CN117414379A - Application of Alligator Flower Extract in Preparing Medications for Preventing or Treating Ulcerative Colitis - Google Patents

Abstract

The invention discloses the application of the extract of Alligator's Mouth Flower in the preparation of medicines for preventing or treating ulcerative colitis. By establishing a mouse model of ulcerative colitis induced by Dextran Sulfate Sodium (DSS), the results showed that alligator flower extract can down-regulate the levels of inflammatory factors such as IL-6 and TNF-α, and inhibit IKKβ/ The expression of inflammatory pathway proteins such as IKKα/NF‑κB and JAK2/STAT3 upregulates the expression of intestinal tight junction proteins ZO1, Claudin1, Occludin and the key protein MUC2 for intestinal mucosal barrier homeostasis. At the same time, it also inhibits further damage to colon tissue caused by oxidative stress, thereby maintaining the integrity of the epithelial cell barrier, restoring the activity of intestinal epithelial cells, exerting a therapeutic effect on ulcerative colitis, and providing new drug research for the clinical treatment of ulcerative colitis. Drug source.

Description

Application of Alligator Flower Extract in Preparing Medications for Preventing or Treating Ulcerative Colitis

Technical field

The present invention relates to the technical field of traditional Chinese medicine, and specifically relates to the application of crocodile flower extract in the preparation of medicines for preventing or treating ulcerative colitis.

Background technique

Inflammatory bowel disease (IBD) is a diffuse and relapsing intestinal inflammatory disease. Environmental factors act on genetically susceptible individuals, causing intestinal immune imbalance and intestinal mucosal barrier damage with the participation of intestinal microorganisms, leading to persistent inflammation. Among them, there are mainly two phenotypes: ulcerative colitis (UC) and Crohn's disease (CD). IBD is most common in North America, Western and Northern Europe, Australia, and New Zealand. As of 2020, the prevalence exceeds 0.5% and is as high as 0.75% in Canada and Scotland. However, the incidence has increased rapidly in parts of Asia and South America in recent years, and IBD has evolved become a global disease.

The inflammation of UC is mainly limited to the mucosa, and ulcers, edema, and bleeding of varying severity occur in the colon. Histopathological findings reveal acute and chronic mucosal inflammation caused by granulocytes and monocytes, leading to crypt abscesses, mucosal glandular deformation, and loss of goblet cells. Currently, conventional treatments for UC mainly include anti-tumor necrosis factor (anti-TNF) drugs, 5-aminosalicylic acid (5-ASA), salicylazole-sulfapyridine (SASP), corticosteroids, and immunomodulators (such as thiopurines). , methotrexate). 5-ASA is widely used to treat mild to moderate UC and as maintenance therapy. Anti-inflammatory steroid drugs may be used for acute IBD attacks but not for maintenance treatment. Immunosuppressants are mainly used for maintenance treatment, but their efficacy is limited and they have many adverse side effects. Therefore, finding effective methods to treat UC has become an urgent problem for the medical community. Different from traditional therapeutic drugs, Chinese herbal medicines have the characteristics of multiple targets and low toxicity. They have potential value in treating various complex diseases and can be used as a potential source for the development of new drugs to prevent and treat UC.

Clinacanthus nutans Lindau (CN), also known as Clinacanthus nutans Lindau, also known as Clinacanthus nutans Lindau, is a plant of the genus Clinacanthus in the Acanthaceae family. The whole plant is used as medicine. It has the effects of regulating menstruation, reducing swelling, removing blood stasis, relieving pain, and setting bones. It can treat bruises, anemia, jaundice, rheumatism and other diseases. Compounds currently isolated and identified from crocodile flowers include sterols, triterpenoids, flavonoid glycosides, sulfur-containing glycosides, cerebrosides, chlorophylls, megastigmanes, benzenoids and alkaloids. The main pharmacological effects include antiviral, antioxidant, antitumor, anti-inflammatory, antibacterial and immune regulation. Studies have shown that crocodile flower extract can inhibit the activation of TLR-4 in the RAW264.7 inflammation model and exert anti-inflammatory effects. In addition, the methanol extract of Alligator's Flower has anti-tumor and antioxidant activities in 4 T1 tumor-bearing mice. However, there have been no literature reports on the use of Alligator Flower extract in the preparation of drugs for the prevention or treatment of ulcerative colitis.

