Use of ethyl acetate extract of curcuma zedoary in preparation of medicine for treating acute ulcerative colitis

Abstract

This invention belongs to the field of novel pharmaceutical uses, and more specifically, relates to the application of ethyl acetate extract of turmeric in the preparation of drugs for treating acute ulcerative colitis. The preparation method of the extract is as follows: turmeric slices are soaked overnight in 10-20 times their weight in a 60%-80% ethanol solution, refluxed at 80-90°C for 2-4 times, each time for 1-3 hours. The extracts are combined and concentrated until no alcohol odor remains, then dried to constant weight to obtain the total extract. The total extract is reconstituted with water and extracted 2-4 times with ethyl acetate at an equal volume ratio. The extracts are combined and dried to constant weight to obtain the final product. Experimental results show that this extract can significantly improve weight loss, disease activity index, colonic shortening, and histopathological damage in model mice, and effectively upregulate intestinal tight junction proteins and downregulate the expression of pro-inflammatory factors and pro-apoptotic proteins. This extract can be formulated into various dosage forms, providing a new natural drug option for the treatment of UC.

Description

Technical Field

[0001] This invention belongs to the field of new uses of pharmaceuticals, and more specifically, relates to the application of ethyl acetate extract of turmeric in the preparation of a drug for treating acute ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC) is a diffuse, nonspecific inflammatory bowel disease affecting the colorectal region, commonly found in the rectum and sigmoid colon. Its etiology and pathogenesis are closely related to genetic factors, immune abnormalities, environmental factors, psychological factors, and gut microbiota imbalance. Clinical symptoms of UC include diarrhea, abdominal pain, and bloody, mucous stools, which are closely related to the severity of intestinal inflammation. In the early stages of inflammation, intermittent abdominal pain and loose stools may occur. Under long-term inflammatory stimulation, a large amount of inflammatory mediators accumulate in the intestinal mucosa and submucosa, damaging intestinal mucosal epithelial cells, disrupting the normal intestinal structure, and causing pathological changes such as intestinal mucosal edema, fibrous tissue hyperplasia, polyp formation, and ulceration, increasing the risk of colorectal cancer. Studies have shown that long-term unhealthy lifestyle habits significantly increase the incidence of UC, and the disease is gradually becoming more prevalent in younger populations. Furthermore, UC is prone to recurrence, has a high risk of cancerous transformation, and has a slow prognosis; clinical treatment often focuses on controlling inflammation and preventing recurrence.

[0003] Natural medicines are pharmacologically active substances obtained from natural resources. Their sources are diverse, including plants, animals, minerals, and microorganisms. Furthermore, natural medicines contain abundant chemical components that exhibit anti-inflammatory, anti-tumor, antioxidant, antibacterial, and antiviral biological activities. Their rich chemical structures and biological activities can provide new ideas for developing novel drugs and improve clinical treatment levels.

[0004] As a representative herbal medicine, turmeric contains abundant natural chemical products, including terpenes, curcuminoids, and alkaloids, which have pharmacological effects such as anti-tumor, anti-inflammatory, and antioxidant properties. However, its complex chemical composition, unclear active ingredients, and unknown regulatory mechanisms severely restrict the clinical application and further in-depth research of turmeric and its active components. Summary of the Invention

[0005] The purpose of this invention is to provide the application of ethyl acetate extract of turmeric in the preparation of a drug for treating acute ulcerative colitis.

[0006] This invention provides the application of ethyl acetate extract of turmeric in the preparation of a drug for treating acute ulcerative colitis. The preparation method of the ethyl acetate extract of turmeric is as follows: turmeric is chopped and soaked overnight in 10-20 times its weight of a 60%-80% ethanol solution. It is then refluxed at 80-90°C for 2-4 times, each time for 1-3 hours. The extracts are combined and concentrated until no alcohol odor remains. The extract is dried to constant weight to obtain the total extract. The total extract is reconstituted with water and extracted 2-4 times with ethyl acetate at an equal volume ratio. The extracts are combined and dried to constant weight to obtain the ethyl acetate extract of turmeric.

