CN117414379A - Application of crocodile flower extract in preparation of medicines for preventing or treating ulcerative colitis - Google Patents
Application of crocodile flower extract in preparation of medicines for preventing or treating ulcerative colitis Download PDFInfo
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- CN117414379A CN117414379A CN202311554027.0A CN202311554027A CN117414379A CN 117414379 A CN117414379 A CN 117414379A CN 202311554027 A CN202311554027 A CN 202311554027A CN 117414379 A CN117414379 A CN 117414379A
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/19—Acanthaceae (Acanthus family)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
Abstract
The invention discloses application of crocodile flower extract in preparation of a medicament for preventing or treating ulcerative colitis. The results of establishing a dextran sodium sulfate (Dextran Sulfate Sodium, DSS) induced ulcerative colitis mouse model show that the crocodile flower extract can down regulate the level of inflammatory factors such as IL-6, TNF-alpha and the like, inhibit the expression of inflammatory pathway proteins such as IKKKbeta/IKKKalpha/NF-kappa B, JAK2/STAT3 and the like, and up regulate the expression of intestinal canal zonula occludens ZO1, claudin1, occludin and intestinal mucosa barrier steady-state key proteins MUC 2. Meanwhile, the further damage of oxidative stress to colon tissues is inhibited, so that the integrity of an epithelial cell barrier is maintained, the activity of the intestinal epithelial cells is recovered, the treatment effect of ulcerative colitis is exerted, and a new medicine source is provided for the medicine research of clinical treatment of ulcerative colitis.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicines, in particular to application of crocodile flower extract in preparation of a medicament for preventing or treating ulcerative colitis.
Background
Inflammatory bowel disease (inflammatory bowel disease, IBD) is a diffuse and recurrent inflammatory disease of the intestinal tract. The genetic susceptibility is affected by environmental factors, intestinal immune imbalance is caused under the participation of intestinal microorganisms, and intestinal mucosa barrier is damaged, so that persistent inflammation is caused. Among these, the two phenotypes, ulcerative colitis (ulcerative colitis, UC) and Crohn's Disease (CD), are mainly involved. IBD is most common in north america, western and northern europe, australia and new zealand, with prevalence of over 0.5% and up to 0.75% in canada and scotland, but in recent years the prevalence has rapidly increased in parts of asia and south america, and IBD has evolved into a global disease.
Inflammation of UC is mainly limited to the mucous membranes, with ulcers, oedema and bleeding of varying severity at the colonic site. Histopathologically, acute and chronic mucosal inflammation caused by granulocytes and monocytes is found, resulting in crypt abscess, mucosal gland deformation and goblet cell disappearance. Currently, conventional treatments for UC mainly include anti-tumor necrosis factor (anti-TNF) drugs, 5-aminosalicylic acid (5-ASA), salicylzolsulfapyridine (SASP), corticosteroids, and immunomodulators (e.g., thiopurine, methotrexate). 5-ASA is widely used to treat mild to moderate UC and as a maintenance therapy. Anti-inflammatory steroid drugs are useful in the onset of acute IBD, but are not suitable for maintenance therapy. Immunosuppressants are mainly used for maintenance therapy, but have limited efficacy and various adverse side effects. Therefore, finding an effective method for treating UC is a problem that needs to be solved in the medical community. Different from the traditional therapeutic drugs, the Chinese herbal medicine has the characteristics of multiple targets, low toxicity and the like, has potential value in the aspect of treating various complex diseases, and can be used as a potential source for developing new drugs for preventing and treating UC.
Crocodile flower Clinacanthus nutans Lindau (CN), called Clinopodium polycephalum and Philippine, is a plant of crocodile flower of Acanthaceae, and has effects of regulating menstruation, detumescence, removing blood stasis, relieving pain, and promoting bone fracture, and can be used for treating traumatic injury, anemia, jaundice, rheumatism, etc. The compounds separated and identified from crocodile flower at present comprise sterols, triterpenes, flavone carbosides, sulfur-containing glycoside, cerebrosides, chlorophyll, megastigmanes, benzenoids, alkaloids and the like. The main pharmacological actions include antivirus, antioxidation, anti-tumor, anti-inflammatory, antibiosis, immunoregulation and the like. Studies have shown that crocodile flower extracts can inhibit the activation of TLR-4 in RAW264.7 inflammation models to exert anti-inflammatory effects. In addition, crocodile methanol extract has anti-tumor and antioxidant activity in 4 T1 tumor-bearing mice. However, the literature report of crocodile flower extract in preparing medicines for preventing or treating ulcerative colitis is not known at present.