Contents of the invention

In view of this, the object of the present invention is to provide the application of Alligator's Mouth Flower extract in the preparation of medicines for preventing or treating ulcerative colitis.

The Alligator's Mouth Flower extract of the present invention is a Alligator's Mouth Flower water extract.

Preferably, the preparation method of the Alligator's Mouth flower water extract includes the following steps:

Powder the dried leaves of Alligator's Mouth Flower, pass through a 60-mesh sieve, soak in water for 1-2 hours, add water and heat under reflux for extraction 1-2 times, 0.5-1 hour each time, combine the extracts, filter and concentrate to obtain.

Ulcerative colitis (UC) is a chronic, relapsing, non-specific inflammatory disease of the colon mucosa, and its pathogenesis remains to be fully elucidated. It is currently believed that the causes of this disease include immune system abnormalities, genetic factors, intestinal microbiota imbalance and environmental factors = many aspects. In clinical medicine, mucosal healing in UC patients is receiving more and more attention. The mucosal barrier is a protective barrier established by IECs tightly arranged between the harmful external environment and the internal environment, and is an important line of defense for the body. The mucosal barrier is composed of IECs and their secretions, in which the mucin MUC2 is a key component of mucus and prevents large particles, including bacteria, from directly contacting the epithelial cell layer. In addition to producing the mucus layer, IECs also form a continuous physical barrier with the epithelial junction complex formed by tight junctions, adherens junctions, and desmosomes. Therefore, IECs play a key role in the intestinal mucosal barrier, and the death of IECs is the key to the development and progression of UC. Understanding the death mechanism of IECs and how to prevent intestinal cell damage is of great significance for the diagnosis and early treatment of UC. This study found that Alligator flower extract CN can repair the intestinal mucosal barrier damaged by DSS in mice.

More and more studies have shown that oxidative stress plays a certain role in the progression of UC. Oxidative stress induced by abnormally high levels of reactive oxygen species (ROS) in the colon is a characteristic and causative factor of IBD. This was confirmed in the study of the present invention. A large amount of ROS and MDA accumulated in the colon tissue of DSS-induced model mice, and the contents of antioxidant enzymes SOD and GSH were also reduced. Oxidative stress not only promotes inflammation but can also lead to cell death through the oxidation of DNA, proteins, lipids, and virtually any other cellular component. Nrf2 and its endogenous inhibitor Keap1 are ubiquitous and evolutionarily conserved intracellular defense mechanisms against oxidative stress. Under normal circumstances, Keap1 in the cytoplasm sequesters Nrf2 and directs it to the proteasome for degradation. Under conditions of oxidative stress, Nrf2 detaches from Keap1 and translocates to the nucleus, binds to the antioxidant response element (ARE), and recruits transcriptional antioxidant key factors (such as HO1, NQO1). Through protein immunoblotting, the present invention verified that CN can regulate oxidative stress through the Nrf2/Keap1 signaling pathway and alleviate UC. In addition, the present invention also noticed that CN also plays a role in repairing oxidative damage. In UC model mice, the DNA damage marker H2AX was down-regulated and the expression level of the DNA repair enzyme OGG1 was up-regulated after treatment. The DNA damage response is initiated after receiving oxidative stress signals, which will be detrimental to cell survival and maintenance of genome stability, and OGG1 can recruit and repair oxidative DNA damage.

The medicine of the present invention contains an effective content of Alligator's Mouth flower extract and a pharmaceutically acceptable carrier. The medicine for preventing or treating ulcerative colitis of the present invention can be prepared into a suitable dosage form by conventional methods in the art. Preferably, the dosage form of the drug is a mixture, tablet, capsule, pill, powder or granule.

