Application of valeriana jatamansi jones extract in preparation of medicine for treating and preventing colitis

Abstract

The invention discloses an application of a valeriana jatamansi jones extract in preparation of a medicine for treating and preventing colitis, and provides an application of the valeriana jatamansi jones extract in preparation of a medicine for treating and preventing colitis, which discovers that the valeriana jatamansi jones extract can improve the intestinal function of a mouse with colitis and has an anti-colitis effect. The valeriana jatamansi jones extract with increased dosage increases the number of goblet cells of mice, improves the distribution of mucoprotein, and improves the damage condition of the intestinal mucous layer of the mice with colitis.

Description

Technical Field

[0001] The invention belongs to the technical field of drugs for treating colitis, and in particular relates to use of a spider fragrance extract in preparing drugs for treating and preventing colitis. Background Art

[0002] The traditional Chinese medicine spider incense is the dried rhizome and root of the spider incense Valeriana jatamansi Jones of the family Valeriana jatamansi Jones, which can regulate the nervous and digestive systems. Spider incense was first recorded in the "Southern Yunnan Materia Medica" in the Ming Dynasty in China. The "Chinese Pharmacopoeia" (2020 edition) points out that spider incense has the effects of regulating qi and relieving pain, digesting food and stopping diarrhea, dispelling wind and dampness, and calming the nerves. It is used to treat abdominal distension and pain, indigestion, diarrhea, dysentery, rheumatic pain, soreness of the waist and knees, and insomnia. Pharmacological studies have shown that spider incense has anti-inflammatory, analgesic and sedative effects.

[0003] Ulcerative colitis (UC) is an intestinal disease characterized by chronic recurrent inflammation, with abdominal pain, diarrhea and mucus, purulent stools as the main clinical manifestations. Colonoscopy shows intestinal mucosal congestion, edema, granular changes and ulcers. Histologically, it is characterized by widespread ulcer distribution, accompanied by neutrophil infiltration and epithelial cell necrosis. At present, the treatment methods for UC are mainly aminosalicylic acid preparations, immunosuppressants, hormones, biological agents, etc., such as tumor necrosis factor (TNF) inhibitors and Janus kinase inhibitors have been increasingly used to treat patients with ulcerative colitis, but about one-third of patients do not respond to initial treatment, half of patients lose response over time, and there are serious side effects, which bring great distress to patients. Traditional Chinese herbal medicines are increasingly attracting attention at home and abroad for their unique multi-component, multi-target and low side effects in the prevention, treatment and diagnosis of diseases. Therefore, finding effective, low-toxic and low-cost Chinese medicines for the treatment of UC based on Chinese medicine has become a research goal now and in the future. However, whether spider fragrance extract has a therapeutic effect on ulcerative colitis has not been reported yet. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems and provide a use of a spider fragrance extract in preparing a medicine for treating and preventing colitis.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The invention comprises the use of the spider fragrance extract in preparing medicine for treating and preventing colitis.

[0007] A pharmaceutical composition comprising a spider fragrance extract.

[0008] In the pharmaceutical composition, the spider fragrance extract is provided in the form of a monomer, or in the form of a plant extract containing the spider fragrance extract.

[0009] Furthermore, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier or excipient.

[0010] Furthermore, the pharmaceutically acceptable carrier or excipient is selected from at least one of a diluent, a disintegrant, a precipitation inhibitor, a surfactant, a glidant, a binder, a lubricant, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative, a stabilizer, a hydrating agent, an emulsification accelerator, a buffer, an absorbent, a colorant, a flavoring agent, a sweetener, an ion exchanger, a release agent, a coating agent, a flavoring agent and a coating material.

[0011] The pharmaceutical composition is used in preparing or treating colitis drugs.

