Application of aconite polysaccharide in preparing medicine for treating inflammatory bowel disease and intestinal mucositis

Abstract

The present invention relates to the field of medical technology, and discloses the use of aconite polysaccharide in the preparation of drugs for treating inflammatory bowel disease and intestinal mucositis. The present invention conducts a series of studies on aconite polysaccharide using a mouse model, and finds that aconite polysaccharide can significantly improve the intestinal microbial diversity of mice, and at the same time, can improve intestinal flora disorder and make it tend to normal, and provides a new use of aconite polysaccharide-the use in the preparation of drugs for treating inflammatory bowel disease and intestinal mucositis. For example, aconite polysaccharide is used for drug treatment of ulcerative colitis mouse model and 5-Fu-induced intestinal mucositis mouse model, and both can significantly improve their pathological damage.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to application of aconite polysaccharide in preparing medicines for treating inflammatory bowel disease and intestinal mucositis. Background Art

[0002] Aconite is a processed product of the root of Aconitum Carmichaeli Dexb, a plant of the Ranunculaceae family. It has the functions of restoring yang and rescuing adverse conditions, replenishing fire and supporting yang, dispersing cold and relieving pain. It is used for yang-deficiency collapse, cold limbs and weak pulse, impotence, cold uterus, cold pain in the heart and abdomen, vomiting and diarrhea due to deficiency and cold, and edema due to yin and cold. "Tangye Bencao" mentioned: "Aconite is a drug that enters the Shaoyang triple burner and the gate of life. It floats in the middle and reaches everywhere. It is pungent and hot. It is the yang among the yang"; "Bencao Jingdu" also mentioned: "Aconite is pungent and warm in temperature. It has a quick fire nature and reaches everywhere. Therefore, it is the first-class medicine for restoring yang and rescuing adverse conditions"; "Compendium of Materia Medica" records: "Aconite is heavy and stagnant in nature, warms the spleen and drives away cold". In modern times, aconite is often used to treat chronic heart failure, shock, rheumatoid arthritis, enteritis and diarrhea.

[0003] The main chemical components of Radix Aconiti Lateralis are alkaloids, polysaccharides, glycosides, etc. The research on Radix Aconiti Lateralis at home and abroad mainly focuses on the alkaloid components, while the research on polysaccharides in Radix Aconiti Lateralis is relatively small. Polysaccharides are a class of natural high-molecular compounds with multiple physiological activities. Chinese medicine polysaccharides have become one of the key directions of current drug research and health care product development. Existing studies have shown that Radix Aconiti Lateralis polysaccharides have strong physiological activity and no obvious toxicity. They have the effects of regulating immunity, anti-tumor, protecting myocardial cells, and lowering blood lipids. Their high efficiency and low toxicity have gradually attracted people's attention.

[0004] Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is a group of chronic intestinal inflammatory diseases of unknown cause. Due to the increasing incidence, it has become a common digestive system disease in my country and is also listed as one of the modern intractable diseases by the WHO. At present, the mainstream clinical treatments include aminosalicylate, corticosteroids, immunosuppressants and biological agents, but there are still many problems such as limited efficacy, high recurrence rate, long treatment cycle, easy to develop drug resistance, and often accompanied by serious side effects. Therefore, it is still of great clinical value to find cheaper and less toxic, efficient and orally administrable natural medicines. Traditional Chinese medicine has the advantages of less toxic side effects, low cost, multiple targets and multiple pathways, and has great clinical application prospects. Therefore, it is of great social and economic significance to further explore efficient and low-toxic traditional Chinese medicines and their effective ingredients in the field of UC treatment and fully clarify their mechanisms of action.

[0005] Intestinal mucositis (IM) is also defined as structural, functional and immunological changes caused by inflammation of the mucosa lining the digestive tract. IM is clinically manifested by diarrhea, abdominal pain, blood in the stool, nausea and vomiting, and various serious complications may also occur. The onset of these symptoms, the cycle of onset and the clinical manifestations are associated with factors such as the patient's age, race and gender. At the same time, the symptoms of IM depend on the type and course of the cancer being treated. Intestinal mucositis is divided into the following five stages: initiation, primary mucosal injury, inflammatory response signal amplification, ulceration and initiation of healing. Apoptosis, inflammatory cytokines, intestinal flora, direct cytotoxicity and reactive oxygen species are considered to be key pathogenic determinants. Summary of the invention

[0006] In order to solve the technical problems of developing the above-mentioned drugs for treating inflammatory bowel disease and intestinal mucositis, the present invention provides the use of aconite polysaccharide in preparing drugs for treating inflammatory bowel disease and intestinal mucositis. The present invention has conducted a series of studies on aconite polysaccharide and found that aconite polysaccharide can regulate intestinal flora, provides the use of aconite polysaccharide in preparing drugs for treating inflammatory bowel disease and intestinal mucositis, and provides a new use of aconite polysaccharide.