[0007] This invention stems from the urgent clinical need for safe and effective new drugs for ulcerative colitis. Addressing the unclear specific active components of turmeric, the focus shifted to finding solutions within the natural drug turmeric. First, a systematic extraction and separation process (ethanol extraction followed by sequential extraction with petroleum ether, ethyl acetate, and n-butanol) transformed the complex chemical composition of turmeric into a parallel screening of the efficacy of components with different polarities. Then, a DSS-induced acute UC mouse model was established for multi-level validation from phenotype to mechanism: Phenotypic indicators such as body weight, DAI score, colon length, and tissue H&E / AB-PAS staining were used to initially screen the ethyl acetate component (EA) as the key effective site; Western blotting was then used to delve into the molecular mechanism, confirming that EA bidirectionally regulates—downregulating pro-inflammatory factors (iNOS, IL-1β, IL-6, TNF-α) and pro-apoptotic proteins (Bax, Cleaved Caspase9, Cleaved PARP1) while upregulating intestinal barrier tight junction proteins (ZO-1, Occludin) and anti-apoptotic proteins (Bcl-2). This study followed the logic of "extraction and separation - in vivo efficacy screening - preliminary elucidation of molecular mechanisms", which not only clarified the therapeutic effect of EA components, but also confirmed its safety through histological evaluation of the heart, liver, spleen, lungs and kidneys, providing a complete chain of experimental evidence for the development of turmeric into an anti-UC drug.

[0008] Furthermore, the drug is used to relieve colonic and rectal shortening caused by acute ulcerative colitis.

[0009] Furthermore, the drug is used to increase the expression of tight junction proteins ZO-1 and Occludin in the colon of patients with acute ulcerative colitis, thereby reducing intestinal barrier damage.

[0010] Furthermore, the drug is used to inhibit the expression of inflammatory factors iNOS, IL-1β, IL-6 and TNF-α in the lesioned colon of acute ulcerative colitis.

[0011] Furthermore, the content of ethyl acetate extract of turmeric in the drug is 0.1 wt% to 99 wt%.

[0012] Furthermore, the drug is made from the ethyl acetate extract of turmeric tablets and pharmaceutically acceptable excipients.

[0013] Furthermore, the excipients include any one or more of non-toxic fillers, stabilizers, diluents, and adjuvants.

[0014] Furthermore, the diluent is either water or physiological saline.

[0015] The beneficial effects of this invention are as follows: This invention involves extracting turmeric slices, followed by alcohol extraction to obtain a total extract, and further extraction with petroleum ether, ethyl acetate, and n-butanol to obtain petroleum ether, ethyl acetate, and n-butanol components. Finally, the aqueous phase is dried to obtain the aqueous phase. An acute ulcerative colitis (UC) model was established in mice. From an in vivo experimental perspective, the therapeutic effects of the total turmeric extract and its components on DSS-induced UC in mice were evaluated using multiple indicators, including mouse body weight, disease activity index score, intestinal tissue pathological structure, and expression levels of inflammatory factors. This invention found that the ethyl acetate component of turmeric slices significantly reduced the rate of body weight loss, increased DAI score, shortened colorectal length, and massive infiltration of inflammatory cells in the intestinal mucosa caused by DSS in mice, significantly increased the number of goblet cells, and restored intestinal barrier function. Attached Figure Description

[0016] Figure 1 A photo of dried turmeric slices.

[0017] Figure 2 The graph shows the effects of turmeric extract and its components on the basic physiological characteristics of DSS-induced acute ulcerative colitis (UC) mice. A represents the weight change in acute UC mice, B represents the DAI score of acute UC mice, C represents the colon and rectum length of acute UC mice, which is the most characteristic feature of the total turmeric extract and its components, and D represents the statistical graph of colon and rectum length in acute UC mice. *P < 0.05, **P < 0.01, ***P < 0.001 (difference between the treatment group and the model group); ###P < 0.001 (difference between the model group and the blank control group).

[0018] Figure 3 H&E staining images of colorectal tissue from mice with acute UC, including turmeric extract and its components.

[0019] Figure 4 The graph shows the effects of the total extract of turmeric and its components on the heart and liver of mice. In the graph, A is the H&E staining of the heart tissue of mice in each group, B is the statistical graph of the heart-to-body ratio of mice in each group, C is the H&E staining of the liver tissue of mice in each group, and D is the statistical graph of the liver-to-body ratio of mice in each group.

