Application of curcuma wenyujin extract and enema thereof in treatment of ulcerative colitis

Abstract

The invention relates to the technical field of natural medicines, and particularly discloses application of a curcuma wenyujin extract and an enema thereof in treating ulcerative colitis. Experiments show that the curcuma wenyujin extract can repair intestinal epithelial barrier, relieve oxidative stress of intestinal epithelium and reduce generation of neutrophil ROS (reactive oxygen species), and through the synergistic effect of multiple ways, the disease progress of ulcerative colitis is remarkably relieved in vivo, the intestinal barrier function is enhanced, and inflammatory response is controlled. Therefore, the curcuma wenyujin extract has great potential in preparation of medicines for treating ulcerative colitis. The application range of the curcuma wenyujin extract is widened, an experimental basis is provided for the curcuma wenyujin extract and the enema thereof to treat clinical ulcerative colitis, and the curcuma wenyujin extract and the enema thereof have a good clinical transformation prospect.

Description

Technical Field

[0001] The invention relates to the technical field of natural medicines, and specifically provides application of a Curcuma zedoaria extract and an enema thereof in treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a chronic nonspecific inflammatory bowel disease. It can occur in people of any age and is characterized by persistent and diffuse inflammation involving the rectal and proximal colon mucosa and submucosa. Clinical manifestations include abdominal pain, diarrhea, mucus, pus and blood in the stool, and weight loss. Histopathology shows diffuse inflammatory response, reduced goblet cells, and destruction of crypt structure. Ulcerative colitis has a long course, recurrent symptoms that are difficult to cure, seriously interfering with the patient's normal work and life, and there is a risk of cancer. In recent years, its incidence and prevalence have been increasing year by year and becoming younger worldwide, and it is listed as one of the most serious gastrointestinal diseases by the World Health Organization.

[0003] The pathogenesis of ulcerative colitis is very complex and has not yet been clarified. The mainstream view is that it is mostly related to intestinal barrier dysfunction, immune disorders, host susceptibility genes, and intestinal microecological disorders. Among them, intestinal barrier dysfunction is the main cause. The tight junctions between the intestinal mucosal barrier and intestinal epithelial cells are destroyed, resulting in increased paracellular permeability and increased ability to absorb pathogens, thereby triggering a large number of immune cell infiltrations in the lesion site. At the same time, multiple immune-related pathways are activated, resulting in a serious imbalance in the ratio of anti-inflammatory factors and pro-inflammatory factors, further accelerating the development of inflammation.

[0004] The unclear pathogenesis has resulted in no specific treatment for ulcerative colitis. Currently, the commonly used intervention drugs in clinical practice focus on controlling inflammation and alleviating disease progression, mainly including 5-aminosalicylate, steroid hormones, immunosuppressants and monoclonal antibodies. However, the above drugs usually have slow onset, poor efficacy, serious adverse reactions, are prone to drug resistance, have a high recurrence rate after discontinuation of the drug, and are expensive. Therefore, finding highly effective, low-toxic and inexpensive natural drugs for the prevention and treatment of ulcerative colitis has become an urgent problem to be solved in contemporary medicine.

[0005] Using old drugs in new ways is a common strategy for drug development, which can significantly shorten the development cycle of new drugs and reduce scientific research investment. In recent years, studies have found that traditional Chinese medicine has shown great potential in the treatment of ulcerative colitis, with significant effects in relieving intestinal inflammation, repairing mucosal barriers, and regulating immune function. It has long-lasting effects, low prices, and high safety, and has gradually become a research hotspot for new anti-ulcerative colitis drugs.

[0006] Curcuma wenyujin's extract (CWE) is extracted from Curcuma wenyujin (Curcuma wenyujin YH Chen et C. Ling). In this application, the extract of Curcuma wenyujin refers to the oily product obtained by heating and refluxing the dry powder of Curcuma wenyujin, also known as Curcuma wenyujin volatile oil or Curcuma zedoaria volatile oil, which contains a variety of active ingredients such as geranone, β-elemene, curcumol, etc., and has been included in the "Chinese Pharmacopoeia". Wide clinical applications have confirmed that it has strong pharmacological activity and has significant anti-tumor, antiviral, antibacterial, anti-inflammatory and other effects. Among them, many single components have been proven to have the effect of inhibiting inflammation. For example, curcumol can significantly inhibit the increase of inflammatory factors such as interleukin-2 and interleukin-6 in the psoriasis-like auricle tissue of guinea pigs induced by propranolol. However, the pharmacological activity of the extract of Curcuma wenyujin in the treatment of ulcerative colitis has not been reported.