[0007] The specific technical scheme of the present invention is:

[0008] The present invention provides a new use of aconite polysaccharide: use of aconite polysaccharide in preparing a medicine for treating inflammatory bowel disease or intestinal mucositis.

[0009] The researchers of the present invention team conducted a series of studies on aconite polysaccharides using the most commonly used mammalian model mouse model. On the one hand, it was found that feeding aconite polysaccharides to mice with ulcerative colitis can significantly inhibit the shortening of the colon and weight loss of mice with ulcerative colitis, significantly improve the DAI score, and significantly restore the damaged colon pathological tissue; on the other hand, it was found that feeding aconite polysaccharides to mice with 5-Fu-induced intestinal mucositis can reduce the weight loss and shortening of colon length of mice with 5-Fu-induced intestinal mucositis, improve the diarrhea score of mice with intestinal mucositis, and significantly improve the symptoms of mice with intestinal mucositis and pathological damage of the small intestine and colon. Based on this, the researchers of the present invention team conducted in-depth research on the mechanism of aconite polysaccharide treatment to improve inflammatory bowel disease and intestinal mucositis. By feeding mice with aconite polysaccharides, it was found that aconite polysaccharides can significantly improve the intestinal microbial diversity of mice, and at the same time, it can improve intestinal flora disorders and make them tend to normal.

[0010] The researchers of the present invention team conducted a series of studies on aconite polysaccharides using a mouse model, indicating that aconite polysaccharides can be effectively used in the preparation of drugs for the treatment of inflammatory bowel disease and / or intestinal mucositis, providing a new use of aconite polysaccharides.

[0011] The present invention also provides a traditional Chinese medicine composition for treating inflammatory bowel disease or intestinal mucositis, wherein the traditional Chinese medicine composition comprises aconite polysaccharide.

[0012] The present invention also provides a traditional Chinese medicine composition for treating inflammatory bowel disease or intestinal mucositis, comprising aconite polysaccharide. Based on the new use of aconite polysaccharide provided by the present invention in preparing a drug for treating inflammatory bowel disease and / or intestinal mucositis, those skilled in the art, after knowing the new use of aconite polysaccharide, can add it to the traditional Chinese medicine composition without any creative work, and add it as an adjuvant to obtain the traditional Chinese medicine composition for treating inflammatory bowel disease or intestinal mucositis.

[0013] Aconite polysaccharide can be added with pharmaceutically acceptable excipients to prepare one of the following dosage forms: decoction, tea, granules, ordinary tablets, dispersible tablets, effervescent tablets, orally disintegrating tablets, lozenges, chewable tablets, capsules, soft capsules, microcapsules, pills, powders, pellets, sustained-release preparations, controlled-release preparations, oral liquid preparations and injections.

[0014] Specifically, the present invention also provides a method for extracting aconite polysaccharide, comprising the following steps:

[0015] (1) Add 6 to 10 times the volume of ethanol solution to the powdered aconite root, reflux and extract for 1 to 2 hours, filter and take the residue for later use;

[0016] (2) Taking the filter residue of step (1) and adding 6 to 10 times the amount of water by volume, reflux extraction for 1 to 2 hours and then centrifuging, retaining the supernatant, repeating the reflux extraction and centrifugation operations for 2 to 3 times, mixing the supernatants obtained in each time and concentrating to 1 to 3 times the amount of the feed, then adding 2 to 5 times the amount of anhydrous ethanol while stirring, standing, filtering, and drying the filter cake to obtain aconite polysaccharide.