Disclosure of Invention
In view of the above, the present invention aims to provide the use of crocodile flower extract in the preparation of a medicament for preventing or treating ulcerative colitis.
The crocodile flower extract disclosed by the invention is a crocodile flower water extract.
Preferably, 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.
Ulcerative colitis UC is a chronic, recurrent, non-specific inflammatory disease of the colonic mucosa, the pathogenesis of which has yet to be fully elucidated. It is currently believed that the causes of the disease include immune system abnormalities, genetic factors, intestinal microbiota imbalance, and environmental factors, among others = multiple aspects. In clinical medicine, mucosal healing in UC patients is receiving increasing attention. The mucosal barrier is a protective barrier established by the close arrangement of the IECs between the harmful external ring and the internal environment, an important line of defense for the body. The mucosal barrier consists of IECs with their secretions, where mucin MUC2 is a key component of mucus, preventing large particulate matter, including bacteria, from directly contacting the epithelial cell layer. In addition to creating a mucus layer, IECs also form a continuous physical barrier with tight junctions (light junctions), adhesive junctions (adherens junctions), and epithelial junction complexes formed by desmosomes (desmosomes). Therefore, the IECs play a key role in the intestinal mucosal barrier and death of the IECs is key to the development and progression of UC. Understanding the death mechanism of IECs and how to prevent intestinal cell damage is of great importance for diagnosis and early treatment of UC. This study found that alligator extract CN was able to repair the intestinal mucosal barrier of DSS-induced mouse lesions.
More and more studies indicate that oxidative stress plays a role in the progression of UC. Abnormally high levels of Reactive Oxygen Species (ROS) -induced oxidative stress in the colon are characteristic and causative factors of IBD. This was confirmed in the studies of the present invention, where DSS-induced accumulation of large amounts of ROS and MDA in colon tissues of model mice was demonstrated, while the levels of antioxidant enzymes SOD and GSH were also reduced. Oxidative stress not only promotes inflammation, but also causes cell death through oxidation of DNA, proteins, lipids, and almost any other cellular component. Nrf2 and its endogenous inhibitor Keap1 are a ubiquitous, evolutionarily conserved intracellular defense mechanism against oxidative stress. Under normal conditions, keap1 in the cytoplasm sequesters Nrf2 and directs it to degradation in the proteasome. In the case of oxidative stress, nrf2 separates from Keap1 and translocates to the nucleus, where it binds to Antioxidant Response Elements (ARE), recruiting transcription of antioxidant key factors (e.g., HO1, NQO 1). Through western blotting, the invention verifies that CN can regulate and control oxidative stress through Nrf2/Keap1 signal paths to play a role in relieving UC. In addition, the invention also notices that CN also plays a role in oxidative damage repair, the UC model mice are subjected to down regulation of the DNA damage marker H2AX after the administration treatment, and the expression level of DNA repair enzyme OGG1 is also subjected to up regulation. The DNA damage response is initiated after being subjected to oxidative stress signals, which would be detrimental to cell survival and maintenance of genomic stability, while OGG1 is capable of recruiting and repairing oxidative DNA damage.
The medicine disclosed by the invention comprises an effective content of crocodile flower extract and a pharmaceutically acceptable carrier. The medicament for preventing or treating ulcerative colitis can be prepared into a proper dosage form by a conventional method in the field. Preferably, the dosage form of the medicine is mixture, tablet, capsule, pill, powder or granule.
Compared with the prior art, the invention has the following excellent effects:
the invention provides a new application of crocodile extract in preparing medicines for preventing or treating ulcerative colitis, and results show that the crocodile extract can down regulate the level of inflammatory factors such as IL-6, TNF-alpha and the like and inhibit the expression of inflammatory pathway proteins such as IKKbeta/IKKK alpha/NF-kappa B, JAK2/STAT3 and the like, and up regulate the expression of intestinal tight junction proteins ZO1, claudin1, occludin and intestinal mucosa barrier steady-state key proteins MUC2 by establishing a dextran sodium sulfate (Dextran Sulfate Sodium, DSS) induced ulcerative colitis UC mouse model. Meanwhile, the further damage of oxidative stress to colon tissues is inhibited, so that the integrity of an epithelial cell barrier is maintained, the activity of the intestinal epithelial cells is recovered, the UC treatment effect is exerted, and a new medicine source is provided for the medicine research of clinically treating ulcerative colitis.
