Use of an alkaloid compound in the preparation of a drug for resisting colitis

Abstract

The application belongs to the technical field of effective components of traditional Chinese medicine, and particularly relates to application of an indole alkaloid compound in preparation of an anti-colitis medicine, a structure formula of the alkaloid compound is shown as formula I, and it is found that Isovoacangine has a good treatment effect on colitis, and the specific performance is that, compared with a model group, the body weight of an Isovoacangine group is obviously increased, a DAI index is obviously improved, the length and weight of the colon are obviously increased, the damage degree is lower, the morphology of the colon tissue is obviously recovered, the IL-1beta, IL-6 and TNF-alpha contents of the colon tissue are obviously reduced, and the IL-10 content is increased.

Description

Technical Field

[0001] This invention belongs to the technical field of the use of effective components of traditional Chinese medicine, specifically relating to the application of an indole alkaloid compound in the preparation of anti-colitis drugs. Background Technology

[0002] The statements in this section are merely background information related to the present invention and do not necessarily constitute prior art.

[0003] Inflammatory bowel disease (IBD) is an idiopathic inflammatory bowel disease affecting the ileum, rectum, and colon. Clinical manifestations include diarrhea, abdominal pain, and even bloody stools. This disease includes ulcerative colitis (UC) and Crohn's disease (CD). Ulcerative colitis is a continuous inflammation of the colonic mucosa and submucosa, usually starting in the rectum and gradually spreading to the entire colon. Crohn's disease can affect the entire digestive tract, presenting as a discontinuous, full-thickness inflammation, most commonly affecting the terminal ileum, colon, and perianal area. The incidence of ulcerative colitis has been increasing in recent years. The etiology and pathogenesis of ulcerative colitis are not fully understood. It is known that an abnormal reaction of the intestinal mucosal immune system plays an important role in the pathogenesis of IBD, which is believed to be caused by the interaction of multiple factors, mainly including environmental, genetic, infectious, and immune factors. Current clinical treatment mainly uses anti-inflammatory drugs such as 5-aminosalicylic acid preparations and immunosuppressants such as glucocorticoids and azathioprine. Ulcerative colitis often recurs, thus requiring long-term maintenance therapy. Although many beneficial explorations have been made in recent years regarding new treatments for ulcerative colitis, shortcomings remain, such as the difficulty in achieving a complete cure and the lack of targeted drugs. Traditional Chinese medicine (TCM) treatment for ulcerative colitis has a strong regulatory effect on patients' immune imbalances and is characterized by its multi-faceted, multi-dimensional, and multi-target approach. Furthermore, TCM treatment for ulcerative colitis has fewer side effects, high clinical efficacy, and is easily accepted by patients. In particular, recent years have seen the discovery of good efficacy of *Cynanchum paniculatum* and its preparations for ulcerative colitis, although the specific active ingredients remain unclear.

[0004] *Tabernaemontana divaricata* (Linnaeus) R. Brown ex Roemer & Schultes is a plant belonging to the Apocynaceae family and the *Tabernaemontana* genus. Modern pharmacological studies have shown that *Tabernaemontana divaricata* possesses anti-inflammatory, anti-tumor, anti-addictive, and anti-acetylcholinesterase biological activities. Its leaves can be used medicinally, having the effect of lowering blood pressure, and are commonly used in folk medicine to treat eye diseases, sores, mastitis, and bites; the roots can be used to treat headaches and fractures.

[0005] Indole alkaloids have been shown to have antibacterial properties and to inhibit LPS-induced expression of cytokines. Patent WO03059343A2 discloses that indole alkaloids can be used to treat AIDS; CA3186900A1 discloses that indole alkaloids can be used to treat mental disorders; WO2022125370A8 discloses that indole alkaloids can be used to improve symptoms of heart failure. However, currently, no research has shown the application of indole alkaloids in the treatment of colitis. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, this invention has discovered an alkaloid compound with anti-colitis activity. This invention also provides the application of an indole alkaloid compound in the preparation of anti-colitis drugs, specifically Isovoacangine, which has the following structural formula:

[0007]

[0008] Specifically, the present invention is achieved through the following technical solution:

[0009] In a first aspect, the present invention provides the use of the indole alkaloid compound Isovoacangine in the preparation of an anti-colitis drug;

[0010] In one or more embodiments, the colitis is ulcerative colitis.