[0017] The present invention extracts and prepares aconite polysaccharide by an alcohol precipitation and water-soluble method, and the preparation process is simple and easy to operate. Specifically, step (1) removes the lipid components of aconite powder by alcohol reflux extraction, and then extracts aconite polysaccharide from aconite by reflux in step (2) water. The alcohol precipitation and water-soluble method of the present invention can extract the aconite polysaccharide, a water-soluble component in aconite, by alcohol extraction and water extraction, and separate alcohol-soluble components, such as most lipids, while extracting the aconite polysaccharide, a water-soluble component in aconite, so as to obtain an extract with higher purity, which is conducive to further separation and purification, and at the same time, the extraction efficiency is higher.

[0018] As a preferred embodiment of the above technical solution, in step (1), the volume concentration of the ethanol solution is 95%.

[0019] As a preferred embodiment of the above technical solution, in step (2), the concentration method is reduced pressure concentration.

[0020] As a preferred embodiment of the above technical solution, in step (2), the standing time is 10 to 15 hours and the temperature is 0 to 5°C.

[0021] After adding anhydrous ethanol to the supernatant, the supernatant is allowed to stand at a temperature of 0 to 5°C for 10 to 15 hours, which is beneficial to improving the separation efficiency and the purity of aconite polysaccharide.

[0022] At the same time, the present invention also provides a method for purifying the above-mentioned aconite polysaccharide, comprising the following steps:

[0023] Take aconite polysaccharide, add water to dissolve it, then add a mixed solution of dichloromethane and n-butanol, centrifuge, remove the lower layer and the middle layer, repeat the operation 4 to 7 times, take the upper aqueous solution and freeze-dry it to obtain purified aconite polysaccharide.

[0024] As a preferred embodiment of the above technical solution, the amount of water added is 2 to 3 times the amount of aconite polysaccharide by volume.

[0025] As a preferred embodiment of the above technical solution, the volume ratio of dichloromethane to n-butanol in the mixed solution is 5 to 4:1.

[0026] As a preferred embodiment of the above technical solution, the volume ratio of the mixed solution of dichloromethane and n-butanol to water is 1:1 to 1.5.

[0027] Compared with the prior art, the present invention has the following technical effects:

[0028] The present invention conducted a series of studies on aconite polysaccharide using a mouse model, and found that aconite polysaccharide can significantly improve the intestinal microbial diversity of mice, and at the same time, can improve intestinal flora disorders and make them tend to normal, and provided a new use of aconite polysaccharide - application in the preparation of drugs for treating inflammatory bowel disease and intestinal mucositis. For example, aconite polysaccharide is used for drug treatment of ulcerative colitis mouse model, which can significantly inhibit the shortening of the colon and weight loss of ulcerative colitis mice, significantly improve the DAI score, and significantly restore the damaged colon pathological tissue; it is used for the treatment of 5-Fu-induced intestinal mucositis mouse model, which can reduce the weight loss and shortening of colon length of 5-Fu-induced intestinal mucositis mice, improve the diarrhea score of intestinal mucositis mice, and significantly improve the symptoms of intestinal mucositis mice and pathological damage of the small intestine and colon.

[0029] The present invention also provides a method for preparing aconite polysaccharide, wherein the lipid components of aconite powder are removed by alcohol reflux extraction, and then the aconite polysaccharide is extracted from the aconite by reflux in water. The alcohol precipitation and water dissolution method of the present invention can extract the aconite polysaccharide, a water-soluble component in aconite, and separate the alcohol-soluble components, such as most lipids, while extracting the aconite polysaccharide, a water-soluble component in the aconite, through alcohol extraction and water extraction in sequence, so as to obtain an extract with higher purity, which is beneficial to further separation and purification, and at the same time, the extraction efficiency is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a diagram showing the structural identification results of aconite polysaccharide in Example 4 of the present invention;

[0031] Figure 2 This is a graph showing the therapeutic effect of aconite polysaccharide on DSS-induced UC mice in Example 5 of the present invention;

[0032] Figure 3 This is a graph showing the therapeutic effect of aconite polysaccharide in Example 6 of the present invention on mice with intestinal mucosal inflammation induced by 5-fluorouracil;

[0033] Figure 4 This is a graph showing the therapeutic results of aconite polysaccharide in Example 7 of the present invention on pathological lesions of the small intestine and colon in mice with mucositis;

[0034] Figure 5 This is a diagram showing the results of the analysis of the diversity of differential microbial communities in Example 8 of the present invention;

[0035] Figure 6 This is a diagram of the flora analysis results in Example 8 of the present invention. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with embodiments and drawings.

