Use of hyperforin in the preparation of a medicament for the treatment of inflammatory bowel disease

Abstract

The application belongs to the technical field of medicines, and particularly relates to application of hyperforin in preparation of medicines for treating inflammatory bowel disease, and proves that hyperforin treatment can increase the colon length of a DSS-induced mouse colitis model, improve the pathological structure and clinical symptoms of the colon tissue of the model mouse, and significantly up-regulate ZO-1 protein and improve the levels of related inflammatory factors such as TNF-alpha, IL-6 and IL-1beta, and has significant anti-ulcerative colitis activity, and the hyperforin can provide a new approach for preparing medicines for treating ulcerative colitis in the future.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and specifically relates to the application of hypericin in the preparation of drugs for treating inflammatory bowel disease. Background Technology

[0002] Inflammatory bowel disease (IBD) is a general term for chronic, nonspecific, relapsing inflammatory diseases of the gastrointestinal tract with unknown etiology. It is not directly caused by a specific pathogen, but rather by an abnormal attack on the gut by the immune system. IBD includes ulcerative colitis and Crohn's disease. Ulcerative colitis is a chronic, relapsing gastrointestinal inflammation characterized by diffuse inflammation of the colonic and rectal mucosa, manifesting as bloody diarrhea, abdominal pain, and systemic complications. Treatment of ulcerative colitis follows a stepwise strategy. For mild to moderate cases, aminosalicylic acid preparations are the first-line basic drugs for inducing and maintaining remission, with local rectal administration showing significant efficacy in distal colitis. If ineffective, glucocorticoids are used to rapidly control acute inflammation, but due to their severe systemic side effects, long-term use for maintenance therapy is strictly prohibited. For patients unresponsive to, dependent on, or with moderate to severe cases, the core of treatment shifts to biologics such as the anti-TNF-α infliximab, the intestinal-selective anti-integrin vedolizumab, and small molecule drugs such as the JAK inhibitor tofacitinib. These drugs target and inhibit key immune pathways, achieving deep remission. For patients with acute and severe illness, those who have failed medical treatment, or those who have developed cancer, the final course of treatment is surgery.

[0003] Although the incidence of ulcerative colitis has been rising globally in recent decades, mortality rates have decreased due to the use of steroids and immunosuppressants, as well as advancements in colectomy. However, ulcerative colitis remains a refractory disease, with low drug response rates and adverse reactions severely impacting patients' physical and mental health. Therefore, there is an urgent need to develop new treatments for ulcerative colitis.

[0004] St. John's wort is a widely studied medicinal plant whose phytochemicals include various bioactive compounds such as hypericin, flavonoids, pseudohypericin, and various phenolic compounds. Since ancient times, hypericin has been used in traditional medicine preparations to aid wound healing and treat anxiety, depression, and liver diseases. With further scientific research, researchers have also discovered that hypericin can treat amyotrophic lateral sclerosis (ALS) (see patent CN119700727A) and promote the browning of white adipose tissue and enhance the activity and function of brown adipose tissue (see invention patent CN109044999A). Clinically, hypericin is currently used to treat mild to moderate-to-severe depression, but its application in the treatment of inflammatory bowel disease has not been reported. Summary of the Invention

[0005] To address the aforementioned technical problems, the purpose of this invention is to provide a novel drug for treating ulcerative colitis, specifically the application of hypericin in the preparation of a drug for treating ulcerative colitis.

[0006] The primary objective of this invention is to provide the application of hypericin in the preparation of drugs for treating inflammatory bowel disease, wherein the structural formula of hypericin is shown in formula (I):

[0007] (I).

[0008] Preferably, the inflammatory bowel disease includes ulcerative colitis and / or Crohn's disease.

[0009] Preferably, the inflammatory bowel disease is ulcerative colitis.

[0010] A second object of the present invention is to provide a medicament for treating inflammatory bowel disease, the medicament comprising hypericin and excipients, wherein the structural formula of hypericin is shown in formula (I):

[0011] (I).

[0012] Preferably, the drug preparation is any one of liquid formulation, solid formulation, semi-solid formulation, and gaseous formulation.

[0013] Preferably, the dosage form of the drug specifically includes any one of drops, oral liquid, tablets, capsules, granules, films, gels, powders, emulsions, pellets, or solutions.

[0014] Preferably, the drug is any one of liquid preparations, solid preparations, semi-solid preparations, and gaseous preparations.