[0007] In view of this, the present invention applies the extract of Curcuma wenyujin to the research of preparing the drug for treating ulcerative colitis, and finds that it can significantly alleviate the disease process, enhance the intestinal barrier function, control the inflammatory response, etc., and provides an experimental basis for the development and application of the extract of Curcuma wenyujin as an anti-ulcerative colitis drug. In addition, Curcuma wenyujin is low in price, high in safety, and has a relatively simple extraction process, which can significantly reduce the scientific and technological investment and the medical burden of patients, and has considerable clinical application prospects. Summary of the invention

[0008] The invention proves that the enema of Curcuma wenyujin extract can effectively treat ulcerative colitis, and provides an experimental basis for the development of Curcuma wenyujin extract as a potential drug for treating ulcerative colitis.

[0009] The present invention has obtained through a large number of pharmacological experimental studies that the enema of Curcuma aromatica extract has a significant effect of antagonizing dextrose sodium sulfate (DSS)-induced ulcerative colitis in mice, can effectively improve weight loss and diarrhea and bloody stools, significantly improve pathological changes, and significantly reduce inflammatory responses.

[0010] In order to achieve the above technical objectives and the above therapeutic effects, the present invention adopts the following technical solutions: An enema for treating ulcerative colitis is composed of an active ingredient, Curcuma zedoaria extract, and an inactive ingredient, a pharmaceutical excipient.

[0011] Specifically, the mass content of the Curcuma aromatica extract in the enema is 0.1-10%.

[0012] Specifically, the mass content of the Curcuma aromatica extract in the enema is 0.5-5%.

[0013] Specifically, the mass content of the Curcuma aromatica extract in the enema is 0.5-2%.

[0014] An enema for treating ulcerative colitis is composed of an active ingredient, Curcuma zedoaria extract, and an inactive ingredient, a pharmaceutical excipient.

[0015] Some specific enema preparations for treating ulcerative colitis, wherein the Curcuma aromatica extract is prepared by a reflux extraction method, comprising the following steps: Place the dry powder of Curcuma aromatica medicinal material in a three-necked flask, add water to soak it, connect a volatile oil extractor to the three-necked flask, add water to the extractor until it just flows into the flask, install a condensing device, heat the flask to boiling, and stop heating when no oil droplets flow out of the extractor after reflux, release the water in the extractor, and take it.

[0016] Specifically, the dried powder of Curcuma aromatica medicinal material is mixed with water in a weight ratio of 1:10-50 for reflux extraction.

[0017] In some embodiments, the extraction conditions include: 10-50 parts by weight of Curcuma aromatica dry powder and 100-500 parts by weight of water.

[0018] In some specific enema preparations for treating ulcerative colitis, the dosage of the Curcuma aromatica extract is no less than 40 mg / kg each time.

[0019] In some specific enema preparations for treating ulcerative colitis, the dosage of the Curcuma aromatica extract is no less than 50 mg / kg each time.

[0020] In some specific enema preparations for treating ulcerative colitis, the dosage of the Curcuma aromatica extract is 40-100 mg / kg each time.

[0021] In some specific enema preparations for treating ulcerative colitis, the dosage of the Curcuma aromatica extract is 50 mg / kg each time.

[0022] The enema is in foam or liquid form.

[0023] The enema is an aqueous or oily solution, emulsion, gel or suspension.