[0020] Figure 5The figures show the effects of the total extract of turmeric and its components on the lungs and kidneys of mice. In the figures, A is the H&E staining of lung tissue in each group of mice, B is the statistical graph of the lung-to-body ratio in each group of mice, C is the H&E staining of kidney tissue in each group of mice, and D is the statistical graph of the kidney-to-body ratio in each group of mice.

[0021] Figure 6 The figures show the effects of the total extract of turmeric and its components on mouse kidneys. A shows the spleen size of mice in each group, B shows the spleen-to-body ratio of mice in each group, and C shows the H&E staining of spleen tissue of mice in each group.

[0022] Figure 7 This image shows AB / PAS staining of colorectal tissue from mice with acute UC, along with extracts of turmeric and their components.

[0023] Figure 8 The graph shows the effects of total extract of turmeric and its components on the expression of iNOS and IL-1β in the colon of mice with acute UC. In the graph, A is the Western blot map, B is the statistical graph of IL-1β protein expression, and C is the statistical graph of iNOS protein expression.

[0024] Figure 9 The graph shows the effects of total extract of turmeric and its components on the expression of IL-6 and TNF-α in the colon of mice with acute UC. In the graph, A is the Western blot map, B is the statistical graph of IL-6 protein expression, and C is the statistical graph of TNF-α protein expression.

[0025] Figure 10 The graph shows the effects of the total extract of turmeric and its components on the expression of ZO-1 and Occludin in the colon of mice with acute UC. In the graph, A is the Western blot map, B is the statistical graph of ZO-1 protein expression, and C is the statistical graph of Occludin protein expression.

[0026] Figure 11 The graph shows the effects of the total extract of turmeric and its components on the expression of apoptotic proteins Bax, Bcl-2, Cleaved Caspase 9, and Cleaved PARP1 in the colorectal region of mice with acute UC. In the graph, A is the Western blot map, B is the statistical graph of Bax protein expression, C is the statistical graph of Bcl-2 protein expression, D is the statistical graph of Cleaved Caspase 9 protein expression, and E is the statistical graph of Cleaved PARP1 protein expression. Detailed Implementation

[0027] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0028] Example 1: Extraction and separation of Rhizoma Curcumae Longae and its components.

[0029] The raw material Rhizoma Curcumae Longae used in this example was purchased from the GAP base of Chinese medicinal materials of Wenyujin in Ruian City, Wenzhou City, Zhejiang Province in December 2023. Figure 1 It is a dried decoction piece of Rhizoma Curcumae Longae. The outer skin of the Rhizoma Curcumae Longae decoction piece is yellow, the surface is rough and covered with wrinkles, the cut surface is yellowish white to brownish yellow, obvious annulations and numerous vein dots can be seen, and their shapes are various, most of them are oblong or irregular flakes. The size of the decoction piece is 6 - 9 cm. The texture of the decoction piece is firm and brittle, with a strong smell and a slightly bitter taste.

[0030] Weigh 2 kg of Rhizoma Curcumae Longae decoction pieces, chop them and put them into a round - bottom flask, add an ethanol solution with a volume fraction of 70% and a quantity 15 times the mass of the Rhizoma Curcumae Longae decoction pieces to submerge the decoction pieces, and soak overnight. Heat - reflux extract at 85 °C for 3 times (2 h each time), combine the filtrates, then concentrate the filtrates until there is no alcohol smell, and dry to constant weight to obtain 309.66 g of the total extract (TE). After redissolving with water, extract with petroleum ether, ethyl acetate, and n - butanol reagents successively according to the ratio (1:1, v / v) for 3 times each. After combining the filtrates, dry to constant weight to obtain 109.4 g of the petroleum ether fraction (PE), 31.05 g of the ethyl acetate fraction (EA), 35.22 g of the n - butanol fraction (BU), and finally dry the aqueous layer part to obtain 112.62 g of the aqueous phase fraction (AF).

[0031] Example 2: In - vivo effect of Rhizoma Curcumae Longae extract on dextran sulfate sodium (DSS) - induced ulcerative colitis in mice.

[0032] I. Experimental materials and design.