[0015] The beneficial effects of this invention are as follows: This invention provides the application of hypericin in the preparation of drugs for treating inflammatory bowel disease. Using a DSS-induced ulcerative colitis model, the therapeutic effect of hypericin on ulcerative colitis was evaluated by gavage. The results showed that hypericin can effectively reduce the mortality rate of mice with ulcerative colitis and reduce the levels of TNF-α, IL-6, IL-1β and ZO-1 in the colon tissue of mice with ulcerative colitis. Attached Figure Description

[0016] Figure 1 The effect of hypericin on colon length in mice with DSS-induced ulcerative colitis; Note: Compared with the DSS model group, **P<0.01.

[0017] Figure 2The effect of hypericin on body weight in mice with DSS-induced ulcerative colitis; Note: Compared with the DSS model group, **P<0.01.

[0018] Figure 3 The effect of hypericin on the disease activity index (DAI) in DSS-induced ulcerative colitis mice; Note: Compared with the DSS model group, **P<0.01.

[0019] Figure 4 The effect of hypericin on the pathological structure of colon tissue in mice with DSS-induced ulcerative colitis; Note: Compared with the DSS model group, **P<0.01.

[0020] Figure 5 The effect of hypericin on ZO-1 protein in colon tissue of mice with DSS-induced ulcerative colitis; Note: Compared with the DSS model group, **P<0.01.

[0021] Figure 6 The effect of hypericin on TNF-α in colon tissue of mice with DSS-induced ulcerative colitis; Note: Compared with the DSS model group, **P<0.01.

[0022] Figure 7 The effect of hypericin on IL-6 protein in colon tissue of mice with DSS-induced ulcerative colitis; Note: Compared with the DSS model group, **P<0.01.

[0023] Figure 8 The effect of hypericin on IL-1β protein in colon tissue of mice with DSS-induced ulcerative colitis; Compared with the DSS model group, **P<0.01. Detailed Implementation

[0024] The scope of protection of the present invention will be described in detail below with reference to specific embodiments. It should be noted that the scope of protection of the present invention is not limited by the following embodiments.

[0025] It should be noted that, unless otherwise specified, the methods described in the following embodiments are all conventional methods, and the reagents described are all commercially available.

[0026] Sodium dextran sulfate (DSS) is a water-soluble, negatively charged sulfated polysaccharide that can induce intestinal inflammation by disrupting the intestinal mucosal barrier. Its induced colitis model is reproducible and controllable. Among various chemically induced colitis models, DSS-induced mouse colitis shows the highest similarity to human ulcerative colitis in terms of pathological features. This invention establishes a DSS-induced ulcerative colitis mouse model and systematically evaluates the preventive and therapeutic effects of hypericin on ulcerative colitis by measuring colon length, levels of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and the expression of tight junction protein (ZO-1).

[0027] In this invention, the molecular weight of the hypericin is 536.793 g / mol, and its molecular formula is C1. 35 H 52 O4, the structural formula is as follows: .

[0028] Example 1: Application of Hypericin in the Preparation of Drugs for Treating Ulcerative Colitis 1. Experimental Methods 1.1 Animal grouping and administration Hypericin (Shanghai Yuanye Biotechnology Co., Ltd.); DSS reagent (Shanghai Yisheng Biotechnology Co., Ltd.); Immunostaining blocking solution and DAB colorimetric reagent kit (Shanghai Beyotime Biotechnology Co., Ltd.); IL-1β, IL-6, and TNF-α detection ELISA kit (Wuhan Baikangdi Biotechnology Co., Ltd.).

[0029] All experimental animals were male C57BL / 6 black mice (8 weeks old) from the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, production license: SCXK (Gan) 2020-0002. Throughout the experiment, the mice were housed in a standardized environment within an SPF-grade barrier system. The housing environment was strictly controlled with constant temperature and humidity (25±1℃) and a 12h / 12h light-dark cycle. Feed, drinking water, and sterile bedding were changed regularly.

[0030] After one week of acclimatization, the mice were randomly divided into 6 groups (n=8): control group, model group, low-dose hypericin group (HPF-L, 25 mg / kg / d), medium-dose hypericin group (HPF-M, 50 mg / kg / d), and high-dose hypericin group (HPF-H, 100 mg / kg / d).

[0031] 1.2 Construction of a mouse model of ulcerative colitis and evaluation of DAI Mice in the normal control group were given purified water, while mice in the other groups were given a 2.5% (w / v) sodium dextran sulfate solution to induce an ulcerative colitis model for 7 consecutive days. From day 1 to day 9, the normal control group and the model group were administered physiological saline by gavage, while the positive control group was given the corresponding dose of hypericin solution, once daily at a volume of 10 mL / kg. On day 9, mice were fasted for 12 hours. On day 10, blood was collected from the eyeballs, and the mice were sacrificed. Colonic tissue was collected and its length measured. Throughout the experiment, daily changes in mouse body weight and fecal characteristics were recorded to evaluate the mice's disease-associated acute inflammation (DAI).