[0024] The pharmaceutical excipients are selected from solvents (such as purified water, water for injection, soap water, physiological saline, glycerol, vegetable oil, dimethyl sulfoxide, isopropanol, polyethylene glycol 400, polyethylene glycol 600), stabilizers (such as Tween-80, poloxamer, cyclodextrin, nicotinamide, calcium glucarate), cosolvents (such as sodium glutamate, tyrosine), preservatives (such as benzoic acid, sodium benzoate, benzalkonium bromide, paraben, ethyl paraben, propoxyethanol), antioxidants (such as tocopherol, tea polyphenols, Butylated hydroxyanisole, isoascorbic acid and its salts, butylated hydroxytoluene, tert-butyl paraben, propyl gallate, potassium metabisulfite, sodium metabisulfite), antibacterial agents (such as benzalkonium chloride, ethyl hydroxybenzoate), pH adjusters (such as glacial acetic acid, sodium acetate, potassium acetate, potassium dihydrogen phosphate, sodium hydroxide, sulfuric acid, hydrochloric acid, citric acid, sodium citrate, ammonia solution, DL-malic acid, L-malic acid, DL-tartaric acid, fumaric acid, potassium bicarbonate, aminomethyl propanol), thickeners (such as glycerol, Propylene glycol, alginic acid, sodium alginate, carbomer, polycarbophil, xanthan gum, povidone, chitosan), emulsifiers (such as petrolatum, castor oil, egg yolk lecithin, soy lecithin, poloxamer 188, gum arabic, triethanolamine, sorbitan oleate, oleoyl polyoxyethylene glyceride, polysorbate 20), suspending agents (such as guar gum, sodium cholate, sodium carboxymethyl cellulose, agar, aluminum sulfate, polyvinyl alcohol), buffers (such as phosphate buffered saline, ammonium sulfate, borax), penetration enhancers One or more of the following: (such as urea, laurocapram, dimethyl sulfoxide, tetradecanol, caprylic acid, oleic acid), gelling agent (such as pectin), ointment base (such as ethyl oleate, yellow vaseline, stearic acid, polyethylene glycol 1500, polyethylene glycol 4000, polyethylene glycol 6000), lubricant (such as liquid paraffin, magnesium stearate, talc, polyethylene glycol 1000), osmotic pressure regulator (such as potassium nitrate, sodium sulfate, calcium chloride, potassium chloride, magnesium chloride), diluent (such as calcium sulfate).

[0025] A specific enema for treating ulcerative colitis, the enema is composed of a Curcuma aromatica extract, Tween-80 and normal saline, wherein the mass content of the Curcuma aromatica extract is 0.5-5%, and the mass content of Tween-80 is 5%-25%.

[0026] The packaging body of the enema for treating ulcerative colitis.

[0027] For some specific packages, the dosage is not less than 40 mg / kg each time.

[0028] For some specific packages, the dosage is 40 to 100 mg / kg each time.

[0029] The invention discloses an application of Curcuma aromatica extract only as an active ingredient in the preparation of a medicine for treating ulcerative colitis.

[0030] Here, only the Curcuma Radix extract is used as the active ingredient, which means that except the Curcuma Radix extract, no other active ingredients with therapeutic effects are contained, and other inactive pharmaceutical excipients mentioned above may be contained.

[0031] Furthermore, the Wenyujin extract enema was used as a drug for treating ulcerative colitis to conduct in vivo and in vitro pharmacodynamic evaluation and mechanism exploration using DSS colitis model mice and intestinal epithelial cells.

[0032] Furthermore, the content of the Curcuma aromatica extract in the drug for treating ulcerative colitis is not less than 30 mg / kg, that is, the content of the Curcuma aromatica extract is prepared according to the patient's weight.

[0033] The present invention has the following advantages: 1. The present invention not only broadens the application scope of Curcuma wenyujin extract, but also discovers potential candidate drugs for treating ulcerative colitis in natural products; 2. It has a more obvious advantage to use the extract of Curcuma wenjin as an enema to directly reach the affected area through local enema therapy. On the one hand, it can fully contact the lesion, quickly absorb and increase the drug concentration in the lesion, and achieve precise and efficient treatment; on the other hand, direct absorption through the colon affected area can significantly reduce the gastrointestinal tract's damage to the active ingredients of the drug, so that the drug can play a more effective role; 3. The present invention uses a DSS-induced mouse ulcerative colitis model to evaluate and confirm that Curcuma aromatica extract can effectively antagonize the disease progression of DSS colitis mice, alleviate weight loss, colon atrophy, and inflammatory cell infiltration in the lesion site, reduce the disease activity index (DAI), significantly enhance the intestinal barrier function, and control the inflammatory response; 4. The present invention uses human colorectal adenocarcinoma cell line (Caco-2) and mouse primary neutrophils as in vitro evaluation models, and found that the extract of Curcuma wenyujin can effectively protect the Caco-2 monolayer cell model in vitro from cytotoxicity, oxidative stress, and barrier damage caused by exposure to DSS, relieve the oxidative stress of inflammatory cells represented by neutrophils, and reduce inflammatory response; 5. The Curcuma wenyujin used in the present invention has a wide range of sources, is easy to collect, has low cost, and has a simple process flow for extraction and preparation. Clinical applications have confirmed that the Curcuma wenyujin extract has high biological safety;

[0034] 6. The Curcuma wenjin extract and its enema of the present invention have considerable market demand and prospects as anti-ulcerative colitis drugs, are suitable for industrial production, and have huge social and economic effects;

[0035] 7. The enema provided by the present invention uses only the Curcuma aromatica extract as an active ingredient to effectively treat and control ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a schematic diagram of the body weight of DSS colitis model mice after treatment with Curcuma aromatica extract enema according to the embodiment of the present invention.