[0037] Example 1 Preparation of Aconite Polysaccharide

[0038] 1.1 Extraction:

[0039] By volume, add 8 times the amount of 95% ethanol solution to the aconite powder in the extraction bottle, reflux extraction for 1.5 hours, and filter; add 8 times the amount of 95% ethanol solution to the residue, continue to reflux extraction for 1.5 hours, filter, and take the residue for later use.

[0040] Take the above-mentioned filter residue, add 8 times the amount of water, reflux extraction for 1.5 hours, centrifuge, and take the supernatant; repeat this operation 3 times and combine the supernatants of 3 times.

[0041] The supernatant was concentrated under reduced pressure to twice the amount of the feed, and 3 times the amount of anhydrous ethanol was added while stirring. The mixture was allowed to stand at 4°C for 12 hours, taken out, filtered, and the filter cake was dried to obtain crude aconite polysaccharide.

[0042] 1.2 Purification:

[0043] Take the crude aconite polysaccharide obtained in step 1.1, add water to dissolve it, then add a mixed solution of dichloromethane and n-butanol in a volume ratio of 5:1, the volume ratio of the crude aconite polysaccharide, water, and the mixed solution is 1:2:2, centrifuge, remove the lower organic layer and the middle layer, repeat the operation 6 times until the protein is completely removed, and freeze-dry the upper aqueous solution to obtain aconite polysaccharide ACP.

[0044] Example 2 Preparation of Aconite Polysaccharide

[0045] 2.1 Extraction:

[0046] According to the volume fraction, add 6 times the amount of 95% ethanol solution to the aconite powder in the extraction bottle, reflux extraction for 1 hour, and filter; add 6 times the amount of 95% ethanol solution to the residue, continue to reflux extraction for 1 hour, filter, and take the residue for later use.

[0047] Take the above-mentioned filter residue, add 10 times the amount of water, reflux extraction for 1 hour, centrifuge, and take the supernatant; repeat this operation 4 times and combine the 4 supernatants.

[0048] The obtained supernatant was concentrated under reduced pressure to 3 times the amount of the feed, and 3 times the amount of anhydrous ethanol was added while stirring, and the mixture was allowed to stand at 0°C for 15 hours, taken out, filtered, and the filter cake was dried to obtain crude aconite polysaccharide.

[0049] 2.2 Purification:

[0050] Take the crude polysaccharide of aconite obtained in step 2.1, add water to dissolve it, then add a mixed solution of dichloromethane and n-butanol in a volume ratio of 5:1, the volume ratio of crude polysaccharide of aconite, water, and mixed solution is 1:2:3, centrifuge, remove the lower organic layer and the middle layer, repeat the operation 4 times until the protein is completely removed, and freeze-dry the upper aqueous solution to obtain aconite polysaccharide ACP.

[0051] Example 3 Preparation of Aconite Polysaccharide

[0052] 3.1 Extraction:

[0053] According to the volume ratio, add 10 times the amount of 95% ethanol solution to the aconite powder in the extraction bottle, reflux and extract for 2 hours, filter and take the residue for later use.

[0054] Take the above-mentioned filter residue, add 6 times the amount of water, reflux extraction for 2 hours, centrifuge, and take the supernatant; repeat this operation 5 times and combine the supernatants of 5 times.

[0055] The obtained supernatant was concentrated under reduced pressure to twice the amount of the feed, and 5 times the amount of anhydrous ethanol was added while stirring, and the mixture was allowed to stand at 5°C for 10 hours, taken out, filtered, and the filter cake was dried to obtain crude aconite polysaccharide.

[0056] 3.2 Purification:

[0057] Take the crude aconite polysaccharide obtained in step 3.1, add water to dissolve it, then add a mixed solution of dichloromethane and n-butanol in a volume ratio of 4:1, the volume ratio of the crude aconite polysaccharide, water, and the mixed solution is 1:3:3, centrifuge, remove the lower organic layer and the middle layer, repeat the operation 7 times until the protein is completely removed, and freeze-dry the upper aqueous solution to obtain aconite polysaccharide ACP.