[0032] 1.3 HE staining of colon tissue Mouse colon tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and then sectioned. The sections were dewaxed, hydrated, and stained with hematoxylin and eosin. After staining, the sections were dehydrated with graded alcohols, cleared with xylene, and mounted with neutral resin. Histopathological changes were observed under an optical microscope and photographed.

[0033] 1.4 Immunohistochemistry of tight junction protein ZO-1 in colonic tissue Paraffin-embedded sections were incubated with 3% hydrogen peroxide for 10 minutes, followed by blocking with immunostaining blocking solution to prevent nonspecific antigen binding for 15 minutes at room temperature. Subsequently, the sections were incubated overnight at 4°C with primary antibody: ZO-1 (1:1000; Wuhan Sanying Biotechnology Co., Ltd.), and then incubated at 37°C for 1 hour. After washing, the sections were incubated at 37°C with appropriate biotinylated secondary antibody for 1 hour. Finally, staining was performed using a DAB kit.

[0034] 1.5 ELISA Detection Weigh 100 mg of colon tissue sample, add lysis buffer at a ratio of m / v=1:9, and homogenize using a homogenizer on ice. Centrifuge the prepared homogenate at 5000 g for 10 min, and use the supernatant for TNF-α, IL-6, and IL-1β ELISA kit detection. Refer to the ELISA kit instructions for specific operating procedures and precautions. A simplified operating procedure is as follows: Add the sample to be tested or standard solutions of different concentration gradients to a 96-well ELISA plate, then add biotinylated antibody working solution, incubate at room temperature for 1 h, wash thoroughly and discard the sample and biotinylated antibody working solution. Add HRP-labeled secondary antibody to all wells except the blank wells, and incubate at room temperature for 30 min. Discard the secondary antibody solution, wash thoroughly, add chromogenic substrate, and incubate at room temperature in the dark for 15 min. Add stop solution and measure the absorbance at 450 nm. Calculate the content of inflammatory factors by plotting a standard curve. All experiments were performed in triplicate.

[0035] 2. Experimental Results 2.1 Changes in the length of mouse colon tissue In a mouse model of ulcerative colitis, colon length can reflect the severity of intestinal inflammatory damage, such as... Figure 1 The colon length bar chart showed that the colon length in the model group was significantly shorter than that in the normal group. After intervention with different doses of HPF, the colon length increased significantly and in a dose-dependent manner. The high-dose group showed the best recovery effect, indicating that HPF can improve DSS-induced colonic injury.

[0036] 2.2 Changes in mouse body weight and evaluation of DAI When evaluating the therapeutic effect of drugs on ulcerative colitis, mouse body weight and DAI (diastolic blood glucose level) are important indicators. Body weight fluctuations reflect the impact of the drug on the overall health of the mice, while the DAI score can determine the severity of ulcerative colitis. In the normal group, mice showed normal weight gain and DAI throughout the experiment, while in the model group, mice began to lose weight on the third day after modeling. On the fourth day, some mice showed loose stools and mild fecal occult blood. On the fifth day, some mice developed diarrhea, and fecal occult blood was common. On the sixth day, mice showed significant weight loss, lethargy, and widespread diarrhea and significant bloody stools. This demonstrates that 2.5% DSS in drinking water successfully established an ulcerative colitis model. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) Figure 2 It was found that after intervention with different doses of hypericin, weight loss in all treatment groups was inhibited in a dose-dependent manner, indicating that hypericin can improve the symptoms of DSS-induced weight loss. The DAI score results are as follows: Figure 3 The results showed that the DAI scores of all mice after drug intervention were lower than those of the model group. The severity of diarrhea, stool characteristics, and bloody stool were significantly reduced in the drug intervention group, with the high-dose group showing the most significant improvement. The body weight changes and DAI scores of all hypericin-treated groups were improved compared to the model group, indicating that hypericin can improve the clinical symptoms of DSS-induced ulcerative colitis in mice.