[0037] Figure 2 This is a schematic diagram of the disease activity index (DAI) score of the DSS colitis model mice after treatment with Curcuma aromatica extract enema according to the embodiment of the present invention.

[0038] Figure 3 The figure is a schematic diagram of the colon length of the DSS colitis model mice after treatment with the Curcuma extract enema according to the embodiment of the present invention, wherein figure a is a photo of the colon length, and figure b is a bar graph of the difference in colon length.

[0039] Figure 4 This is a schematic diagram of the H&E staining of the colon to evaluate the histomorphological changes of the DSS colitis model mice after treatment with the Curcuma extract enema according to the embodiment of the present invention (×20). Among them, Figure a is the negative control group, Figure b is the DSS colitis model group, and Figure c is the Curcuma extract enema group.

[0040] Figure 5 This is a schematic diagram of the tight junctions of the colon intestinal epithelium of the DSS colitis model mice after treatment with Curcuma aromatica extract enema according to the example of the present invention (×10).

[0041] Figure 6 This is the effect of the Curcuma aromatica extract described in the examples of the present invention on the growth of Caco-2 cells.

[0042] Figure 7 The ability of the Curcuma zedoaria extract described in the embodiment of the present invention to inhibit the production of intracellular reactive oxygen species (ROS). Among them, Figure a is a Caco-2 cell line, and Figure b is a mouse primary neutrophil.

[0043] Figure 8 Schematic diagram of the inhibition of JAK / STAT pathway in Caco-2 cells by the Curcuma wenjin extract according to the embodiment of the present invention. Among them, Figure a is a schematic diagram of the determination of protein expression by protein immunoblotting, and Figure b is a schematic diagram of the qualitative analysis of protein expression by immunofluorescence (×20).

[0044] Fig. 9 The figure is a schematic diagram of the effect of the extract of Curcuma wenyujin on the tight junction of Caco-2 monolayer cells according to the embodiment of the present invention. Among them, figure a is a schematic diagram of the protein expression level measured by protein immunoblotting, and figure b is a bar graph of resistance value. DETAILED DESCRIPTION

[0045] In order to illustrate the technical solution of the embodiment of the present invention, the present invention will be further described below in conjunction with the data of the experimental example. The following experimental example is only used to illustrate the present invention and is not intended to limit the scope of application of the present invention. Various changes, modifications and modifications made to the present invention all belong to the scope limited by the rights of the present invention.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0047] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources or prepared according to conventional methods.

[0048] The enema of Curcuma extract used in the following examples is a liquid solution obtained by mixing Curcuma extract, Tween-80 and saline. The dosage is 50 mg / kg, calculated by the weight of Curcuma extract (50 mg of Curcuma extract is administered per kilogram of body weight).

[0049] The mass content of the Curcuma zedoaria extract in the Curcuma zedoaria extract enema is 1%, the mass content of Tween-80 is 11%, and the balance is normal saline. The Curcuma zedoaria extract is prepared by mixing the Curcuma zedoaria dry powder with water in a weight ratio of 1:10 and performing reflux extraction. Example 1

[0050] DSS is a polyanion derivative and one of the most common and effective drugs for inducing ulcerative colitis. It is widely used because its induced clinical manifestations and pathological changes are similar to those of human colitis. In this example, a mouse ulcerative colitis model was established using a DSS-induced method, and the therapeutic effect of Curcuma extract enema on ulcerative colitis was observed through an animal model experiment.

[0051] 1. Animal grouping and establishment of DSS colitis model Male C57BL / 6 mice aged 6-8 weeks and weighing 20-22 g were used as research subjects. The ulcerative colitis model was established by drinking water with a concentration of 2.5% DSS for 7 consecutive days, normal sterile drinking water for 7 consecutive days, and 2.5% DSS for 7 consecutive days. After purchase, the mice were adapted to a standard environment for 7 days, with no more than 5 mice per cage, and free access to water and food. All animal experiments were approved by the Animal Ethics Committee of Dalian University of Technology, and the relevant experiments were conducted in strict accordance with the principles of experimental animal welfare. The mice were randomly divided into 3 groups according to the standard laboratory method. The specific experimental process and grouping are as follows: Negative control group (Control): normal sterile drinking water.