[0011] In a second aspect, the present invention provides the use of the indole alkaloid compound Isovoacangine in the preparation of a drug for protecting the colonic mucosal epithelium;

[0012] Specifically, protecting the colonic mucosal epithelium manifests as protecting the integrity of the mucosal structure, reducing lymphocyte infiltration, and increasing the number of goblet cells.

[0013] In a third aspect, the present invention provides the use of the indole alkaloid compound Isovoacangine in the preparation of a drug for eliminating colonic mucosal edema.

[0014] A fourth aspect of the present invention provides a method for extracting the indole alkaloid compound Isovoacangine according to any one of the first to third aspects described above:

[0015] 1) Crush the dog tooth flower, cold soak it with 95% ethanol, combine the extracts, concentrate under reduced pressure to obtain crude extract;

[0016] 2) Disperse the crude extract obtained in step 1) in water, add 1% dilute hydrochloric acid to adjust the pH to 1-2, pour into a separatory funnel, add ethyl acetate for extraction; add ammonia to the remaining aqueous phase to adjust the pH to 9-10, add chloroform for extraction, and concentrate to obtain the alkaloid phase;

[0017] 3) Pass the alkaloid phase obtained in step 2) through alkaline silica gel column chromatography, using a gradient elution of dichloromethane / methanol solution (5-200:1V / V), and combine the eluents after concentration;

[0018] 4) The combined substances from step 3) were recrystallized from methanol to obtain the indole alkaloid compound Isovoacangine;

[0019] Specifically, the volume ratio of dichloromethane to methanol is 200:1, 100:1, 50:1, 30:1, 10:1, or 5:1.

[0020] In a fifth aspect, the present invention provides a medicament for treating colitis, the medicament comprising an indole alkaloid compound, Isovoacangine, and a pharmaceutically acceptable carrier or excipient; wherein the colitis is ulcerative colitis;

[0021] In one or more embodiments, the dosage form of the drug is a gastrointestinal dosage form and a non-gastrointestinal dosage form; the gastrointestinal dosage form includes powder, tablet, granule, capsule, solution, emulsion or suspension; the non-gastrointestinal dosage form includes injection dosage form, respiratory dosage form, skin dosage form, mucosal dosage form and cavity dosage form;

[0022] In one or more embodiments, the injectable dosage form includes intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, or intracavitary injection.

[0023] The respiratory drug delivery dosage forms include sprays, aerosols, or powder sprays;

[0024] The skin drug delivery dosage forms include topical solutions, lotions, liniments, ointments, plasters, pastes, or patches;

[0025] The mucosal drug delivery dosage forms include eye drops, nasal drops, ophthalmic ointments, mouthwashes, sublingual tablets, adhesive patches, or films;

[0026] The cavity drug delivery dosage forms include suppositories, aerosols, effervescent tablets, drops, or pellets.

[0027] In a sixth aspect, the present invention provides a pharmaceutical composition comprising an indole alkaloid compound, Isovoacangine, and at least one other pharmaceutically active ingredient.

[0028] In a seventh aspect, the present invention provides a health product for colitis, wherein the ingredients of the health product are prepared from the drug described in the fifth aspect or the drug composition described in the sixth aspect and conventional excipients.

[0029] The above one or more technical solutions have the following beneficial effects:

[0030] (1) This invention discovered the alkaloid compound Isovoacangine in the dog tooth flower through separation and extraction.

[0031] (2) This invention demonstrates through experiments that the alkaloid compound Isovoacangine in dog tooth flower has significant anti-enteritis activity, providing a new research direction for the treatment of colitis.