Drawings
FIG. 1 shows the results of CN on reducing the ulcerative colitis of DSS-induced C57BL/6 mice (A is a schematic diagram of a study design; B is weight change of each group of mice; C is disease activity index change; D is colon statistics analysis of each group of mice; E is a colon picture; F is an H & E picture);
FIG. 2 shows results of CN improving intestinal barrier function in UC mice (A is the effect on the level of diamine oxidase (DAO) activity in serum; B is the effect on the level of endothelin 1 (ET-1) activity in serum; C is the effect on the expression of colonic zonal compact protein (including ZO-1, occludin and claudin-1 proteins) in colonic tissue; D is the result of quantitative analysis of WB for colonic compact protein expression);
FIG. 3 shows the results of CN inhibition of DSS-induced colon inflammation in mice model (A-C is the effect of pro-inflammatory cytokines in colon tissue, including IL-1β, IL-6 and TNF-a; D is the effect on the JAK2/STAT3 pathway; E-F is the quantitative analysis of WB for JAK2/STAT3 pathway protein expression; G is the effect on the IKK α/β/IκBα pathway; H is the quantitative analysis of WB for JAK2/STAT3 pathway protein expression; I is a representative image of NLRP3 levels in colon tissue of mice as determined by immunohistochemical staining);
FIG. 4 shows the results of oxidative stress inhibition by CN in UC mice (A is the effect of MDA activity on colon tissue, B is the effect of ROS activity on colon tissue, C is the effect of SOD activity on colon tissue, D is the effect of GSH activity on colon tissue, E is the effect on Keap1-Nrf2 pathway, F is the result of WB quantitative analysis of Keap1-Nrf2 pathway protein expression, G-I is a representative image of Nrf2, OGG1, H2A.X levels in colon tissue of mice as determined by immunohistochemical staining);
note that: #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 Description
The present invention will be further illustrated by the following examples, which are not intended to limit the scope of the invention.
1. Materials and methods
1.1 preparation of reagents and medicaments
Dry crocodile flower leaves (lot number: 20200824) purchased from the company Mo Gu, inc. of Wuzhishan, hainan, jingzhongshan, chinese medical department of Hospital, confucius clever-Angustifolia, identified as crocodile flower of crocodile genus of AcanthaceaeClinacanthus nutans(Burm.f.) Dry leaves of Lindau.
Pulverizing fresh dry crocodile leaves, sieving with 60 mesh sieve, soaking in water for 2 hr, heating in water, and extracting twice by reflux for 1 hr each time. The solid-to-liquid ratio was set to 1 for the first time: 10, set to 1 for the second time: 8. collecting, filtering, mixing filtrates, concentrating to 0.79g/ml, and storing at 4deg.C to obtain crocodile flower extract.
1.2 Experimental design
C57BL/6 mice of 10 weeks of age (, male) were supplied by Guangdong laboratory animal center (Buddha, china). Animals were acclimatized to laboratory conditions (23 ℃, 12 h/12 h light/dark, 50% humidity, free access to food and water) for 2 weeks prior to the experiment, with no animal death prior to the experiment. All mice were free to obtain food and water and maintained under 12 hours of diurnal cycling. Mice were randomly assigned to the 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 the mass of the coarse), and 2.0% dss+cn low dose (CN-L, 3.9 g/kg based on the mass of the coarse). Oral SAPA and CN from day 1 to day 14, mice were observed after 2h termination of CN treatment with 2.0% DSS (W/V) induced mouse colitis model for 7 days (from day 6 to day 14). The experimental design is shown in figure 1 a.
1.3 Disease Activity Index (DAI)
DAI is the combined score of percent weight loss, fecal consistency, and fecal hemorrhage, and the total score of the three results is divided by 3 to give the DAI value, dai= (body mass index + fecal form + bleeding)/3.
1.4 sample collection
Following treatment, anesthetized mice were collected serum and then dissected. Blood was collected and centrifuged at 3500 rpm for 15 min at 4℃to obtain serum. The colon was resected to measure length. Part of the colon tissue was fixed with 4% Paraformaldehyde (PFA) and the rest was stored in a-80 ℃ freezer.
1.5 hematoxylin-eosin (H & E) staining
The collected colon tissue was fixed with 4% paraformaldehyde and paraffin embedded. Paraffin-embedded tissues were sectioned to 4- μm thickness and then stained with hematoxylin and eosin. Morphological changes of the tissue were observed under an optical microscope and photographed.
1.6 ELISA
ELISA kits were obtained commercially and biochemically analyzed 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 10-fold PBS (V/W). Samples were centrifuged at 5000g for 10min at 4C and supernatants were collected for biochemical analysis. The mice were sampled, standing at room temperature until the upper serum layer formed, and then centrifuged to collect the upper layer, which was frozen at-80 ℃.
