainly used for clearing damp heat in upper jiao and is a ministerial drug. The garden burnet root can cool blood and stop bleeding, detoxify and heal sore, remove lower-jiao heat, treat the symptom of hematochezia and urine and stop bleeding; the diarrhea is stopped, and the medicine is used as an adjuvant drug; punica granatum skin astringes the intestines to check diarrhea as a guiding drug. The theory of traditional Chinese medicine is as follows: the lung and large intestine are both exterior and interior, i.e. the lung meridian of hand taiyin and the large intestine meridian of hand yangming are both exterior and interior, and it is considered that the lung disease and the intestinal disease are related to each other. Clinically, patients suffering from fever and pulmonary infection often have gastrointestinal dysfunction and diarrhea and other symptoms; patients with bacillary dysentery and intestinal infection often have fever, shortness of breath and other symptoms. So the four herbs are combined together to achieve the actions of clearing lung-heat, removing dampness-heat, cooling blood and stopping diarrhea, and can be clinically used for bronchitis, diarrhea and other symptoms. However, the former research shows that the composition has good effect of treating colonitis except for inflammation and dysentery, which is a new discovery of the drug effect.

To date, over 66 different animal models of IBD have been established. The DSS induced C57BL/6 mouse IBD mouse is a mature model at present, and can simulate the pathological process of human colon inflammation and immunity, is also highly similar to human colonitis in clinical symptoms, physical signs and pathological manifestations, is easy to operate and good in repeatability, and is widely used for screening new IBD drugs. The present study therefore uses this model as the subject of the study.

Concentration and use time of the DSS solution are important factors for successful induction model, and an ideal model can be constructed by generally using 3% DSS solution for induction for one week. In this application, the applicant successfully established an IBD mouse acute colitis model, with typical clinical manifestations of colitis: diarrhea, bloody stool, weight loss, reduced drinking water, reduced colon and ulcers; the results observed with colon tissue HE sections also correspond to the pathological features of IBD. The medicine composition particles can improve pathological characteristics of IBD mice, inhibit release of pro-inflammatory factors NO, TNF-alpha, IL-1 beta and IL-6, increase secretion of anti-inflammatory factors IL-10, further inhibit activation of NF-kappa B and JAK/STAT3 channels, maintain intestinal tract mechanical barriers by promoting expression of tight junction proteins ZO-1, occludin-1 and Claudin-1, regulate intestinal flora to jointly act for treating IBD, and embody the treatment characteristics of complex disease system regulation and multi-component multi-target synergistic effect of traditional Chinese medicine.

Claims (7)

1. Use of a pharmaceutical composition in the preparation of enteric granules for the prevention and/or treatment of ulcerative colitis; the enteric particles are used for increasing the content of short chain fatty acids in cecum and regulating intestinal flora;

the medicinal composition is prepared from the extracts of pericarpium Granati, radix Scutellariae, radix Sanguisorbae and cortex Phellodendri in parts by weight:

130-190 parts of pericarpium Granati, 120-160 parts of radix scutellariae, 100-140 parts of garden burnet and 100-140 parts of cortex phellodendri;

the preparation method of the enteric granule comprises the following steps:

(1) Weighing pericarpium Granati, pulverizing into coarse powder, adding 55-65v/v% ethanol 4-8 times of pericarpium Granati coarse powder for the first time, soaking, heating and reflux extracting for 10-30 min, filtering to obtain filtrate, adding 55-65v/v% ethanol 3-6 times of pericarpium Granati coarse powder for the filter residue, heating and reflux extracting for 10-30 min, filtering to obtain filtrate, and mixing the two filtrates to obtain filtrate A;

(2) Weighing radix Scutellariae, radix Sanguisorbae and cortex Phellodendri, pulverizing into mixed coarse powder of three materials, adding 55-65v/v% ethanol 4-8 times the weight of the mixed coarse powder for the first time, soaking, heating and reflux extracting for 0.5-2 hr, and filtering to obtain filtrate; adding 55-65v/v% ethanol with 3-6 times of the weight of the mixed coarse powder into the filter residue, heating and reflux extracting for 0.5-2h, filtering to obtain filtrate, and mixing the two filtrates to obtain filtrate B;

(3) Mixing filtrate A and filtrate B, concentrating and recovering ethanol until the relative density is 1.2-1.3, and spray drying to obtain dry extract powder;

(4) And (3) forming a preparation: preparing soft material with 55-65v/v% ethanol, granulating with 16 mesh sieve, drying, grading with 16 mesh sieve, and rolling in coating pan; eudragit L30D-55 is used as enteric coating material, PEG6000 is used as plasticizer, talcum powder is used as anti-adhesion agent, water is added to prepare coating liquid, then the particles are coated, and finally the enteric particles are obtained by drying.

2. The use according to claim 1, characterized in that: the enteric particles are used for inhibiting colon shortening.

3. The use according to claim 1, characterized in that: the enteric particles are used for repairing intestinal mucosa.

4. The use according to claim 1, characterized in that: the enteric-coated particles are used for reducing the content of inflammatory mediators NO in colon tissues.

5. The use according to claim 1, characterized in that: the enteric coated particles are used for down regulating MPO content in colon tissue.

6. The use according to claim 1, characterized in that: the enteric coated particles are used for inhibiting NF-kappa B, JAK/STAT3 pathway activation.

7. The use according to claim 1, characterized in that: the enteric coated particles are used for up-regulating the expression of the tight junction proteins ZO-1, occidin-1 and Claudin-1.

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