[0058] Example 4 Structural Identification of Aconite Polysaccharide

[0059] The prepared aconite polysaccharide in Example 1 was analyzed by FT-IR spectrum, monosaccharide composition spectrum, chromatography, and HPGP spectrum. Figure 1 , where A is the FT-IR spectrum of ACP, B is the monosaccharide composition spectrum of ACP, C is the chromatogram of the monosaccharide standard, and D is the HPGP spectrum of ACP. Table 1 summarizes the peak attributes.

[0060] like Figure 1 The AFT-IR spectrum showed that the ACP was at 3432.67, 2929.34 and 1633.41 cm -1 The stretching vibrations of OH, CH and CO show the characteristic absorption of polysaccharides. -1 The absorption at is attributed to COC and COH in the pyranose ring. Figure 1 B, C and as shown in Table 2, the monosaccharide composition of ACP is mainly composed of arabinose, galactose, glucose, galacturonic acid and mannuronic acid with a molar ratio of 9.64:4.92:22.8:1.64:1.00. Figure 1 The HPGP spectrum of D showed that ACP had a single symmetrical peak, which meant that ACP was a homogeneous polysaccharide with a weight-average molecular weight (Mw) of 1.61×10 5 Da (Mw / Mn=1.01).

[0061] Table 1 Properties of peaks in FT-IR absorption spectra

[0062]

[0063]

[0064] Table 2 Monosaccharide composition of ACP

[0065]

[0066] Example 5 Effects of ACP on Body Weight Change, DAI Score and Pathological Lesions in DSS-Induced UC Mice

[0067] Experimental methods: Thirty (20±2g) male C57BL / 6SPF mice, 6 mice in each group, were randomly divided into 5 groups: control group, model group, SASP group (SASP administration 200mg / kg), ACP group (ACP administration 50mg / kg) and ACP group (ACP administration 100mg / kg). Except for the control group, all mice freely drank 3% DSS solution for 7 days to induce UC. At the same time, the corresponding drugs were gavaged twice a day at a dose of 0.1mL / 10g for 7 consecutive days. The body weight, stool and other abnormal conditions of the mice were recorded daily, and the disease activity index (DAI) score was obtained in a blind manner. On the 8th day, the mice were killed for cervical dislocation and anatomical sampling. The colon was separated, photographed and weighed (unit: grams). The contents of the colon were removed and the colon was divided into two. One part was immediately frozen in liquid nitrogen, and the other part was fixed with 4% paraformaldehyde, embedded, sliced, stained and photographed under a microscope. Pathological changes such as edema, adhesion, ulcer, and necrosis were observed, and histological scores were evaluated.

[0068] Experimental results: The experimental results are as follows Figure 2 As shown, A is the curve of colon length change, B is the curve of body weight change, C is the curve of DAI change, D is the representative picture of H&E staining of colon sections, and E is the morphological score of colon sections. Each point is expressed as mean ± standard deviation (n = 6). Compared with the control group, # p<0.05, ### p<0.001; compared with the model, *p<0.05, **p<0.01, ***p<0.001. (SASP 200mg / kg represents the SASP group (SASP administration 200mg / kg), ACP 50mg / kg represents ACP (ACP administration 50mg / kg), ACP 100mg / kg represents the ACP group (ACP administration 100mg / kg))

[0069] Depend on Figure 2 A and Figure 2 B shows that ACP can significantly inhibit the shortening of the colon and the weight loss of mice, especially in the ACP group (ACP administration 100 mg / kg); Figure 2 C shows that ACP significantly improved the DAI score; Figure 2 D and E show that ACP can significantly restore the damaged colon pathological tissue. In summary, ACP can significantly improve the symptoms and colon pathological damage of UC mice.

[0070] Example 6 Therapeutic effect of ACP on 5-Fu-induced intestinal mucositis in mice Experimental method: SPF-grade C57BL / 6 mice, purchased from Zhejiang Provincial Experimental Animal Center, male, weight: 18-22g. After 1 week of adaptive feeding, 24 C57BL / 6 mice were randomly divided into 4 groups, 6 in each group: control group, model group, ACP 50mg / kg group and ACP 100mg / kg group. Except for the control group, all mice freely drank 5-FU (25mg / mL) solution for 4 days to induce IM. During the entire 4 days, the drug was administered intragastricly at a volume of 10μL·g-1 body weight, once in the morning and once in the evening. Observe and record the diarrhea of ​​mice 24h after the first modeling, and the degree of diarrhea can be measured by the diarrhea score. After the mice were killed on the 5th day, the ileum and colon tissues were immediately collected and then fixed with 4% buffered paraformaldehyde. Among them, the ACP 50mg / kg group was administered with ACP at a dosage of 50mg / kg, and the ACP 100mg / kg group was administered with ACP at a dosage of 100mg / kg.