[0032] (3) There are records of using dog tooth flower and its preparations as treatment for colitis in the prior art. The present invention confirms that Isovoacangine can effectively play a therapeutic role in colitis. Compared with the use of mixtures for treatment, using Isovoacangine as a treatment drug is more convenient and more economical, and has significant progress compared with the prior art.

[0033] (4) The extraction method of this application is simple, has a high yield, is highly practical, and is easy to scale up for production.

[0034] Advantages of additional aspects of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0035] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0036] Figure 1 For Isovoacangine in Example 1 1 H NMR spectrum

[0037] Figure 2 For Isovoacangine in Example 1 13 C NMR spectrum

[0038] Figure 3 The mass spectrum of Isovoacangine in Example 1.

[0039] Figure 4 Figure showing the weight changes of the model mice during drug administration in Example 2.

[0040] Figure 5IDA data of mice after modeling in Example 2, including feces and blood in stool.

[0041] Figure 6 Figure 2 shows the changes in colon length and weight in mice from different experimental groups in Example 2.

[0042] Figure 7 Images of colon tissue and HE staining from the model mice in Example 2.

[0043] Figure 8 This is a diagram showing the expression of cytokines in serum during Example 2.

[0044] Figure 9 This is a graph showing the changes in the proportions of Th1 and Th17 cells in the spleen and mesenteric lymph nodes of mice in each group in Example 2.

[0045] Figure 10 This is a graph showing the changes in the proportion of Treg cells in the spleen and mesenteric lymph nodes of mice in each group during Example 2. Detailed Implementation

[0046] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0047] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0048] In this invention, the term "treatment" has its general meaning, and specifically refers to the treatment of a mammalian individual (preferably a human) with colitis by means of the drug of this invention, with the aim of producing a therapeutic, curative, alleviating, or reducing effect on the disease, and obtaining the desired pharmacological and / or physiological effects.

[0049] "Pharmaceutically acceptable carriers" are recognized in the art and include pharmaceutically acceptable materials, compositions, or carriers suitable for administering the compounds of the present invention to mammals. These carriers include liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials that participate in carrying the host substance or transferring it from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of compatibility with other components in the formulation and harmlessness to the patient. Examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate, powdered tragacanth gum, malt, gelatin, and talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; and other non-toxic and compatible substances used in pharmaceutical preparations.

[0050] The composition may also contain wetting agents, emulsifiers and lubricants such as sodium dodecyl sulfate and magnesium stearate, as well as colorants, releasing agents, coating agents, sweeteners, flavoring agents and aroma agents, preservatives and antioxidants.

[0051] The formulations of this invention can be conveniently available in unit dosage form and can be prepared by any method known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier substance to prepare a single-dose formulation is generally sufficient to produce a therapeutic effect.

[0052] The amount of compound used.

[0053] Experimental procedures not described in detail in this invention are commonly used by those skilled in the art and will not be elaborated upon here.

[0054] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0055] Example 1

[0056] The extraction method for Isovoacangine includes the following steps:

[0057] 1) Crush 13 kg of dog tooth flower, cold soak it with 95% ethanol (25 L × 3 times), combine the extracts, concentrate under reduced pressure to obtain crude extract (1250 g);

[0058] 2) Disperse the *Cynodon dactylon* extract obtained in step 1) in water, add 1% dilute hydrochloric acid to adjust the pH to 1-2, pour into a separatory funnel, add ethyl acetate for extraction; add ammonia to the remaining aqueous phase to adjust the pH to 9-10, add chloroform for extraction, and concentrate to obtain 100g of alkaloid phase.

[0059] 3) The alkaloid phase obtained in step 2) was subjected to alkaline silica gel column chromatography using a gradient elution of dichloromethane:methanol (volume ratios of 200:1, 100:1, 50:1, 30:1, 10:1, and 5:1), with approximately 5 L of eluent for each gradient. Each 500 mL eluent fraction was collected and concentrated using a small rotary evaporator. After TLC analysis, the fractions were combined into 7 fractions (D1-D7).