[0012] In summary:

[0013] The present invention provides a use of a spider incense extract in preparing a medicine for treating and preventing colitis. It is found that the spider incense extract can improve the intestinal function of colitis mice and has an anti-colitis effect. An increased dose of the spider incense extract increases the number of goblet cells in mice, improves the distribution of mucin, and improves the damaged condition of the intestinal mucus layer in colitis mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a mouse collection location map provided by the present invention;

[0015] Among them: Figure (1A) Schematic diagram of the construction of DSS-induced colitis mouse model and the oral administration of spider incense;

[0016] Figure (1B) Disease activity index score of mice;

[0017] Figure (1C) Mouse body weight changes;

[0018] Figure (1D) Representative images of mouse colon length;

[0019] Figure (1E) Statistical graph of mouse colon length;

[0020] Figure (1F) Levels of inflammatory factors INF-, II-1β, and IL-6 in mouse serum;

[0021] Figure (1G) Levels of serum inflammatory factors INF-, II-1β, and IL-6 in mice;

[0022] Figure (1H) Levels of inflammatory factors INF-, II-1β, and IL-6 in mouse serum;

[0023] Figure 2is a mouse curve graph provided by an embodiment of the present invention;

[0024] in: Figure 2 (A) Hematoxylin-eosin immunofluorescence staining (40x) and Alzheimer's blue immunofluorescence staining (100x) of colon tissue sections of mice in each group, scale bar: 100m;

[0025] Figure 2 (B) The number of goblet cells in the tissue sections of each group of mice;

[0026] Figure 2 (C) Muc2 mRNA levels in colon tissue;

[0027] Figure 2 (D) Protein immunoblotting analysis of colon tissue revealed differential expression of Occludim, E-cadhein, and Z0-1 proteins;

[0028] Figure 2 (E) Statistical graph of Western blotting analysis results. DETAILED DESCRIPTION

[0029] The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0030] Case Study 1: Construction of Mouse Model

[0031] The experimental animals used were 6-8 week old male C57BL / 6J mice, weighing 20-22 g, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and kept in the Animal Center of Tianjin University of Traditional Chinese Medicine, license number SYXK (Tianjin) 2020-0005, SPF animal breeding room, sterilized standard mouse food and sterilized water free access during the experiment, temperature controlled at 24 ± 1 ° C, humidity controlled at 50 ± 5%, and lighting system set to 12 hours / day. All animal experiments were carried out in strict accordance with the "Guide for the Care and Use of Laboratory Animals", and all experimental protocols were approved by the Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (Tianjin, China).

[0032] Medicinal materials: The spider incense medicinal materials were purchased from Guizhou and identified by Professor Chang Yanxu of Tianjin University of Traditional Chinese Medicine as the roots and rhizomes of the spider incense Valeriana jatamansi Jones. of the Valeriana family.

[0033] Preparation of spider perfume extract: 1 kg of spider perfume medicinal materials are appropriately chopped, added to 10 L of pure water, soaked for 2 hours, heated under reflux for extraction for 2 hours, filtered through gauze to obtain the first extract, the residue is added to 8 L of pure water, heated under reflux for extraction for 2 hours, filtered through gauze to obtain the first extract, the two extracts are combined, recovered under reduced pressure to obtain a water extract concentrate, and freeze-dried to obtain 247 g of spider perfume extract with a yield of 24.7%.

[0034] The UC mouse modeling method is as follows: C57BL / 6J mice were adaptively fed for one week and adaptively fed for 7 days. 72 mice were randomly divided into 6 groups, with 12 mice in each group. The model group, positive drug group, and drug administration group were given sterile water containing 2.5% DSS to drink freely for 7 days to construct a colitis model. On the 8th to 10th day, sterile water was replaced for drinking. The specific grouping is shown in each embodiment. The blank group was given sterile water for drinking for 10 days. The daily liquid drinking amount of each group of mice was precisely controlled, 5mL / mouse / day.

[0035] 72 mice were randomly divided into 6 groups, 12 mice in each group. Blank control group (CON): normal drinking water, eating, and 0.5% CMC-Na was administered by gavage; DSS group (DSS): modeled according to the above method, 0.5% CMC-Na was administered by gavage; positive drug group (sulfasalazine): modeled according to the above method, sulfasalazine (500 mg / kg) was administered by gavage. The spider incense low, medium, and high dose groups were administered spider incense extract 2g / kg, 4g / kg, and 8g / kg by gavage, respectively.