[0052] DSS colitis model group (DSS): Drinking water containing 2.5% DSS for 7 consecutive days, changed to normal sterile drinking water for 7 consecutive days from the 8th day, and changed to drinking water containing 2.5% DSS for 7 consecutive days from the 15th day.

[0053] Curcuma wenyujin extract enema group (DSS+CWE): Drinking water containing 2.5% DSS for 7 consecutive days, changed to normal sterile drinking water for 7 consecutive days from the 8th day, changed to drinking water containing 2.5% DSS for 7 consecutive days from the 15th day, among which enema was given every other day from the 7th day, with a dose of 50 mg / kg, calculated as Curcuma wenyujin extract.

[0054] 1.1 Disease Activity Index (DAI) score During the experiment, the weight changes, fecal characteristics and blood in the stool of mice were recorded at a fixed time every day. The DAI score was performed according to the following standards. The weight change score was: 0-1% weight loss, recorded as 0 points; 1-5% weight loss, recorded as 1 point; 5-10% weight loss, recorded as 2 points; 10-20% weight loss, recorded as 3 points; and a decrease of more than 20% weight loss, recorded as 4 points. Stool characteristics score: normal, 0 points; loose but shaped, 1 point; loose, 2 points; very loose and moist, 3 points; diarrhea, 4 points. Fecal occult blood score: negative, 0 points; weak positive, 1 point; positive strong positive, 2 points; bloody stool, 3 points; obvious bloody stool, 4 points. After scoring, the total score of the three parts was calculated and the average was obtained.

[0055] 1.2 Sampling and colon length observation After the experiment, the mice were euthanized and the colon tissue was removed. The colon length was measured and photographed for comparison. After taking pictures, the samples were kept and placed in 4% paraformaldehyde and made into paraffin sections for H&E staining to detect microscopic histopathological changes; or embedded in frozen section embedding medium for immunofluorescence analysis.

[0056] 1.3 Colon histopathological observation and scoring Paraffin sections were dewaxed to water using conventional xylene, various levels of ethanol, and distilled water (xylene I 10 min-xylene II 10 min-absolute ethanol 5 min-95% ethanol 5 min-80% ethanol 5 min-70% ethanol 5 min-distilled water 10 min); H&E staining: hematoxylin staining for 10 min, rinse with running water, eosin staining for 2 min, rinse with running water. After dehydration, the sections were sealed with neutral resin, and pathological histological changes were observed using a research upright metallographic microscope. The histopathological evaluation criteria for colon sections were evaluated based on structural integrity, epithelial damage, and the degree of mucosal inflammatory cell infiltration.

[0057] 1.4 Expression of colonic tight junction-related proteins To further evaluate the protective effect of Curcuma wenyujin extract on colonic barrier function, immunofluorescence staining of ZO-1, Occludin, and Claudin 1 proteins was performed. Conventional immunofluorescence staining was performed on frozen sections, and changes in protein expression were observed using a fluorescence microscope.

[0058] 2. Experimental results 2.1 Effect of Curcuma zedoaria extract on body weight of mice with DSS colitis The weight line graph of each group of mice is shown in Figure 1 As shown, the weight of mice in the negative control group did not decrease during the experimental period. Compared with the negative control group, the weight of mice in the DSS colitis model group showed a significant downward trend during the experimental period. Compared with the DSS colitis model group, the weight loss trend of mice in the Wenyujin extract enema group during the experimental period was effectively controlled. This shows that Wenyujin extract can slow down weight loss and alleviate the progression of ulcerative colitis.

[0059] 2.2 Effect of Curcuma wenjin extract on the disease activity index (DAI) score of DSS colitis mice The DAI scores of mice in each group are shown in Figure 2 As shown, compared with the negative control group, the DAI score of mice in the DSS colitis model group was significantly increased, reaching 3.39±0.44 on the 21st day, and the loose stools and bloody stools were aggravated; the Wenyujin extract enema group could alleviate the rising trend of DAI score, which was only 2.67±0.42 on the 21st day, which was significantly different from the score of mice in the DSS colitis model group (P=0.0159), indicating that Wenyujin extract enema can significantly improve the symptoms of loose stools and bloody stools in ulcerative colitis and alleviate disease progression.