[0071] Experimental results: The experimental results are as follows Figure 3 As shown, A is the curve of mouse weight change, B is a representative picture of colon morphology change, C is the curve of colon length change, and D is the statistical result of diarrhea index. Each point is expressed as mean ± standard deviation (n = 6). Compared with the control group, # p<0.05, ### p<0.001; *p<0.05, **p<0.01, ***p<0.001 compared with the model.

[0072] Depend on Figure 3 A shows that ACP (50 and 100 mg / kg) groups can reduce the weight loss of mice with intestinal mucosal inflammation induced by 5-Fu; Figure 3 B and C show that ACP (50 and 100 mg / kg) groups can inhibit the shortening of colon length; Figure 3 D shows that ACP (50 and 100 mg / kg) groups can significantly improve the diarrhea scores of mice. In summary, ACP (50 and 100 mg / kg) can significantly improve the symptoms of intestinal mucositis mice and pathological damage of the small intestine and colon.

[0073] Example 7 ACP improves ileum and colon tissues of mice with 5-Fu-induced intestinal mucosal inflammation Experimental method: The treatment of mice was the same as in "Experimental Example 6". After cervical dislocation, the colon tissues of all mice were separated, measured and photographed as soon as possible, the colon contents were taken out, and the colon was divided into two. One part was immediately frozen in liquid nitrogen, and the other part was fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm thick sections, and stained with hematoxylin-eosin (HE). Images of each intestinal segment were taken under an optical microscope (Olympus, Tokyo, Japan) (magnification 20 times). The villi and crypts were measured, the ileal villus height (VH) and the colon crypt depth (CD) were calculated, and the pathological damage of the samples was analyzed. Pathological changes such as edema, adhesions, ulcers, and necrosis were evaluated, and histological scores were evaluated.

[0074] Experimental results: The experimental results are as follows Figure 4 As shown, A is a representative picture of H&E staining of small intestine sections of 4 groups of mice in Example 6, B is a representative picture of H&E staining of colon sections of mice, C is the height of ileal villi, D is the depth of ileal crypts, and E is the depth of colonic crypts. Each point is expressed as mean ± standard deviation (n = 6). Compared with the control group, ## p<0.01, ### p<0.001; *p<0.05, **p<0.01, ***p<0.001 compared with the model.

[0075] Depend on Figure 4 A and Figure 4 As shown in B, the control group mice did not show any inflammatory cell infiltration in the ileum and colon, the crypt structure remained intact and arranged in an orderly manner, no inflammatory cell infiltration was found, the crypt structure remained intact, and the arrangement was regular and orderly; in the small intestine and colon tissues of the model group mice, epithelial cells were necrotic, the crypt structure was destroyed, and a large number of infiltrating inflammatory cells infiltrated into the mucosal layer; compared with the model group, the ACP (50, 100 mg / kg) group showed less inflammatory cell infiltration, more complete tissue structure, more regular cell arrangement, and the morphology and distribution of ileum and colon epithelial cells and crypts were restored. Figure 4 As shown in C, the height of ileal villi in the model group mice was significantly reduced, while the height of ileal villi in the ACP group mice was significantly restored. Figure 4 D and Figure 4 As shown in E, the crypt depths of the ileum and colon of mice in the model group were significantly reduced, while the crypt depths of mice in the ACP group were significantly restored. In summary, ACP can repair 5-FU-induced intestinal mucosal damage and improve the histopathological score of intestinal mucositis.

[0076] Example 8 ACP regulates intestinal microbiota disturbance in mice with 5-Fu-induced intestinal mucosal inflammation

[0077] Experimental methods:

[0078] ① DNA extraction: DNA of the four groups of mice in Example 6 was extracted using the EZNA feces DNA kit, and total DNA was eluted using 50 μL elution buffer for PCR detection.

[0079] ②PCR amplification and 16S rRNA sequencing: Primers 341F (5'-CCTACGGGNGGCWGCAG-3') and 805R (5'-GACTACHVGGGTATCTAATCC-3') (where N, W, H, and V are annexed bases, representing N: A / T / C / G; W: A / T; H: A / T / C; V: G / A / C) were used to amplify the V4 region of the 16S rRNA gene. PCR amplification was performed using 25 ng of template DNA, 12.5 μL of PCR premix, and 2.5 μL of each primer.