[0033] 1. Experimental animals: Male C57BL / 6J mice at 6 weeks of age (SPF grade), purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and raised in the animal experiment center of Wenzhou Medical University (animal implementation license number: SYXK (Zhe) 2021 - 0020) in a specific - pathogen - free (SPF - grade) clean animal house. The temperature is maintained at 25 ± 2 °C, the relative humidity is 40% - 70%, and a 12 - h light / dark cycle is satisfied. All experiments strictly comply with the relevant regulations of the "Regulations on the Administration of Laboratory Animals", and the animal experiment ethics batch number: xmsq2024 - 0437.

[0034] 2. Experimental Design: After one week of acclimatization, mice were randomly divided into 13 groups: blank control group (Ctrl), DSS model group (M), sulfasalazine enteric-coated tablets positive control group (P), high-dose turmeric total extract group (TEH), low-dose turmeric total extract group (TEL), high-dose turmeric petroleum ether component group (PEH), low-dose turmeric petroleum ether component group (PEL), high-dose turmeric ethyl acetate component group (EAH), low-dose turmeric ethyl acetate component group (EAL), high-dose turmeric n-butanol component group (BUH), low-dose turmeric n-butanol component group (BUL), high-dose turmeric aqueous component group (AFH), and low-dose turmeric aqueous component group (AFL). The high-dose groups received 300 mg / kg, and the low-dose groups received 150 mg / kg. Sulfasalazine enteric-coated tablets were administered at a dose of 125 mg / kg.

[0035] All components were thoroughly dissolved using a 1% Tween-80 aqueous solution, and the solution was prepared fresh each time it was used. During the experiment, the body weight of mice in each group was recorded daily. Mice in the Ctrl group were given normal drinking water, with the volume of water consumed calculated based on their body weight. The drug-treated groups were administered the drugs by gavage to mice sequentially according to their daily body weight.

[0036] Except for the Ctrl group, all treatment groups were given a 3% (w / v) DSS aqueous solution, administered daily by gavage in an equal volume to the drug for 10 days to induce acute UC. On day 15, blood was collected from the eyeballs of mice in each group, and blood, heart, liver, spleen, lung, and kidney tissues were collected and weighed, and the relevant data were recorded. The colorectal tissue from the ileocecal valve to the anus was separated, the length of the mouse colorectal tissue was recorded, and 1 cm of colonic tissue was harvested from the cecum and completely immersed in 4% paraformaldehyde tissue fixative. The mouse colorectal tissue was longitudinally dissected, the intestinal contents were washed away with physiological saline, the surface moisture of the colorectal tissue was drained, and it was immediately flash-frozen in dry ice and then stored at -80°C.

[0037] II. Measurement of physiological indicators.

[0038] 1. Experimental Methods: During the experiment, the mental and limb status, fur condition, and water intake of mice in each group were closely observed daily. Daily changes in body weight, fecal characteristics, and fecal blood were recorded in detail. The Disease Activity Index (DAI) was calculated as (weight loss score + fecal characteristics score + occult blood score) / 3. DAI was used as the scoring standard, as shown in Table 1.

[0039] Table 1: Disease Activity Index Scoring Criteria (1) The weight loss rate is based on the previous day's weight, according to Weight loss (%) = (W (n+1)d -W nd The weight loss is calculated using the formula 100% × 100%, and the degree of weight loss is scored according to the DAI scoring table.

[0040] (2) Stool viscosity: The stool characteristics are scored according to the characteristics of the stool, which mainly include normal formed stool, soft stool, and watery stool. The scores are based on Table 1.

[0041] (3) Fecal occult blood test: Fecal occult blood was detected in mouse feces using a fecal occult blood qualitative test kit. Take an appropriate amount of feces, add equal amounts of o-toluidine solution and oxidant, and score according to the color change of the feces within 20 seconds. If blood is visible around the anus or there is obvious blood in the stool, the kit test is not required. Refer to Table 1 for DAI score.

[0042] 2. Experimental results: such as Figure 2 As shown in Figure B, compared with the Ctrl group, the DAI index of mice in the M group gradually increased from day 2, and remained at the same level after reaching its maximum score. The M group had the highest DAI value, indicating that the acute UC model was successfully established. Compared with the M group, the DAI index of mice increased slowly after each component intervention. However, the DAI change trends of the TE, PE, BU, and AF groups showed a high degree of overlap. On day 9, the EA group showed a significant difference from the other groups, with its DAI score index showing a significant decrease and gradually approaching that of the Ctrl group. From day 12 to 14, the EAH group had the lowest DAI index (P < 0.01), and the difference was statistically significant. The DAI results indicate that the EA component is a key component of turmeric tablets in improving UC in mice.