[0036] During the experiment, the weight, diarrhea, and blood in the stool of the mice were observed and recorded every day, and the blood in the stool was observed daily or detected using a fecal occult blood qualitative detection kit. The disease activity index (DAI) of colitis mice was scored according to the method established by Kihara N et al. DAI score = (weight score + diarrhea score + blood in the stool score) / 3. The evaluation criteria are shown in Table 1.

[0037] Table 1 DAI scoring criteria

[0038]

[0039] The mice were fasted for 12 hours before sacrifice and were allowed to drink water freely. After isoflurane anesthesia, blood was collected from the eye sockets. After standing for 30 minutes, the serum was separated by low-speed centrifugation and stored in a -80°C refrigerator. After blood collection, the mice were fixed in a supine position, and the colon and cecum were carefully separated. The colon of each group was measured, recorded and photographed. 0.5 cm of colon was taken and fixed in 4% tissue fixative.

[0040] Implementation case 2: Colon histopathological analysis

[0041] The colon tissue was fixed with formalin solution for 48 hours, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (HE).

[0042] 1. Dewax the paraffin sections to water: put the sections into environmentally friendly dewaxing liquid I for 20 minutes, environmentally friendly dewaxing liquid II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, and 75% alcohol for 5 minutes, and wash with tap water.

[0043] Frozen section rewarming and fixation: Take the frozen sections out of the -20° refrigerator and restore them to room temperature. Fix them with tissue fixative for 15 minutes and then rinse with running water.

[0044] 2. Pretreatment: The sections were treated in high-definition constant stain pretreatment solution for 1 min.

[0045] 3. Hematoxylin staining: Stain the sections with hematoxylin solution for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, reblue with bluing solution, and rinse with running water.

[0046] 4. Eosin staining: Dehydrate the sections in 95% alcohol for 1 min, and then stain them in eosin solution for 15 s.

[0047] 5. Dehydration and sealing: The slices were placed in anhydrous ethanol I for 2 min, anhydrous ethanol II for 2 min, anhydrous ethanol III for 2 min, n-butanol I for 2 min, n-butanol II for 2 min, xylene I for 2 min, xylene II for 2 min, and sealed with transparent neutral gum.

[0048] 6. Microscopic examination, image acquisition and analysis. Observe the severity of tissue inflammation, lesion depth, inflammatory cell infiltration and epithelial destruction, and perform pathological scoring (histological index, HI) according to the method established by Melgar S et al.

[0049] Table 2 Scoring criteria for histopathological sections

[0050]

[0051] The colon tissue was fixed with formalin solution for 48 h, embedded in paraffin, sectioned, and stained with Alcian blue (AB).

[0052] 1. Dewax the paraffin sections to water: put the sections into environmentally friendly dewaxing liquid I for 20 minutes, environmentally friendly dewaxing liquid II for 20 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, and 75% alcohol for 5 minutes, and wash with tap water.

[0053] Frozen section rewarming and fixation: Take the frozen sections out of the -20° refrigerator and restore them to room temperature. Fix them with tissue fixative for 15 minutes and then rinse with running water.

[0054] Fixation of cell slides: discard the culture medium, wash with PBS three times, 5 minutes each time, add tissue fixative for 15 minutes, and then wash with distilled water.

[0055] 2. Alcian blue staining: Stain the sections in Alcian blue staining solution for 10-15 minutes, then wash with tap water. Add 1 ml of Alcian blue staining solution into the wells of the cell culture plate and stain for 15 minutes, then wash with distilled water.

[0056] 3. Nuclear staining: Dye with nuclear fast red stain for 3 minutes, wash with tap water. Add 1 ml of nuclear fast red stain into the wells of the cell culture plate for 3 minutes, and wash with distilled water.

[0057] 4. Dehydration and sealing: Put the slices into anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, anhydrous ethanol III for 5 minutes, xylene I for 5 minutes, xylene II for 5 minutes to make it transparent, and seal with neutral gum. Add anhydrous ethanol I to the cell culture plate for 5 minutes and then discard it; anhydrous ethanol II for 5 minutes and then discard it; anhydrous ethanol III for 5 minutes and then discard it. Take out the cover glass with cells and dry it, and seal it on the slide with the cell side facing down with neutral gum.