[0080] ③Data analysis: Samples were sequenced on the Illumina HiSeq platform. Paired end reads were assigned, truncated, and merged using FLASH (version 1.2.8). Chimeric sequences were screened, and 97% similarity was assigned to the same operational taxonomic unit (OTU) using Vsearch software (v2.3.4). Representative sequences were selected for each OTU, and classification data were assigned to each representative sequence using the Ribosomal Database Project (RDP) classifier. Differences in dominant species in different groups were determined using mafft software (V7.310).

[0081] Experimental results: The experimental results are as follows Figures 5-6 As shown in Tables 3 to 5. Figure 5 These are the results of the differential microbial community diversity analysis, where A is the Plelou_e curve, B is the Observed_otus curve, C is the Shannon curve, D is the Chao1 curve, E is the Simpson curve, and F is the Goods_coverage curve. Figure 6 Figure 2 is the result of microbiota analysis, where A is a Venn diagram, B is a principal component analysis diagram, C is a principal coordinate analysis diagram, D is a non-metric multidimensional scaling diagram, E is a cluster analysis of changes in intestinal microbiota at the phylum level, and F is a cluster analysis of intestinal microbiota at the genus level.

[0082] (1) By Figure 5 A and Table 3 show that 5-Fu inhibited the microbial richness compared with the control group. Figure 5 B shows that the observed species index results show that the α diversity between the control group and the model group decreased, and ACP (50 mg / kg) can improve the diversity of intestinal flora to a certain extent. Figure 5 C. Figure 5 D. Figure 5E shows that ACP (50 mg / kg) can improve the diversity of intestinal flora to a certain extent, and Goods_coverage also reaches a plateau. These results indicate that ACP (50 mg / kg) can significantly improve the intestinal microbial diversity of mice with 5-Fu-induced intestinal mucosal inflammation.

[0083] Table 3 α-diversity analysis results

[0084] Control Model ACP_25 Observed_otus 589.17±85.74 559.17±56.80 715.83±186.84 Shannon 6.05±0.94 5.99±0.84 6.53±0.93 Simpson 0.92±0.07 0.93±0.07 0.95±0.06 Chao1 592.67±86.49 561.45±57.22 720.24±188.20 Goods_coverage 1.00±0.00 1.00±0.00 1.00±0.00 Pieloué 0.66±0.09 0.66±0.09 0.69±0.07

[0085] (2) By Figure 6 A Venn diagram showed that there were 537 OTU overlaps in all groups, 711 OTU overlaps in the control group and the model group, and 729 OTU overlaps in the control group and the ACP group; in addition, the number of individual OTUs in the control group, the model group, and the ACP group were 1666, 722, and 786, respectively, suggesting that ACP can improve the diversity of the microbiota to a certain extent. Figure 6 B) Principal coordinate analysis ( Figure 6 C) and nonmetric multidimensional scaling (NMDS) ( Figure 6 The results of D) all showed that the isolated clusters between the control group and the model group were more clearly separated, indicating that the OTUs were very different. However, the ACP group tended to be closer to the control group, suggesting that ACP could improve the intestinal flora disorder to a certain extent. Figure 6 E shows that, in addition, the model group and the control group showed significant changes at the phylum level and the genus level, and ACP reversed the above changes. Tables 4 and 5 show that ACP can significantly improve the changes in various flora caused by 5-FU at the phylum and genus levels, indicating that ACP significantly improved the intestinal flora disorder of IM mice.