[0043] The rate of change in mouse body weight is a key indicator in disease-associated inflammation (DAI), closely related to disease severity and reflecting inflammatory status, providing important information for subsequent research. During the modeling process, changes in mouse body weight are beneficial for evaluating the therapeutic effects of drugs and screening for effective treatments. Figure 2As shown in Figure A, the mouse body weight showed a decreasing trend, with the high-dose group showing significantly lower weight than the low-dose group, indicating a clear dose-dependent relationship between the experimental doses. During the 15-day experimental period, the body weight of the Ctrl group mice showed a steady increasing trend. Compared to the Ctrl group, the body weight of the M group mice decreased sharply and continuously from day 3, indicating the successful establishment of the DSS-induced mouse UC model. Compared to the M group, the body weight decrease in the P, TE, EA, BU, and AF groups was less than that in the M group, suggesting that these components may contain effective components that improve acute UC and can effectively resist DSS-induced inflammatory damage. However, from day 3 to day 13, the body weight of the PE group mice continuously decreased to its lowest point, indicating that this component could not effectively resist the acute inflammatory damage caused by DSS.

[0044] Currently, sulfasalazine enteric-coated tablets are mainly used clinically to treat ulcerative colitis (UC), and this drug was also used as a positive control in experiments. Compared with the positive control, the TEH, EA, BU, and AF groups were able to maintain a stable increase in mouse body weight, while the TEL mice experienced a significant decrease in body weight. This suggests that the TEH, EA, BU, and AF groups can effectively resist inflammatory damage induced by chemical reagents, further indicating that the positive control is not effective in clinical practice and there is an urgent need to find more effective alternative drugs to improve the overall level of clinical treatment.

[0045] Compared to the EA group, the BU group mice experienced a greater decrease in body weight, especially the BUL group, suggesting that the EA group can stabilize mouse body weight and may play a role in alleviating acute UC. From day 3 to 12, the AF group mice showed a sustained and significant decrease in body weight, suggesting that the AF group may contain a large amount of polysaccharides, leading to weight gain. The accumulation of polysaccharides in the mice further exacerbated inflammation.

[0046] In summary, the EA component may be the key to improving acute UC in mice. Compared with other components, the overall evaluation indicators of the PE group were almost not significantly different from those of the M group, suggesting that PE may contain multiple components that cause toxic side effects and inflammatory responses. The overall evaluation indicators of the AF and BU groups were significantly lower than those of the M group, suggesting that they contributed less to the improvement of acute UC by turmeric. Therefore, it is necessary to separate and extract turmeric slices to effectively isolate components with significant toxic side effects, retain the effective components, further improve the content of active ingredients, and ensure the safety and efficacy of turmeric extract.

[0047] III. Effects on colorectal length in mice.

[0048] 1. Experimental Methods: The mouse abdominal cavity was dissected to locate the cecum, colon, and rectum. The colon was carefully separated from the surrounding tissues, and the entire colon was completely freed from the cecum-colonic junction to the end of the rectum (near the anus). Surgical scissors were used to cut the colon at the junction and the end of the rectum, and a complete colon sample was obtained. The processed colon sample was laid flat in a dry, sterile culture dish, ensuring that the intestinal tract was not stretched, bent, or twisted, maintaining its natural physiological morphology. The measurement starting point was the center of the cross-section of the intestinal tract at the cecum-colonic junction, and the measurement endpoint was the center of the cross-section of the intestinal tract near the anus at the end of the rectum. A ruler was used to measure the linear distance along the central axis of the colon, avoiding applying external force to pull the intestinal tract during the measurement, and the values ​​were recorded (unit: cm).

[0049] 2. Experimental Results: Changes in colorectal length are an important indicator for evaluating the severity of acute UC. For example... Figure 2 The results of C and D studies revealed differences in colon and rectal length among the TEH, EAH, EAL, and BUH groups, indicating that the above components can significantly resist pathological damage such as colon and rectal shortening, intestinal wall thickening, and hardening caused by DSS modeling.