[0058] 5. Microscope examination, image acquisition and analysis.

[0059] The serum was taken out from the -80°C refrigerator and the IL-6, TFG-β, and IL-1β levels in the serum were detected by enzyme-linked immunosorbent assay (ELISA) according to the instructions. The corresponding antibodies were included in the kit and the operation was performed according to the instructions.

[0060] Implementation case 3: Western blot analysis

[0061] Weigh 20 mg of colon tissue on ice and place it in a pre-cooled 5 mL centrifuge tube. Add an appropriate amount of protein lysis buffer (TPEB: protease inhibitor: phosphatase inhibitor = 100:1:1) and homogenize on ice. Use an appropriate speed to homogenize up and down, 15 seconds each time, place on ice to cool, and homogenize three times in total. Stand on ice for 30 minutes, centrifuge at 12000×g, 4℃ for 15 minutes. Take out 25uL of supernatant and use the BCA protein quantification kit to detect protein concentration. The detection method is carried out according to the kit instructions.

[0062] The total protein loading amount of colon tissue homogenate is 20ug. Electrophoresis was performed at 100V voltage for about 1h until the bromophenol blue dye reached the bottom of the separation gel. The gel was removed, the excess part was cut off, and it was placed on a PVDF membrane that had been soaked in methanol in advance. The wet transfer method was used and the membrane was transferred at 200mA for 90min. After the transfer, the PVDF membrane was placed in 5% skim milk powder and blocked at room temperature for 3h. The blocking solution was poured out, the diluted primary antibody was added, and the shaking was incubated at 4℃ overnight. After washing with TBST, the secondary antibody solution was added and incubated at room temperature for 2h. After washing with TBST, the PVDF membrane was placed in the ECL luminescent solution for full contact, and the bands were photographed and analyzed in an automatic luminescent gel imager. The gray value of the target band was calculated and divided by the gray value of the control group so that the correction value of the Cont group was 1.

[0063] According to the data obtained from the above experiments, Graphpad Prism 8 software was used for statistical analysis. The data were expressed as mean ± standard deviation (meanSD). One-way ANOVA was used for comparison between groups. A p value less than 0.05 was considered to be a significant difference.

[0064] Experimental Results

[0065] Effects of spider perfume extract on disease phenotypes in mice with DSS-induced ulcerative colitis

[0066] After 2.5% DSS modeling, it was found that from the 4th day of modeling, the mice showed a significant trend of weight loss. Compared with the blank group, the weight of the mice in the model group decreased significantly from the 5th day (P < 0.05); similarly, the spider perfume extract groups at various doses and the sulfasalazine (SASP) group showed a phenomenon of weight loss. From the 8th day, the medium and high dose groups showed significant differences compared with the model group (P < 0.05). On the 10th day, it was found that all the dose groups showed a trend of weight recovery, but the weight of the model group was still decreasing. Figure 1 . The fecal occult blood kit is used to detect blood in the feces that cannot be directly judged by the naked eye, so as to determine whether there is a small amount of bleeding in the digestive tract. This experiment uses the chemical method of the Pyramide hole method for semi-quantitative detection of occult blood. It can be found from the DAI score that the score reached the highest on the 7th day of DSS drinking. At this time, the mice lost weight rapidly, had a lot of blood in the stool, severe diarrhea and other symptoms. When switched to drinking sterile water, the weight still showed a downward trend, but the symptoms of blood in the stool and diarrhea were reduced. Compared with the model group, the scores of the medium and high dose groups of spider incense showed significant differences after the 9th day (P < 0.05), and the SASP group also showed significant differences after the 7th day (P < 0.05). Figure 1After the mice were sacrificed, the part from the cecum to the rectum was taken for photographs. DSS can effectively shorten the length of the colorectum and cause redness in the intestine. The SASP group can effectively alleviate the shortening of the colorectum, but cannot alleviate the weight loss of colitis mice. The low, medium and high doses of spider perfume can significantly alleviate the shortening of the colorectum (P < 0.05). In contrast, spider perfume extract has the most significant protective effect on DSS-induced colitis, see Figure 1 .