Compared with the existing technology, the present invention has the following excellent effects:

The present invention provides a new use of crocodile flower extract in preparing drugs for preventing or treating ulcerative colitis. By establishing a UC mouse model of ulcerative colitis induced by dextran sulfate sodium (DSS), the results It has been shown that crocodile flower extract can down-regulate the levels of inflammatory factors such as IL-6 and TNF-α, inhibit the expression of inflammatory pathway proteins such as IKKβ/IKKα/NF-κB, JAK2/STAT3, and up-regulate intestinal tight junction proteins ZO1 and Claudin1. , Occludin and the expression of MUC2, a key protein in intestinal mucosal barrier homeostasis. At the same time, it also inhibits further damage to colon tissue caused by oxidative stress, thereby maintaining the integrity of the epithelial cell barrier, restoring the activity of intestinal epithelial cells, exerting a therapeutic effect on UC, and providing a new drug source for clinical drug research on the treatment of ulcerative colitis.

Description of the drawings

Figure 1 shows the results of CN reducing ulcerative colitis induced by DSS in C57BL/6 mice (A is a schematic diagram of the research design; B is the weight change of mice in each group; C is the change in disease activity index; D is the statistical analysis of the colon of mice in each group ;E is the colon picture; F is the H&E picture);

Figure 2 shows the results of CN improving intestinal barrier function in UC mice (A is the effect on the activity level of diamine oxidase (DAO) in serum; B is the effect on the activity level of endothelin 1 (ET-1) in serum; C is the effect on the expression of colonic tight junction proteins (including ZO-1, occludin and claudin-1 proteins) in colon tissue; D is the result of WB quantitative analysis of colonic tight junction protein expression);

Figure 3 shows the results of CN inhibiting DSS-induced colon inflammation in UC model mice (A-C are the effects on pro-inflammatory cytokines in colon tissue, including IL-1β, IL-6 and TNF-a; D is the effect on the JAK2/STAT3 pathway Effect; E-F are the results of WB quantitative analysis of JAK2/STAT3 pathway protein expression; G is the effect on IKKα/β/IκBα pathway; H is the result of WB quantitative analysis of JAK2/STAT3 pathway protein expression; I is the immunohistochemical staining Representative images of determination of NLRP3 levels in mouse colon tissue);

Figure 4 shows the results of CN inhibiting the oxidative stress response in UC mice (A is the effect on MDA activity in colon tissue; B is the effect on ROS activity in colon tissue; C is the effect on SOD activity in colon tissue; D is the effect on colon tissue The effect of tissue GSH activity; E is the effect on the Keap1-Nrf2 pathway; F is the WB quantitative analysis results of Keap1-Nrf2 pathway protein expression; G-I is the determination of Nrf2, OGG1, and H2A.X in mouse colon tissue by immunohistochemical staining. horizontal representative image);

Note: #p<0.05, ##p<0.01, ###p<0.001 vs. the control group; *p<0.05, **p<0.01, ***p<0.001 vs. the DSS group.

Detailed ways

The present invention will be further described below through specific embodiments. The following examples are specific embodiments of the present invention, but the embodiments of the present invention are not limited by the following examples.

1. Materials and methods

1.1 Reagents and drug preparation

The dried leaves of Clinacanthus nutans (batch number: 20200824) were purchased from Hainan Wuzhishan Wanjiabao Technology Co., Ltd. and were identified as Clinacanthus nutans (Burm.f) by Zeng Congyan, director of the Department of Pharmacy, Zhongshan Hospital of Traditional Chinese Medicine. .)Dried leaves of Lindau.

Powder the fresh dried leaves of Alligator's Mouth Flower, pass through a 60-mesh sieve, soak in water for 2 hours, heat in water, and extract twice by refluxing for 1 hour each time. The solid-liquid ratio is set to 1:10 for the first time and 1:8 for the second time. Collect and filter, mix the filtrate, concentrate to 0.79g/ml, and store at 4°C to obtain the alligator flower extract.

1.2 Experimental design

Ten-week-old C57BL/6 mice (♂) were provided by Guangdong Experimental Animal Center (Foshan, China). Before the experiment, the animals were adapted to the laboratory conditions (23°C, 12 h/12 h light/dark, 50% humidity, free access to food and water) for 2 weeks, and no animals died before the experiment. All mice had free access to food and water and were maintained on a 12-h day-night cycle. Mice were randomly assigned to control group, 2.0% DSS (model), 2.0% DSS+SAPA (200 mg/kg, positive control), 2.0% DSS+CN high dose (CN-H, 7.8 g/kg, based on crude substance amount) and 2.0% DSS+CN low dose (CN-L, 3.9 g/kg, based on crude substance amount) group. SAPA and CN were administered orally from day 1 to day 14, and the mouse colitis model was induced with 2.0% DSS (W/V) for 7 days (from day 6 to day 14). The mice were observed 2 hours after cessation of CN treatment. The experimental design is shown in Figure 1, A.