[0050] Compared with the Ctrl group, the M group mice showed a significantly shorter colon and rectum (P < 0.001). Compared with the M group, the EA group significantly reduced the colon and rectum shortening induced by the modeling drug. The EAH group, in particular, showed the longest colon and rectum with a thin, soft, and elastic intestinal wall. Among the drug-treated groups, the TE and BU groups had longer colon and rectums than the other groups. However, compared with the EA group, they were less effective in resisting the colon and rectum shortening induced by DSS and were not the optimal effective component for alleviating acute UC.

[0051] In summary, the analysis of key indicators such as weight loss rate, DAI index score, and colorectal length in mice shows that the EA group is the most effective part of turmeric tablets. In contrast, the PE component has strong toxic side effects, and the BU and AF components have weaker therapeutic effects and are not the key components for improving UC in mice.

[0052] IV. H&E staining analysis of mouse colorectal tissue and organs.

[0053] 1. Experimental Methods: H&E staining of mouse colorectal tissue: First, the contents of the cecal 1 cm colon tissue in each component were thoroughly washed with pre-cooled PBS solution. The cecum was then longitudinally cut and placed in 4% neutral paraformaldehyde tissue solution for fixation for 24 h. After removal, the tissue was washed with a gradient ethanol system and then cleared twice with xylene solution, 20 min each time. First, xylene I solution was used to remove ethanol from the tissue to achieve initial clearing, followed by treatment with xylene II solution to ensure complete clearing. The tissue was then transferred to an embedding cassette, filled with paraffin, and allowed to cool into blocks. These blocks were then cut to appropriate thicknesses and mounted on glass slides for subsequent staining. After dewaxing and hydration, the paraffin sections were stained with hematoxylin for 5 min, followed by rinsing with running water for 1 min to thoroughly remove excess staining agent. Next, the sections were differentiated using 1% hydrochloric acid-ethanol solution for 3-5 s to further enhance staining contrast. Subsequently, the slides were placed in a 0.2% ammonia solution (v / v), and a bluing reaction occurred after approximately 30 seconds, enhancing the staining effect on cell nuclei. Finally, the slides were thoroughly rinsed with running water. The slides were then immersed in eosin staining solution for approximately 30 seconds, followed by rinsing off excess stain with running water. The stained slides were dehydrated and cleared again, and finally mounted with neutral resin. A coverslip was gently pressed to remove air bubbles and avoid interfering with the experimental results. The pathological morphology of mouse colorectal tissue was evaluated under an optical microscope. Images were collected using a slide scanner and viewed and analyzed using CaseViewer software.

[0054] 2. Experimental Results: From Figure 3 It was found that the colon and rectum of mice in the Ctrl group were normal in morphology, with intact colonic mucosa, no abnormalities in crypt structure, no inflammatory cell infiltration, and a large number of goblet cells. Compared with the Ctrl group, the M group showed a large number of inflammatory cells, granulation tissue proliferation, accompanied by new capillaries and fibroblasts, irregular crypt surface with a villous appearance, and local atrophy and tortuosity. Moreover, the number of goblet cells decreased significantly, cell mucus secretion was reduced, and the colonic mucosa was severely damaged.

[0055] Compared to group M, the degree of colorectal damage in groups TE, PE, BU, and AF was similar, with no statistically significant difference. Furthermore, group P showed a large accumulation of basal lymphocytes, disordered crypt structure, and glandular atrophy in the colorectal tissue, indicating that the positive control drug exerts some pharmacological activity in improving acute UC, but it cannot cure the disease, and long-term use may worsen acute UC lesions. This further demonstrates that turmeric slices may contain components with toxic side effects, requiring further in-depth research.

[0056] In the treatment groups, the colorectal tissue of the EA group showed a relatively regular mucosal gland structure, intact and unbranched crypt surfaces, and a large number of goblet cells with minimal inflammatory cell infiltration. In particular, EAH showed the most ideal effect with significant difference (P < 0.001), suggesting that the EA group can significantly alleviate intestinal barrier damage caused by DSS and is the best active component for improving acute UC.

[0057] Analysis of H&E staining results of turmeric slices and their components revealed that the EA group exhibited the most significant therapeutic effect in improving acute UC, with its histological characteristics closely resembling those of the Ctrl group. In contrast, the section results of other treatment groups showed obvious inflammatory invasion, indicating that the components were not significantly effective in treating acute UC.