[0067] Effects of spider perfume extract on inflammatory factors in mice with DSS-induced ulcerative colitis

[0068] ELISA was used to detect the levels of IL-6, IL-1β, and TGF-β in mouse serum. Compared with the control group, the levels of the three inflammatory factors in the model group increased significantly, indicating that the mice in the model group had a severe inflammatory response (P < 0.01). Compared with the model group, the SASP group, the medium-dose spider incense group, and the high-dose spider incense group could significantly reduce the levels of IL-6, IL-1β, and TGF-β (P < 0.05), and the low-dose spider incense group could significantly reduce the levels of IL-6 and IL-1β (P < 0.05). Although it could reduce the content of TGF-β, there was no significant difference, see Figure 1 .

[0069] Effects of spider perfume extract on colonic histopathology in mice with DSS-induced ulcerative colitis

[0070] HE staining and pathological observation and scoring of colon tissue showed that the ulcerative colitis mice induced by DSS had changes in colon morphology and structure, the integrity of colon tissue mucosa was destroyed, most of the glands were destroyed and arranged in disorder, and there was severe crypt destruction and inflammatory cell infiltration. The mice in the SASP group and the low, medium and high dose groups of spider incense significantly reduced the infiltration of inflammatory cells in the submucosal layer, which could effectively protect the mucosal structure, the glands were neatly arranged, and the histopathological score was significantly reduced (P < 0.05). Figure 2 Effects of spider perfume extract on colonic mucus layer in mice with DSS-induced ulcerative colitis

[0071] The mucus layer is the first barrier of the colon against harmful substances and microorganisms. The mucosa of the colon is composed of two layers, a solid inner layer (composed of polymerized MUC2) and a loose outer layer (formed by hydrolysis of MUC2 protein). The polymerized MUC2 network structure prevents microorganisms from invading the colon epithelium and plays an important role in maintaining intestinal homeostasis. MUC2 is a type of glycosylated protein produced by goblet cells in the intestine. Therefore, Alcian blue staining was used to analyze the distribution and quantity of mucin MUC2. The results showed that compared with the blank group, the colon mucus layer in the model group was significantly damaged, and mucin was significantly reduced. The SASP group and the low, medium and high dose groups of spider incense significantly increased the number of goblet cells, improved the distribution of mucin, and improved the damage to the intestinal mucus layer of colitis mice. Figure 2 .

[0072] Effects of spider perfume extract on tight junction proteins in intestinal mucosa of mice with DSS-induced ulcerative colitis

[0073] like Figure 2 As shown in the figure, Western Blot results of colon tissue showed that the expression of Occludin, E-cadherin and ZO-1 proteins in the model group was significantly reduced compared with the normal group, indicating that DSS-induced ulcerative colitis destroyed the tight junctions of colon epithelial cells, leading to damage of intestinal epithelial cells. The expression of Occludin, E-cadherin and ZO-1 proteins in the SASP group and the high-dose group of spider incense increased compared with the model group.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of spider fragrance extract in preparing medicine for treating and preventing colitis.

2. A pharmaceutical composition comprising a spider fragrance extract.

3. The pharmaceutical composition according to claim 2, wherein The spider fragrance extract is provided in the form of a monomer, or in the form of a plant extract containing the spider fragrance extract.

4. The pharmaceutical composition according to any one of claims 2 to 3, wherein The pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

5. The pharmaceutical composition according to claim 4, wherein The pharmaceutically acceptable carrier or excipient is selected from at least one of a diluent, a disintegrant, a precipitation inhibitor, a surfactant, a glidant, a binder, a lubricant, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative, a stabilizer, a hydrating agent, an emulsification accelerator, a buffer, an absorbent, a colorant, a flavoring agent, a sweetener, an ion exchanger, a release agent, a coating agent, a flavoring agent, and a coating material.

6. Use of the pharmaceutical composition according to any one of claims 2 to 5 in the preparation or treatment of colitis drugs.

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