1.3 Disease Activity Index (DAI)

DAI is a comprehensive score that combines weight loss percentage, stool consistency and stool bleeding. The total score of the three results is divided by 3 to obtain the DAI value, that is, DAI = (body mass index + stool shape + bleeding) / 3.

1.4 Sample collection

After treatment, mice were anesthetized to collect serum and then dissected. Blood was collected and centrifuged at 3500 rpm for 15 min at 4°C to obtain serum. The colon was removed to measure the length. Part of the colon tissue was fixed with 4% paraformaldehyde (PFA), and the remaining tissue was stored in a -80°C refrigerator.

1.5 Hematoxylin-eosin (H&E) staining

The collected colon tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Paraffin-embedded tissues were sectioned into 4-μm-thick sections and then stained with hematoxylin and eosin. Observe the morphological changes of the tissue under an optical microscope and take pictures.

1.6 Enzyme-linked immunosorbent assay

ELISA kits were obtained commercially, and biochemical analyzes were performed according to the manufacturer's instructions. The colon was washed with cold phosphate buffered saline (PBS, 0.01 M, pH 7.4) and then homogenized with 10x PBS (V/W). The samples were centrifuged at 5000g for 10 min at 4C, and the supernatant was collected for biochemical analysis. Collect mouse blood samples and stand at room temperature until the upper layer of serum is formed, then centrifuge, collect the upper layer, and freeze at −80°C.

1.7 Western blot analysis

Western blotting was performed under standard procedures. Colon tissue protein samples were lysed on ice for 30 min with RIPA lysis buffer added with protease inhibitors and phosphatase inhibitors (RIPA lysis buffer: protease inhibitor: phosphatase inhibitor = 50:1:1). Collect the protein supernatant, centrifuge at 15,000 rpm for 10 min at 4°C, and determine the protein concentration using a BCA protein detection kit. After separation on an 8%-12% sodium dodecyl sulfate polyacrylamide gel, the protein (40 μg) was transferred to a PVDF membrane. After blocking with 5% skim milk, the PVDF membrane was incubated with primary antibodies in a cold room overnight. After washing three times with phosphate buffered saline Tween (PBST) for 15 min, the PVDF membrane was incubated with primary antibody. After washing with phosphate buffered saline Tween (PBST) for three times for 15 min, the PVDF membrane was incubated with secondary antibody. Use ECL kit to observe immunoreactive protein bands. Images were acquired using a Biospectroscopy gel imaging system.

1.8 Immunohistochemistry (IHC)

Embed in paraffin, section with a thickness of 4 m, bake the sections in an oven at 70°C for 1 hour, dewax with xylene, dehydrate with graded ethanol, add 3% H ₂ O ₂ and protect from light for 10 minutes to inactivate endogenous enzymes, citrate repair solution (pH 6.0 ) Heat repair in a pressure cooker for 3 minutes, add NLRP3, Nrf2, MUC2, H2A.X, and OGG1 antibodies dropwise, and incubate in the refrigerator at 4°C overnight. After washing with phosphate buffer saline (PBS) the next day, dropwise add goat anti-rat/rabbit immunoglobulin G polymer, incubate at room temperature for 30 minutes, develop color with DAB, and control the reaction time under a microscope. After staining is completed, Rinse with tap water, counterstain with hematoxylin, dehydrate and clear, and seal with neutral gum. and take pictures using an optical microscope.

1.9 Statistical analysis

Statistical analysis was performed using GraphPad Prism software (version 8). All data are expressed as mean ± standard error of the mean (SEM). Statistical differences between the two groups were compared using Student's t test and one-way analysis of variance (ANOVA) with multiple comparisons. Differences were considered statistically significant with a p value of <0.05.