[0060] 4) The D2 region from step 3) was recrystallized from methanol to obtain the compound Isovoacangine (800 mg).

[0061] Structural identification: The isolated monomeric components were analyzed using an Agilent 1200RRLC-6410 Triple Quad LC / MS mass spectrometer and a Varian INOVA 600 nuclear magnetic resonance spectrometer for MS and NMR spectrometry, respectively (see appendix for details). Figure 1-3 The NMR data are shown below, which helps to identify the structure of this alkaloid compound.

[0062] Isovoacangine: White crystals. It shows an orange-red positive result in the modified potassium bismuth iodide reaction, indicating that the compound is an alkaloid. 1 The molecular formula of this compound was determined to be C13NMR and 13CNMR combined with mass spectrometry data. 22 H 28 N2O3. 1 H-NMR (CD3OD, 600MHz) δ H : 7.13 (1H, d, J = 8.7Hz, H-9), 6.90 (1H, d, J = 2.4Hz, H-12), 6.69 (1H, dd, J = 8.7, 2.4Hz, H-10), 3.80 (3H, s, C 11-OCH3), 3.69 (3H, s, COOCH3), 3.59 (1H, brs, H-21), 3.38 (1H, m, H-5b), 3.07 (1H, m, H-5a), 3.06 ( 1H, m, H-6b), 2.99 (1H, m, H-6a), 2.92 (1H, m, H-3b), 2.78 (1H, dt, J=8.5, 1.8Hz, H-3a), 2.70 (dt, J=13.5, 2.2Hz, H-17b), 1.92 (1H, m, H-17a), 1.84 (1H, m, H-14), 1.76 (1H, m, H-15b), 1.56 (1H, m, H-19b), 1.44 (1H, m, H-19a), 1.37 (1H, m, H-20), 1.13 (1H, m, H-15a), 0.90 (3H, t, J=7.4Hz, H-18).

[0063] 13 C-NMR (CD3OD, 150MHz) δ C :176.3(COOCH3), 154.9(C-11), 139.3(C-2), 132.8(C-8), 129.9(C-7), 112. 3(C-10), 112.2(C-9), 110.6(C-13), 101.1(C-12), 57.9(C-21), 56.4(C-16),

[0064] 56.3(C 11 -OCH3), 54.8(C-5), 54.2(C-3), 52.9(COOCH3), 40.1(C-20), 37.1(C-17), 33.2(C-15), 28.9(C-14), 27.9(C-19), 22.8(C-6), 12.1(C-18).

[0065] Example 2

[0066] Animal experiments have demonstrated that the compound Isovoacangine can inhibit the occurrence and development of colitis.

[0067] 1. Animal modeling experiments

[0068] 1.1 Establishment of a C57BL / 6J mouse colitis model

[0069] Dextran sulfate sodium (DSS) was dissolved in distilled water to prepare a 3.5% DSS (w / v) solution, which was then given to C57BL / 6J mice (8-10 weeks old) for free access to drinking water for 7 days, with the solution changed daily to induce an IBD model. Normal mice were given normal drinking water. The mice were fed SPF-grade experimental breeding mouse food (provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.) and allowed free access to the food.

[0070] 1.2 Animal administration

[0071] C57BL / 6J mice were divided into three groups: normal group, model group, and Isovoacangine 30mg / kg group, with eight female mice in each group.

[0072] The control group drank sterilized water continuously for 10 days;

[0073] The model group was given 3.5% DSS and allowed free access to drink for seven consecutive days, then switched to sterile water and allowed free access to drink until day 10; from day 1, the model group was given 0.2 mL of 0.5% sodium carboxymethyl cellulose by gavage.