[0060] 2.3 Effect of Curcuma zedoaria extract on colon tissue length in mice with DSS colitis The colon of mice with ulcerative colitis induced by DSS atrophied and shortened. Figure 3 As shown. The colon length of mice in the negative control group was 7.89±0.29 cm, while that in the DSS colitis model group was significantly shortened to 4.35±0.20 cm (P<0.0001), and the intestinal wall was fragile and easy to break, the feces in the intestinal cavity had no complete shape, and obvious blood in the stool was visible. The colon length of the Wenyujin extract enema group was significantly increased compared with the DSS model group, reaching 5.76±0.38 cm (P<0.0001), and the feces were formed, and no obvious blood in the stool was seen, indicating that Wenyujin extract enema can slow down the shortening of the colon in model mice.

[0061] 2.4 Effect of Curcuma zedoaria extract on colon histopathological scores in mice with DSS colitis The results of H&E staining of the colon of each group of mice are shown in Figure 4 As shown (×20). Figure a is the negative control group, in which the colon tissue structure is intact, the epithelial cells, villi, and mucosal goblet cells are neatly arranged and morphologically intact, there is no edema in the submucosal layer, and no inflammatory cell infiltration; Figure b is the DSS colitis model group, in which the integrity of the colon tissue structure is significantly damaged, the glandular structure basically disappears, there is a large number of inflammatory cells infiltrating, the mucosal layer cells are necrotic and detached, and the edema of the muscle layer tissue increases; Figure c is the Wenyujin extract enema group, which shows that the above-mentioned colon lesions are significantly improved and the pathological changes are significantly alleviated. It shows that Wenyujin extract enema can maintain the integrity of the colon tissue structure of mice with ulcerative colitis and avoid inflammatory cell infiltration.

[0062] 2.5 Effect of Curcuma zedoaria extract on colonic epithelial tight junctions in mice with DSS colitis In the treatment of ulcerative colitis, repair of intestinal mucosal damage is a key link in promoting healing. Studies have shown that the expression of tight junction proteins plays an important role in the repair of intestinal mucosal damage and can maintain barrier function and the integrity of tight junctions, including three transmembrane proteins: zonula occluden (ZO), occludin, and claudin. The results of immunofluorescence staining of tight junction proteins in the colon of each group of mice are shown in Figure 2. Figure 5 As shown (×10). The fluorescence intensity of ZO-1, Occludin, and Claudin 1 in the DSS colitis model group was significantly weakened, indicating that the protein expression was significantly decreased and the intestinal mucosal barrier was severely damaged; the expression of the three proteins in the Wenyujin extract enema group was significantly increased compared with the DSS colitis model group, indicating that after treatment with Wenyujin extract enema, the tight junctions of the colon epithelium of the model mice can be effectively preserved and the repair of intestinal mucosal damage can be effectively promoted. Example 2

[0063] Studies have shown that DSS-induced Caco-2 monolayer cells are closely related to the early inflammatory response of colitis. When Caco-2 monolayer cells are exposed to a certain amount of exogenous DSS, tight junction damage and epithelial barrier dysfunction will occur. This in vitro model has been used to study DSS-induced cytotoxicity, intestinal barrier damage, cytokine release, cell signal transduction, etc., which is helpful for in-depth research on the treatment and pathogenesis of ulcerative colitis. In this experiment, the therapeutic effect of Curcuma wenyujin extract on ulcerative colitis was observed by establishing an in vitro Caco-2 monolayer cell model and neutrophils.

[0064] 1. In vitro model establishment and experiments 1.1 Protective effect on DSS-damaged Caco-2 The MTT test was used to determine the effects of different concentrations of Curcuma aromatica extract on the growth of Caco-2 cells. Caco-2 cells in the logarithmic growth phase and in good growth condition were selected for digestion and the cell density was adjusted to 8×10 4 The concentration of 100 μl / well was evenly inoculated into a 96-well plate (100 μl / well) and cultured until the cells adhered to the wall. Except for the negative control group, 2% DSS was added to the wells for 8 hours to induce inflammation. Then, the concentrations of Curcuma zedoaria extract were set to 0 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, and 400 μg / ml, and incubated for 16 hours. After drug withdrawal, 100 μl of MTT (0.5 mg / ml) was added to each well and incubated for 4 hours. After the incubation, 200 μl of dimethyl sulfoxide was replaced, and the absorbance of each well at 570 nm was measured using an enzyme reader.

[0065] 1.2 Reactive oxygen species (ROS) scavenging ability The reactive oxygen species probe DCFH-DA was used to detect the ability of Curcuma wenyujin extract to remove ROS in neutrophils and Caco-2 cells. Caco-2 cells were adjusted to 3×10 5 cell / ml, treated with 2% DSS for 8 hours, and then treated with 0μg / ml, 25μg / ml, 50μg / ml, and 100μg / ml of Curcuma zedoaria extract for 16 hours. The drug was withdrawn and 10nM DCFH-DA was added to the digested cells and incubated for 20 min, and analyzed by flow cytometry.