1.7 Western blot analysis
Immunoblotting was performed under standard procedures. Colon tissue protein samples were lysed on ice with the addition of protease inhibitor and phosphatase inhibitor with RIPA lysis buffer for 30 min (RIPA lysis buffer: protease inhibitor: phosphatase inhibitor=50:1:1). Protein supernatants were collected and centrifuged at 15000rpm at 4℃for 10min and protein concentrations were determined using the BCA protein assay kit. After separation with 8% -12% sodium dodecyl sulfate polyacrylamide gel, the protein (40 μg) was transferred onto PVDF membrane. After blocking with 5% skim milk, PVDF membranes were incubated with primary antibodies overnight in the cold room. After three washes with Phosphate Buffered Saline Tween (PBST) for 15 min, the PVDF membrane was incubated for the first antibody, and after three washes with Phosphate Buffered Saline Tween (PBST) for 15 min, the PVDF membrane was incubated for the second antibody. Immunoreactive protein bands were observed with ECL kit. Image acquisition uses a biological spectroscopy gel imaging system.
1.8 Immunohistochemistry (IHC)
Paraffin embedding, slicing with thickness of 4m, baking at 70deg.C for 1 hr, dewaxing with xylene, gradient ethanol dehydration, adding 3%H 2 O 2 Inactivating endogenous enzyme in dark for 10min, performing autoclave thermal repair with citrate repair solution (pH 6.0) for 3 min, dripping NLRP3, nrf2, MUC2, H2A.X, OGG1 antibody, and incubating overnight at 4deg.C. After washing with phosphate buffer (phosphate buffer saline, PBS) the next day, goat anti-rat/rabbit immunoglobulin G polymer was added dropwise, incubated at room temperature for 30 min, DAB developed, reaction time was controlled under a microscope, tap water washing, hematoxylin counterstaining, dehydration permeation and neutral resin sealing. And photographed 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 a one-way anova (anova) for multiple comparisons. Differences at p values <0.05 were considered statistically significant.
2. Experimental results
2.1 CN can relieve DSS induced ulcerative colitis
The body weight of the DSS group mice significantly decreased from day 4 of DSS administration and had a decreasing trend compared to the control group (B in fig. 1). The DAI score increased significantly starting on day 9, as well as diarrhea and blood in the stool (C in fig. 1). At the same time, the length of the colon is also shortened (D-E in FIG. 1). These clinical manifestations are similar to those of UC patients. Following CN and SASP administration, weight loss was reversed, DAI scores decreased significantly, and colon length recovered (B-E in fig. 1). Furthermore, H & E staining results showed (F in fig. 1) that the colon structure of DSS mice was significantly impaired compared to the control group, and that a large number of neutrophils and lymphocytes infiltrate into the lamina propria and submucosa, indicating an increased permeability of the intestinal mucosal barrier, and an impaired intestinal mucosal barrier function. After SASP group, CN-L group and CN-H group, inflammatory cell infiltration is reduced, intestinal mucosa epithelial cell integrity is improved, and colonic mucosa injury is reduced. Indicating that CN can restore colon structure and inhibit damage of DSS induced colon inflammation to intestinal epithelial cells.
2.2 CN can improve mucosal barrier of UC mice
More and more studies have demonstrated that impaired mucosal barriers and increased intestinal permeability play an important role in the pathogenesis of ulcerative colitis. There is growing evidence that disruption of the mucosal barrier and increase in intestinal permeability play a critical role in the occurrence of UC. The present invention evaluates the changes in the respective groups of Diamine Oxidase (DAO) and Endothelin-1 (ET-1). High levels of ET-1 in the colon can lead to the accumulation of microcirculation disorders, exacerbating the development of colonic inflammation. DAO is an enzyme involved in the breakdown and clearance of biogenic amines in the gut and plays an important role in maintaining the integrity of the intestinal mucosal barrier. Under DSS induction, DAO and ET-1 expression levels were elevated in the DSS group. However, after CN treatment, DAO and ET-1 expression was significantly reversed (a in fig. 2). There are studies showing that the decrease in light junction-associated proteins (occludin, ZO-1, claudin-1) is associated with neutrophil infiltration, in which the expression of the zonulin ZO1, occludin and claudin1 were all significantly decreased after DSS induction, suggesting that the intestinal barrier was severely compromised. While the expression of ZO1, occludin and claudin1 was significantly increased after CN treatment (D-E in FIG. 2), indicating that CN has a good protective effect on the intestinal barrier of IBD mice. The invention also uses IHC experiment to detect the change of the key protein MUC2 secreted by the intestinal goblet cells. The results indicate that CN treatment reversed the phenomenon of reduced MUC2 secretion in DSS-induced IBD mice (F in the figure). This demonstrates that CN is able to repair the intestinal barrier and maintain the integrity of the intestinal mucosa.