[0086] Table 4 Cluster analysis results at the phylum (p_) level

[0087] Phylum Control Model ACP_25 Firmicutes 53.95 22.53 41.84 Bacteroidota 31.53 41.16 33.21 Proteobacteria 3.82 22.18 14.60 Verrucomicrobiota 0.13 7.68 0.52 Fusobacteriota 0.11 0.09 0.23

[0088] Table 5 Cluster analysis results at the genus (g_) level

[0089] Genus Control Model ACP_25 Muribaculaceae_unclassified 19.63 10.84 13.84 Escherichia-Shigella 1.06 16.03 8.88 Lachnospiraceae_NK4A136_group 7.75 1.51 12.86 Clostridiales_unclassified 6.45 3.01 8.90 Parabacteroides 0.93 8.58 6.27 Alloprevotella 1.69 5.80 1.45 Bacteroides 1.19 5.76 1.67 Akkermansia 0.12 7.66 0.52 Lachnospiraceae_unclassified 2.47 1.39 3.93 Klebsiella 0.07 1.99 0.26

[0090] The present invention conducted a series of studies on aconite polysaccharide using a mouse model, and found that aconite polysaccharide can significantly improve the intestinal microbial diversity of mice, and at the same time, can improve intestinal flora disorders and make them tend to normal, and provided a new use of aconite polysaccharide - application in the preparation of drugs for treating inflammatory bowel disease and intestinal mucositis. For example, aconite polysaccharide is used for drug treatment of ulcerative colitis mouse model, which can significantly inhibit the shortening of the colon and weight loss of ulcerative colitis mice, significantly improve the DAI score, and significantly restore the damaged colon pathological tissue; it is used for the treatment of 5-Fu-induced intestinal mucositis mouse model, which can reduce the weight loss and shortening of colon length of 5-Fu-induced intestinal mucositis mice, improve the diarrhea score of intestinal mucositis mice, and significantly improve the symptoms of intestinal mucositis mice and pathological damage of the small intestine and colon.

[0091] Based on the new use of aconite polysaccharide provided by the present invention in the preparation of a drug for treating inflammatory bowel disease and / or intestinal mucositis, those skilled in the art, after knowing the new use of aconite polysaccharide, can add it to a traditional Chinese medicine composition as an adjuvant without any creative work to obtain a traditional Chinese medicine composition for treating inflammatory bowel disease or intestinal mucositis.

[0092] The 95% ethanol solution used in the present invention is an ethanol solution with a volume fraction of 95%.

[0093] The aconite polysaccharide used in Examples 5 to 8 of the present invention is the aconite polysaccharide extracted, purified and structurally confirmed in the present invention.

[0094] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0095] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. Use of aconite polysaccharide in the preparation of a medicament for treating inflammatory bowel disease or intestinal mucositis, characterized in that: The extraction method of aconite polysaccharide comprises the following steps: (1) Add 6-10 times the volume of ethanol solution to the powdered aconite root, reflux and extract for 1-2 hours, filter and take the residue for later use; wherein the volume concentration of the ethanol solution is 95%; (2) Taking the filter residue of step (1), adding 6 to 10 times the amount of water by volume, reflux extraction for 1 to 2 hours, centrifuging, retaining the supernatant, repeating the reflux extraction and centrifugation operation for 2 to 3 times, mixing the supernatants obtained in each time, and concentrating to 1 to 3 times the amount of the feed, then adding 2 to 5 times the amount of anhydrous ethanol while stirring, standing, filtering, and drying the filter cake to obtain aconite crude polysaccharide; The method for purifying crude aconite polysaccharide comprises the following steps: Take the crude polysaccharide of Aconitum carmichaelii, add water to dissolve it, then add a mixed solution of dichloromethane and n-butanol, centrifuge, remove the lower layer and the middle layer, repeat the operation 4 to 7 times, take the upper aqueous solution and freeze-dry it to obtain purified Aconitum carmichaelii polysaccharide.

2. The use according to claim 1, characterized in that: In step (2), the concentration method is concentration under reduced pressure.

3. The use according to claim 1, characterized in that: In step (2), the standing time is 10 to 15 hours and the temperature is 0 to 5°C.

4. The use according to claim 1, characterized in that: In the purification method, the amount of water added is 2 to 3 times the amount of aconite polysaccharide by volume.

5. The use according to claim 1, characterized in that: In the purification method, the volume ratio of dichloromethane to n-butanol is 5-4:

1.

6. The use according to claim 1, characterized in that: The dosage form of the medicine is tea, granules, tablets, capsules, pills, powders, sustained-release preparations, controlled-release preparations, oral liquid preparations and injections.

7. The use according to claim 6, characterized in that: The tablets are dispersible tablets, effervescent tablets, orally disintegrating tablets, lozenges, and chewable tablets.

8. The use according to claim 6, characterized in that: The capsule is a soft capsule or a microcapsule.

9. The use according to claim 6, characterized in that: The pills are drop pills.

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