[0066] Neutrophils from bone marrow were extracted and activated with 100 ng / ml LPS for 4 hours, then 0 μg / ml, 25 μg / ml, 50 μg / ml, and 100 μg / ml of Curcuma aromatica extract were added for 4 hours, and 10 nM DCFH-DA was added and incubated for 20 minutes before analysis by flow cytometry.

[0067] 1.3 Inhibition of JAK / STAT pathway Western blotting and immunofluorescence were used to detect the inhibitory effect of Curcuma zedoaria extract on JAK / STAT pathway. Caco-2 cells were adjusted to 3×10 5cell / ml, treated with 2% DSS for 8 hours, and then treated with 0μg / ml, 25μg / ml, 50μg / ml, and 100μg / ml of Curcuma wenyujin extract for 16 hours. Caco-2 cells were lysed with RIPA lysis buffer, and proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. JAK2, p-JAK2, STAT3, p-STAT3, and GAPDH were labeled with specific antibodies, and finally imaged and analyzed using ECL matrix. Conventional immunofluorescence staining was performed on p-JAK and p-STAT3, and changes in their expression levels were observed using a fluorescence microscope.

[0068] 1.4 Protective effect on tight junctions of monolayer cells Western blotting was used to detect the protective effect of Curcuma wenyujin extract on the tight junctions of Caco-2 monolayer cells. Caco-2 cells were adjusted to 3×10 5 cell / ml, treated with 2% DSS for 8 hours, and then treated with 0μg / ml, 25μg / ml, 50μg / ml, and 100μg / ml of Curcuma zedoaria extract for 16 hours. Caco-2 cells were lysed and then electrophoresed, transferred to membranes, and blocked. ZO-1 and Occludin proteins were labeled, imaged and analyzed using ECL matrix. ZO-1 was subjected to conventional immunofluorescence staining, and its expression changes were observed using a fluorescence microscope.

[0069] 1.5 Protective effect on monolayer cell damage Transepithelial electrical resistance (TEER) was measured to assess damage to Caco-2 monolayers. Cells were adjusted to 2.5 × 10 5 cell / ml, inoculated into transwell chambers, and allowed to grow and differentiate for 21 days. TEER was measured regularly using a Millicell ERS-2 epithelial transmembrane cell resistance meter. Monolayer cells were treated with 2% DSS for 8 hours, then replaced with 0μg / ml, 25μg / ml, 50μg / ml, and 100μg / ml of Curcuma longifolia extract for another 16 hours, and TEER changes were monitored.

[0070] 2. Experimental results 2.1 Effect of Curcuma zedoaria extract on the growth of Caco-2 cells Effects of Curcuma zedoaria extract on the growth of Caco-2 cells Figure 6As shown. Compared with the negative control group without any treatment, the viability of Caco-2 cells treated with DSS was significantly reduced to 80.46±3.82% of the untreated negative control group (P<0.0001), while the cell growth was significantly promoted after treatment with 100μg / ml of Wenyujin extract, and the cell survival rate reached 99.42±7.65% relative to the negative control group (P<0.0001). It proves that Wenyujin extract has a protective effect against DSS-induced damage.

[0071] 2.2 Ability of Curcuma longa extract to scavenge intracellular ROS Studies have found that ROS is involved in the progression of colitis. ROS is an important regulator in the progression of inflammation. Excessive ROS will induce damage to lipids, proteins, and DNA by targeting the cysteine ​​redox switch, leading to functional damage to the colon and immune system, further exacerbating ulcerative colitis. In addition, patients with ulcerative colitis often have neutrophil infiltration in the colon. Activated neutrophils will produce excessive ROS in the colon tissue, further damaging the colon mucosa. Therefore, supplementing with antioxidants is beneficial to alleviate the progression of ulcerative colitis. The ability of Curcuma extract to clear ROS in Caco-2 cells and neutrophils is shown in Figure 2. Figure 7 shown.

[0072] The results showed that after exposure to DSS, the ROS in Caco-2 cells increased significantly, reaching 115.30±2.88% of the untreated negative control group (P=0.0008), while the addition of Curcuma aromatica extract significantly inhibited the production of ROS, especially at a concentration of 100 μg / ml, which was significantly reduced to 1 / 2 of the DSS exposure group at 0 μg / ml (P=0.0051).