2.3 CN can inhibit inflammation and relieve UC
Inflammation plays a critical role in the occurrence and progression of Ulcerative Colitis (UC). Upon inflammation, activated NLRP3 signals downstream IL-1β, promoting the expression of inflammatory factors IL-6 and TNF- α. Simultaneously, inflammatory related pathways such as IKKbeta/IKKalpha/NF-kappa B, JAK2/STAT3 are activated. The results are shown in FIG. 3 as A, B, C, and the expression levels of LPS, IL-6 and TNF-alpha in colon tissues of mice in DSS group are significantly increased compared with those in Control group. After CN treatment, the expression level of LPS, IL-6 and TNF-alpha is obviously reversed, which proves that CN effectively inhibits the production of inflammatory factors. Furthermore, the present invention evaluates the expression changes of inflammatory pathway related proteins using WB and IHC. In fig. 3D, E, the phosphorylation of JAK2 and STAT3 occurred in the DSS group compared to the Control group. After CN drug treatment, the expression levels of phosphorylated JAK2 and STAT3 were significantly reduced. F, G in FIG. 3 shows that the occurrence of UC also activates the IKKbeta/IKKalpha/NF- κB pathway. Compared with the Control group, the expression level of p-IKKbeta/alpha and p-Iκ B, p-NF- κB of the DSS group is obviously increased, and the expression level of p-IKKbeta/alpha and p- κ B, p-NF- κB is obviously reduced after CN administration treatment. Taken together, it is demonstrated that the UC mouse colon tissue is in an inflammatory state and that the inflammatory-related signaling pathways of the body of IKKKbeta/IKKalpha/NF- κB and JAK2/STAT3 are activated. CN may alleviate damage to tissues from excessive inflammatory responses by inhibiting phosphorylation of IKKbeta/IKKalpha/NF- κB and JAK2/STAT3 inflammatory pathway-related proteins.
2.4 CN can enhance the antioxidation capability of UC mice
Oxidative stress plays an important role in the pathogenesis and progression of IBD. Oxidative stress occurs due to excessive oxidative load resulting from increased ROS production or reduced reduction reactions, resulting in increased intestinal permeability, promotion of inflammatory reactions, and resulting in lipid and protein modifications, DNA damage, apoptosis, etc. The influence of CN on oxidative stress in colon tissues of a colitis mouse induced by DSS is researched through ELISA, western blot and IHC detection. As shown in figure 4, A, B, C, D, the two important antioxidant parameters glutathione peroxidase (GSH) and superoxide dismutase (SOD) levels 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 Western blot experiments, as shown by the results of E, F in FIG. 4, DSS significantly up-regulates Keap1 expression, down-regulates Nrf2 and its downstream factors HO1, NQO1 and Trx. After CN drug treatment, keap1 expression is significantly reduced, while HO1, trx and NQO1 expression levels are significantly increased, indicating that CN has good regulation effect on oxidative stress. Through IHC experiments, the expression level of a DNA damage marker H2A.X in colon tissues of mice of a UC model induced by DSS is increased, and the expression of Nrf2 and DNA oxidative damage repair enzyme OGG1 is reduced. After CN administration, nrf2 and OGG1 expression levels were up-regulated in the colon, while h2a.x expression was inhibited (G, H, I in fig. 4). This suggests that CN plays a role in oxidative damage repair. Taken together, CN may alleviate ulcerative colitis in mice by enhancing the antioxidant activity of the colon to enhance the mediated intestinal barrier.
The above-mentioned studies show that: the crocodile flower extract can down regulate IL-6, TNF-alpha and other inflammatory factors, inhibit the expression of IKKKbeta/IKKalpha/NF-kappa B, JAK2/STAT3 and other inflammatory pathway proteins, and up regulate the expression of intestinal canal compact junction protein ZO1, claudin1, occludin and intestinal mucosa barrier steady-state key protein MUC 2. Meanwhile, the further damage of oxidative stress to colon tissues is inhibited, so that the integrity of an epithelial cell barrier is maintained, the activity of intestinal epithelial cells is recovered, the UC treatment effect is exerted, and an effective candidate medicine is provided for treating 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.
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