[0074] The Isovoacangine 30 mg / kg group was given 3.5% DSS and allowed free access to drinking water for seven consecutive days, then switched to sterile water; from day one, each mouse in the Isovoacangine 30 mg / kg group was given 0.2 mL of Isovoacangine solution by gavage daily for 10 consecutive days.

[0075] Preparation method of Isovoacangine solution: Isovoacangine is prepared into a homogeneous solution with a final concentration of 3 mg / ml using 0.5% sodium carboxymethyl cellulose (CMC-Na) solution, and then mixed by ultrasonic vibration before use.

[0076] 1.3 Animal model indicators

[0077] 1.3.1 Changes in mouse body weight and disease activity index (DAI)

[0078] Mice were weighed daily, and their water intake, body weight, fecal characteristics, and any instances of blood in the stool were recorded. DAI scores were also calculated. 1.3.2 Length and weight of the mouse colon.

[0079] Mice were euthanized by cervical dislocation, and the abdomen was dissected. The colon from the anus to the ileocecal junction was harvested, and its length was measured. The entire colon segment was weighed. The colon length was recorded in detail during dissection of each mouse. Studies have shown that the DSS colitis model is characterized by colonic edema and shortening, and the shortening of colon length can, to some extent, serve as an indicator of the severity of inflammation. 1.3.3 HE staining to observe morphological changes in the colon.

[0080] After the above experiment was completed, the mice were dissected, and a 0.5 cm section of the middle colon was cut off. After being washed with physiological saline, it was quickly fixed in 4% paraformaldehyde, embedded in paraffin, stained with hematoxylin and eosin (HE), and photographed and observed under a microscope.

[0081] 1.3.4 Measurement of Cytokine Expression

[0082] Before euthanizing mice by cervical dislocation, blood was expelled from the orbits and allowed to stand at room temperature for 30 minutes. The mice were then centrifuged at 3000 rpm for 20 minutes, and the supernatant was collected as serum. The expression levels of cytokines such as IL-1β, IL-10, IL-6, and TNF-α in the serum were detected according to the instructions of the ELISA kits (purchased from Hangzhou Lianke Biotechnology Co., Ltd., kit number EK201B for IL-1β, EK210 for IL-10, EK206 for IL-6, and EK282 for TNF-α).

[0083] 1.3.5 Lymphocytes were isolated and extracted from the spleen and mesenteric lymph nodes of mice in each group, and the changes in the proportions of Th1, Th17, and Treg cells in the spleen and mesenteric lymph nodes were detected by flow cytometry.

[0084] Mice were dissected, and the spleen and mesenteric lymph nodes were removed and placed in pre-cooled RPMI-1640 medium. The cells were ground and passed through a 70 μm cell sieve to obtain a single-cell suspension. The suspension was centrifuged at 400 g, 4 °C, for 6 min. The cells were washed twice with PBS, resuspended in PBS with 2% FBS, and counted. Splenic cells required lysis using erythrocyte lysate (BD Biosciences, catalog number 555899) followed by mesenteric lymph node lysis to prepare a single-cell suspension. Depending on the cell type to be tested, flow cytometry antibodies with different fluorescent labels were added and incubated on ice for 45 min. The flow cytometry antibodies included anti-mouse CD3 (AF700), anti-mouse B220 / CD45R (APC-Cy7), anti-mouse CD4PE-Cy7, anti-mouse CD8a (BV605), anti-mouse CD44 (FITC), anti-mouse CD62L (BV421), anti-mouse IFN-γ (PE-CF594), anti-mouse IL-17A (AF647), anti-mouse CD25 (PE), anti-mouse Foxp3 (AF647), and 7-amino-actinomycin D (7-AAD), purchased from Biolegend Systems, Inc., USA. 2. Experimental Results

[0085] 2.1 Changes in mouse body weight

[0086] like Figure 4 As shown, after DSS modeling, mice began to lose weight from day 3 until the end of the modeling process. Compared with the normal group, the mice in the model group and the Isovoacangine group had significantly less weight, indicating successful modeling. After the end of DSS drinking, compared with the model group, the mice in the Isovoacangine group had a significantly increased average weight, with significant differences in weight on days 8, 9, and 10 of the experiment (p<0.05).