[0073] Similar results were shown in neutrophils. After LPS stimulation, the mean fluorescence intensity of DCF increased to 3.37 times that of the unstimulated negative control group (P=0.0018), while ROS decreased in activated neutrophils treated with Curcuma zedoaria extract, especially at a concentration of 100 μg / ml, which was significantly reduced to 1 / 2 of the mean fluorescence intensity after LPS stimulation (P=0.0032). It was confirmed that Curcuma zedoaria extract can act as an antioxidant to remove ROS produced by cells after DSS damage and exposure to immunogenic stimulants, thereby avoiding further damage.

[0074] 2.3 Effects of Curcuma zedoaria extract on JAK / STAT signaling pathway The JAK / STAT pathway is generally considered to be a pro-inflammatory signaling pathway for colitis. STAT3 is highly phosphorylated in the colon tissue of patients with ulcerative colitis. Currently, JAK inhibitors have been approved for the clinical treatment of ulcerative colitis. Therefore, the inhibitory effect of Curcuma longifolia extract on the JAK / STAT signaling pathway was evaluated in vitro. Figure 8 As shown. After exposure to DSS, p-JAK2 and p-STAT3 increased significantly, indicating that the pro-inflammatory signaling pathway was activated. The immunofluorescence results showed the same trend. The extract of Curcuma zedoaria at a concentration of 100 μg / ml can reduce the phosphorylation of JAK2 and STAT3. It is confirmed that the extract of Curcuma zedoaria can inhibit the JAK / STAT signaling pathway, thereby reducing the transduction of inflammatory signals and avoiding the release of downstream inflammatory factors leading to further recruitment of immune cells.

[0075] 2.4 Effect of Curcuma wenyujin extract on tight junctions in Caco-2 monolayer cell model The tight junction proteins of Caco-2 monolayer cells reflect the integrity of the cell monolayer and are closely related to the ability of the intestinal epithelium to resist external hazards such as pathogenic microorganisms, antigens and toxins. Fig. 9 As shown. After exposure to DSS, the expression of ZO-1 and Occludin proteins decreased, and the expression increased after administration of Curcuma zedoaria extract, especially the concentration of 100μg / ml showed the best protective activity. Similarly, the TEER of the monolayer cell model exposed to DSS decreased significantly to 66.55±3.61% of the basal TEER (P=0.0029), indicating that the integrity of the monolayer was destroyed and the permeability increased, while the 100μg / ml Curcuma zedoaria extract had the most significant ability to resist damage, which increased the TEER to 113.75±16.38% of the basal TEER (P=0.0002). This suggests that Curcuma zedoaria extract can reduce epithelial tight junction damage after DSS exposure and maintain normal intestinal barrier function.

Claims

1. An enema for treating ulcerative colitis, characterized in that: The enema is composed of an active ingredient, Curcuma zedoaria extract, and inactive ingredients, pharmaceutical excipients.

2. The enema for treating ulcerative colitis according to claim 1, characterized in that: The mass content of the Curcuma aromatica extract in the enema is 0.5-5%.

3. The enema for treating ulcerative colitis according to claim 2, characterized in that: The Curcuma Radix extract is prepared by a reflux extraction method, wherein Curcuma Radix dry powder is mixed with water in a weight ratio of 1:10-50, and reflux extraction is performed until no oil beads flow out of the extractor, and heating is stopped to collect the Curcuma Radix extract.

4. The enema for treating ulcerative colitis according to claim 1, characterized in that: The dosage of the Curcuma aromatica extract is no less than 40 mg / kg each time.

5. An enema for treating ulcerative colitis according to claim 1, characterized in that: The dosage of the Curcuma aromatica extract is 40-100 mg / kg each time.

6. The enema for treating ulcerative colitis according to claim 1, characterized in that: The dosage of the Curcuma aromatica extract is 50 mg / kg each time.

7. The enema for treating ulcerative colitis according to any one of claims 1 to 6, characterized in that: The enema is in foam or liquid form.

8. A package of an enema for treating ulcerative colitis according to any one of claims 1 to 6.

9. Use of Curcuma aromatica extract in the preparation of a drug for treating ulcerative colitis using only Curcuma aromatica extract as an active ingredient.

10. The use according to claim 9, characterized in that: The Curcuma aromatica extract has at least one of the following effects: repairing the intestinal epithelial barrier, alleviating intestinal epithelial oxidative stress, reducing neutrophil ROS production, alleviating the disease progression of ulcerative colitis, enhancing intestinal barrier function, and controlling inflammatory response.

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