2. Experimental results

2.1 CN can alleviate DSS-induced ulcerative colitis

Compared with the control group, the body weight of the mice in the DSS group began to decrease significantly from the 4th day after DSS administration, and showed a downward trend (B in Figure 1). The DAI score began to increase significantly on day 9, and diarrhea and blood in the stool became increasingly severe (Figure 1, C). At the same time, the length of the colon was also shortened (D-E in Figure 1). These clinical manifestations are similar to those of UC patients. After administration of CN and SASP, the weight loss was reversed, the DAI score was significantly reduced, and the colon length was restored (Fig. 1, B-E). In addition, H&E staining results showed (F in Figure 1) that compared with the control group, the colon structure of the mice in the DSS group was significantly damaged, and a large number of neutrophils and lymphocytes infiltrated into the lamina propria and submucosa, indicating that the intestinal The permeability of the mucosal barrier is increased, and the intestinal mucosal barrier function is impaired. After administration in the SASP group, CN-L group and CN-H group, inflammatory cell infiltration was reduced, intestinal mucosal epithelial cell integrity was improved, and colon mucosal damage was reduced. This shows that CN can restore the colon structure and inhibit the damage to intestinal epithelial cells caused by DSS-induced colon inflammation.

2.2 CN can improve the mucosal barrier of UC mice

An increasing number of studies have proven that damage to the mucosal barrier and increased intestinal permeability play an important role in the pathogenesis of ulcerative colon disease. Increasing evidence shows that the destruction of mucosal barriers and increased intestinal permeability play a key role in the occurrence of UC. The present invention evaluates the changes in Diamine Oxidase (DAO) and Endothelin-1 (ET-1) in each group. High levels of ET-1 in the colon can lead to the accumulation of microcirculation disorders and exacerbate the development of colon inflammation. DAO is an enzyme involved in the decomposition and clearance of biogenic amines in the intestine and plays an important role in maintaining the integrity of the intestinal mucosal barrier. Under DSS induction, the expression levels of DAO and ET-1 increased in the DSS group. However, the expression of DAO and ET-1 was significantly reversed after CN treatment (Fig. 2, A). Studies have shown that the reduction of tight junction-associated proteins (occludin, ZO-1, claudin-1) is related to the infiltration of neutrophils. In this study, the expression of tight junction proteins ZO1, occludin and claudin1 increased after DSS induction. were significantly decreased, indicating that the intestinal barrier was severely damaged. After CN treatment, the expressions of ZO1, occludin and claudin1 increased significantly (D-E in Figure 2), indicating that CN has a good protective effect on the intestinal barrier of IBD mice. The present invention also uses IHC experiments to detect changes in the key protein MUC2 secreted by intestinal goblet cells. The results showed that CN treatment reversed the decrease in MUC2 secretion in DSS-induced IBD mice (F in the figure). This shows that CN can repair the intestinal barrier and maintain the integrity of the intestinal mucosa.

2.3 CN can inhibit inflammation and relieve UC

Inflammation plays a crucial role in the development and progression of ulcerative colitis (UC). When inflammation occurs, activated NLRP3 transmits signals to downstream IL-1β and promotes the expression of inflammatory factors IL-6 and TNF-α. At the same time, inflammation-related pathways such as IKKβ/IKKα/NF-κB and JAK2/STAT3 are activated. The results shown in Figure 3, A, B, and C, were that the expression levels of LPS, IL-6, and TNF-α in the colon tissue of mice in the DSS group were significantly higher than those in the Control group. After CN treatment, the expression levels of LPS, IL-6, and TNF-α were significantly reversed, indicating that CN effectively inhibited the production of inflammatory factors. In addition, the present invention uses WB and IHC to evaluate the expression changes of inflammatory pathway-related proteins. Figure 3 D and E show that compared with the Control group, the DSS group showed phosphorylation of JAK2 and STAT3. After CN drug treatment, the expression levels of phosphorylated JAK2 and STAT3 decreased significantly. F and G in Figure 3 show that the occurrence of UC also activates the IKKβ/IKKα/NF-κB pathway. Compared with the Control group, the expression levels of p-IKKβ/α, p-IκB, and p-NF-κB in the DSS group were significantly increased. After CN administration, the expression levels of p-IKKβ/α, p-κB, and p-NF-κB were significantly increased. reduce. In summary, it shows that the colon tissue of UC mice is in an inflammatory state, and the IKKβ/IKKα/NF-κB and JAK2/STAT3 inflammation-related signaling pathways are activated. CN may alleviate tissue damage caused by excessive inflammatory response by inhibiting the phosphorylation of proteins related to the IKKβ/IKKα/NF-κB and JAK2/STAT3 inflammatory pathways.