[0087] 2.2 Changes in the DAI index in mice

[0088] like Figure 5 As shown, DAI (Digital Intestinal Adverse Event) curves were plotted based on data such as fecal matter and bloody stools recorded in mice. The results showed that some enteritis changes appeared from day 3, and the feces gradually became looser over time. Bloody stools appeared from day 6, and the intestinal inflammation of the mice worsened until day 7. The Isovoacangine group showed improvement from day 8, 9, and 10 of the experiment. Especially on day 9 and 10, the enteritis status was significantly improved compared with the model group (*, P<0.05), and the loose stools and bloody stools were basically restored.

[0089] 2.3 Changes in mouse colon and weight

[0090] like Figure 6 As shown, compared with the normal group, the length and weight of the colon in mice in the model group and the Isovoacangine group were significantly reduced. However, compared with the model group, the length and weight of the colon in the Isovoacangine group were significantly increased (p<0.01).

[0091] 2.4 HE staining results

[0092] like Figure 7 As shown, the colon tissue of mice in the model group was severely damaged, the basic structure of the mucosa was destroyed, goblet cells were missing in large numbers, lymphocyte infiltration in the lamina propria was obvious, and tissue edema and congestion were visible in the muscularis mucosae. The degree of damage to the colon tissue of mice in the Isovoacangine group was significantly reduced, the tissue structure was more distinct, the infiltration of inflammatory cells in the lamina propria was reduced, the number of goblet cells was relatively increased, no tissue edema and congestion were seen in the muscularis mucosae, and the tissue morphology was significantly restored.

[0093] 2.5 ELISA was used to detect the expression of cytokines in the serum of each group.

[0094] like Figure 8 As shown, compared with the model group, the levels of pro-inflammatory factors such as IL-1β, IL-6 and TNF-α in the colon tissue of the Isovoacangine group were significantly decreased (IL-1β, p<0.01; IL-6, p<0.01; TNF-α, p<0.05), while the levels of anti-inflammatory cytokines such as IL-10 were significantly increased (p<0.01). This indicates that Isovoacangine has a good therapeutic effect on colitis in mice by inhibiting the expression of inflammatory factors and promoting the expression of anti-inflammatory factors to alleviate intestinal inflammation.

[0095] 2.6 Flow cytometry analysis of changes in the proportions of Th1, Th17, and Treg cells in the spleen and mesenteric lymph nodes of mice in each group.

[0096] Flow cytometry was used to detect changes in the proportions of Th1 and Th17 helper T cell subtypes in mesenteric lymph nodes and spleen. Th1 and Th17 cells are cell subtypes that reflect the level of inflammation in the body; an increased proportion of Th1 and Th17 cells indicates an increased level of inflammation, while a decreased proportion indicates an improved inflammatory condition. In this experiment, CD3+CD4+CD44+IFN-γ+ cells were defined as Th1 cells, and CD3+CD4+CD44+IL-17A+ cells were defined as Th17 cells. Figure 9As shown, compared with the model group, the proportions of Th1 and Th17 cells in the spleen and mesenteric lymph nodes of the Isovoacangine group were significantly reduced (p<0.05). The decrease in the proportion of Th1 cells reduced the production of pro-inflammatory factors such as IFN-γ, and the decrease in the proportion of Th17 cells reduced the production of pro-inflammatory factors such as IL-17A, thereby reducing the body's inflammatory response and exerting anti-inflammatory activity. This indicates that Isovoacangine administration can inhibit the level of inflammation in vivo, improve the inflammatory condition in mice, and has a therapeutic effect on enteritis in mice.