[0037] 2.3 HE staining of mouse colon tissue HE-stained colon tissue sections allow for direct observation of pathological changes in a mouse model of ulcerative colitis. HE staining results are shown below. Figure 4As shown, in the normal group, the colonic tissue structure was intact, the mucosal epithelial cells were of normal morphology, the glands were neatly arranged, the crypts were normal, and the goblet cells were abundant, with no obvious inflammatory cell infiltration. In the model group, the colonic tissue pathology was significantly altered, with destruction of the mucosal epithelial structure, severe submucosal edema, disordered gland arrangement, a significant reduction in goblet cells, and disappearance of crypts, accompanied by extensive inflammatory cell infiltration in the mucosa and submucosa, consistent with the pathological characteristics of ulcerative colitis. After intervention with different doses of HPF, the pathological damage to the colonic tissue of ulcerative colitis mice was improved to varying degrees in a dose-dependent manner. The high-dose HPF group showed the most significant improvement in symptoms, and pathological sections showed that the mucosal epithelial structure remained basically intact, the number of goblet cells significantly increased, the glands were neatly arranged, the crypt structure was restored, and there was less inflammatory cell infiltration.

[0038] 2.4 ZO-1 histochemical staining of mouse colon tissue Tight junction proteins are key components of tight junctions between intestinal epithelial cells and are closely related to the tightness and integrity of the intestinal mucosal barrier. Their protein expression levels reflect the severity of intestinal barrier damage in ulcerative colitis mice. Immunohistochemical results are as follows: Figure 5 The results showed that in the normal group, tight junction protein ZO-1 was continuously expressed on the colonic epithelial cell membrane, indicating that tight junctions were intact and the intestinal barrier function was normal. In the model group, the expression of tight junction protein was significantly decreased and its distribution was discontinuous, indicating that the intestinal tight junctions were damaged and the intestinal barrier function was impaired. After treatment with different doses of HPF, the expression of tight junction protein increased compared with the model group. Among them, the expression level of tight junction protein in the colonic tissue of the high-dose HPF group was significantly increased, and the continuity and intensity of the distribution of ZO-1 on the colonic epithelial cell membrane were similar to those of normal tissue. This indicates that when the HPF dose reaches 100 mg / kg, it can promote the expression of tight junction protein and thus effectively repair the intestinal barrier damage in mice with ulcerative colitis.

[0039] 2.5 Expression of inflammatory factors in mouse colon tissue IL-6, TNF-α, and IL-1β are key pro-inflammatory factors in the pathogenesis of ulcerative colitis; elevated levels of these factors reflect the degree of intestinal inflammation and mucosal barrier damage. Figure 6-8 As shown, compared with the normal group, the levels of IL-6, TNF-α, and IL-1β in the colon tissue of the model group mice were significantly increased, indicating a significantly aggravated intestinal inflammatory response. After HPF intervention, the expression levels of pro-inflammatory factors decreased significantly, and the effect of HPF was dose-dependent, indicating that HPF can reduce the expression of pro-inflammatory factors in colon tissue and thus alleviate the colonic inflammation in mice with ulcerative colitis.

[0040] 3. Conclusion Through the above examples, we have preliminarily demonstrated that hypericin at concentrations of 25 mg / kg body weight to 100 mg / kg body weight has a significant protective effect on mice with ulcerative colitis. It significantly increases colon length in ulcerative colitis mice, improves the pathological structure and clinical manifestations of the model mice, while increasing ZO-1 protein expression and decreasing the levels of TNF-α, IL-6, and IL-1β in the abdominal tissue of ulcerative colitis mice. This lays the foundation for the future application of hypericin in the preparation of drugs for treating ulcerative colitis.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of hypericin in the preparation of drugs for treating inflammatory bowel disease, characterized in that, The structural formula of the hypericin described above is shown in formula (Ⅰ): （Ⅰ）。 2. The application as described in claim 1, characterized in that, The inflammatory bowel disease mentioned includes ulcerative colitis and / or Crohn's disease.

3. The application as described in claim 1, characterized in that, The inflammatory bowel disease mentioned is ulcerative colitis.

4. A drug for treating inflammatory bowel disease, characterized in that, The drug comprises hypericin and excipients, and the structural formula of hypericin is shown in formula (I): （Ⅰ）。 5. The drug as described in claim 4, characterized in that, The drug preparation can be any one of liquid formulation, solid formulation, semi-solid formulation, or gaseous formulation.

6. The drug as described in claim 5, characterized in that, The dosage form of the drug specifically includes any one of the following: drops, oral liquid, tablets, capsules, granules, films, gels, powders, emulsions, pellets, or solutions.

7. The drug as described in claim 4, characterized in that, The drug is any one of liquid preparations, solid preparations, semi-solid preparations, or gaseous preparations.

Images