2.4 CN can enhance the antioxidant capacity of UC mice

Oxidative stress plays an important role in the onset and progression of IBD. Oxidative stress occurs due to excessive oxidative load due to increased ROS generation or reduced reduction reaction, which leads to increased intestinal permeability, promotes inflammatory reactions, and leads to lipid and protein modification, DNA damage, apoptosis, etc. The present invention studies the effect of CN on oxidative stress in the colon tissue of mice with DSS-induced colitis through ELISA, Western blot and IHC detection. As shown in A, B, C, and D in Figure 4, the levels of two important antioxidant parameters, glutathione peroxidase (GSH) and superoxide dismutase (SOD), were significantly reduced, and the oxidative stress markers Malondialdehyde (MDA) and ROS were significantly increased after DSS induction. After CN treatment, the effects of DSS on SOD, GSH, MDA and ROS were significantly reversed. In the Western blot experiment, the results of E and F in Figure 4 show that DSS significantly up-regulated the expression of Keap1 and down-regulated Nrf2 and its downstream factors HO1, NQO1 and Trx. After CN drug treatment, the expression of Keap1 was significantly reduced. The expression levels of HO1, Trx and NQO1 were significantly increased, indicating that CN has a good regulatory effect on oxidative stress. Through IHC experiments, it was found that the expression level of DNA damage marker H2A. After CN administration, the expression levels of Nrf2 and OGG1 were up-regulated in the colon, while the expression of H2A.X was inhibited (Figure 4, G, H, I). This suggests that CN plays a role in repairing oxidative damage. In summary, CN can alleviate ulcerative colitis in mice by enhancing the antioxidant activity of the colon to enhance the mediated intestinal barrier.

In summary, the above studies show that: Alligator flower extract can down-regulate the levels of inflammatory factors such as IL-6 and TNF-α, inhibit the expression of inflammatory pathway proteins such as IKKβ/IKKα/NF-κB, JAK2/STAT3, and up-regulate intestinal tight junction proteins. The expression of ZO1, Claudin1, Occludin and the key protein MUC2 for intestinal mucosal barrier homeostasis. At the same time, it also inhibits further damage to colon tissue caused by oxidative stress, thereby maintaining the integrity of the epithelial cell barrier, restoring the activity of intestinal epithelial cells, exerting a therapeutic effect on UC, and providing an effective candidate drug for the treatment of UC.

Claims (8)

1. Application of crocodile flower extract in preparing medicament for preventing or treating ulcerative colitis is provided.

2. The use according to claim 1, wherein the crocodile flower extract is a crocodile flower aqueous extract.

3. The use according to claim 1, wherein the preparation method of the alligator water extract comprises the following steps:

pulverizing dried crocodile leaves, sieving with 60 mesh sieve, soaking in water for 1-2 hr, adding water, heating and reflux extracting for 1-2 times each for 0.5-1 hr, mixing extractive solutions, filtering, and concentrating.

4. The use according to claim 1, wherein said crocodile flower extract is capable of repairing the intestinal barrier and maintaining the integrity of the intestinal mucosa.

5. The use according to claim 1, wherein the crocodile flower extract is capable of inhibiting the phosphorylation of ikkβ/ikkα/NF- κb and JAK2/STAT3 inflammatory pathway related proteins to alleviate damage to tissues by excessive inflammatory responses.

6. The use according to claim 1, wherein the crocodile flower extract is capable of enhancing the antioxidant activity of the colon.

7. The use according to claim 1, wherein the medicament comprises an effective amount of crocodile flower extract and a pharmaceutically acceptable carrier.

8. The use according to claim 1, wherein the medicament is in the form of a mixture, tablet, capsule, pill, powder or granule.