[0097] Flow cytometry was used to detect changes in the proportion of Treg cells, a regulatory T cell subtype with immunosuppressive functions, in mesenteric lymph nodes and spleen. Figure 10 As shown, the proportion of CD3+CD4+CD25+FOXP3+ Treg cells was significantly increased in the model group, both in the mesenteric lymph nodes and spleen. This indicates that the inflammation at the site of inflammation was severe after modeling, inducing more Treg cells with immunosuppressive functions to alleviate the body's excessive immune inflammatory response. In contrast, the inflammation in the mice treated with Isovoacangine was not significantly enhanced. The proportion of Treg cells was slightly increased compared to the normal group, but significantly decreased compared to the model group. This suggests that Isovoacangine administration is beneficial in suppressing the overall immune inflammatory response and promoting the recovery from enteritis.

[0098] 3. Conclusion

[0099] The anti-inflammatory activity of Isovoacangine was investigated using a DSS-induced colitis model. Twenty-four C57BL / 6J mice were randomly divided into three groups (normal group, model group, and Isovoacangine group), with eight mice in each group. After modeling, the Isovoacangine group was administered Isovoacangine 30 mg / kg by gavage, the model group was administered the same dose of sodium carboxymethyl cellulose (CMC-Na) as the Isovoacangine group by gavage, and the normal group was given sterile water throughout. The administration was continued for 10 days. After the administration, the mice's body weight, DAI index, colon length and weight, colonic morphological changes, expression of inflammation-related cytokines, and changes in the proportions of helper T cells (Th1, Th17, and regulatory Treg cells) in the spleen and mesenteric lymph nodes were observed. The results showed that the body weight of mice in all model groups was significantly reduced, while the body weight of mice in the 30 mg / kg Isovoacangine group was significantly increased compared with the model group, and the DAI index of mice was significantly improved. The length and weight of the colon after Isovoacangine administration were also significantly increased compared with the model group. HE staining results showed that the degree of colon tissue damage in the Isovoacangine group was lower than that in the model group, and the morphology of the colon tissue was significantly restored, indicating that Isovoacangine can significantly repair damaged colon tissue. ELISA detection of serum cytokine expression in each group showed that compared with the model group, the levels of IL-1β, IL-6, and TNF-α in the colon tissue of the treated groups were significantly reduced, while the level of the anti-inflammatory factor IL-10 was increased. Flow cytometry analysis revealed that the proportions of Th1, Th17, and Treg cells in the spleen and mesenteric lymph nodes were significantly lower in the Isovoacangine group compared to the model group. While the proportion of Treg cells increased slightly compared to the normal group, the increase was less pronounced than in the model group, indicating an overall decrease in inflammation levels in the mice after administration and a mitigating effect on enteritis. In conclusion, Isovoacangine demonstrates a good therapeutic effect on DSS-induced colitis.

[0100] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of isovoacangine, an indole alkaloid compound, in the preparation of anti-colitis drugs; The structural formula of the alkaloid compound is shown in Formula I. Equation I; The extraction method for the indole alkaloid compound Isovoacangine is as follows: 1) Crush the dog tooth flower, cold soak it with 95% ethanol, combine the extracts, concentrate under reduced pressure to obtain crude extract; 2) Disperse the crude extract obtained in step 1) in water, add 1% dilute hydrochloric acid to adjust the pH to 1-2, pour into a separatory funnel, add ethyl acetate for extraction; add ammonia to the remaining aqueous phase to adjust the pH to 9-10, add chloroform for extraction, and concentrate to obtain the alkaloid phase; 3) The alkaloid phase obtained in step 2) was subjected to alkaline silica gel column chromatography, using a gradient elution of dichloromethane / methanol solution. The volume ratio of dichloromethane to methanol was 200:1, 100:1, 50:1, 30:1, 10:1, or 5:

1. The eluent was concentrated and combined into 7 fractions, namely D1-D7. 4) The D2 region after merging in step 3) was recrystallized from methanol to obtain the indole alkaloid compound Isovoacangine.

2. The application as described in claim 1, characterized in that, The colitis mentioned is ulcerative colitis.

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