[0058] The heart, liver, spleen, lungs, and kidneys are key target organs. H&E staining was used to observe the tissue structure of these organs in mice. This staining method provides accurate and reliable results, enabling precise assessment of the safety of the total extract of turmeric and its components. During the experiment, when the high-dose group (300 mg / kg) and the low-dose group (150 mg / kg) were administered the drug, no serious damage was observed to the heart, liver, spleen, lungs, or kidneys in mice. Therefore, the dosage selected in this experiment can be confirmed as safe and reliable.

[0059] H&E staining provides a direct visual understanding of the degree of damage to the heart caused by total extract of turmeric and its components. Figure 4 Figure A shows the pathological changes in the heart tissue structure of mice after drug intervention. In the Ctrl group, the cardiomyocytes were normal, with oval-shaped, neatly arranged nuclei, and the cells appeared pink. Compared to the Ctrl group, the cardiomyocyte structure of the M group was quite consistent with that of the Ctrl group. After drug administration, the morphology of the cardiomyocytes in the mice was no different from that of the Ctrl group, based on the heart-to-body ratio statistical results (…). Figure 4 (B) There was no significant difference between the treatment group and the Ctrl group.

[0060] Damage to the colorectal mucosa and severe disruption of the intestinal barrier can also impair normal liver function. Figure 4 In group C, the hepatocytes of mice in group Ctrl were morphologically intact, with large, round nuclei and clear lobule boundaries. Compared to group Ctrl, the hepatocyte structure of mice in group M was consistent with that of group Ctrl. Combined with the results of the mouse liver-to-body ratio ( Figure 4 There was no significant difference between the D group and the Ctrl group.

[0061] Based on the H&E staining results of mouse lungs ( Figure 5In group A, the alveoli of mice in the Ctrl group showed an oval structure, with tightly packed and regularly arranged epithelial cells in the alveolar wall and dark purple nuclei, showing no abnormalities. However, the alveolar structure of mice in group M was normal and indistinguishable from that in the Ctrl group. Combined with the results of the mouse lung-to-body ratio ( Figure 5 (B) There was no significant difference between the treatment group and the Ctrl group.

[0062] Based on the H&E staining results of mouse kidneys ( Figure 5 In the C group (Ctrl group), the cell nuclei of kidney tissue cells were small, regular, and tightly arranged in a neat and orderly manner. However, the kidney tissue cell structure of the M group mice was no different from that of the Ctrl group. Combined with the results of the mouse kidney-to-body ratio (…), Figure 5 (D), there was no statistically significant difference between the treatment group and the Ctrl group.

[0063] The spleen is a key organ in the immune response, playing a variety of important functions. Splenomegaly is one of the most representative features of the inflammatory response, accompanied by an increased spleen-to-body ratio. In terms of spleen-to-body ratio, group M showed splenomegaly and congestion (…). Figure 6 The A group showed a significant difference from the Ctrl group (P < 0.001). Figure 6 The B-stain suggests that DSS caused severe spleen tissue damage. In H&E stained sections of mouse spleen (… Figure 6 In the C and Ctrl groups, spleen tissue cells showed clear round island-like structures, a distinct boundary between the red and white medulla, abundant red blood cells in the red medulla (appearing pale red), and rich lymphocytes and macrophages in the white medulla (appearing deep blue), with uniform cell distribution. Compared to the Ctrl group, the M group showed an unclear boundary between the red and white medulla, disordered cell arrangement, red blood cell congestion, reduced white medullary follicle volume, and diffusely widened macrophages in the marginal zone, appearing bluish-purple. Observation of the section results of each treatment group revealed that the PE group was highly consistent with the M group, with blurred red and white medullary boundaries, disordered cell arrangement, and widened marginal zones, indicating that the PE group caused severe damage to the spleen function in mice. In the BU, AF, TEL, and EAL groups, the red and white medullary boundaries were unclear, and the volume and number of red and white medulla were reduced. In the P, TEH, and EAH groups, the red and white medullary boundaries were clear, and the morphology of spleen tissue cells was normal, suggesting that these components could protect the morphology of spleen tissue cells and effectively block the damage of DSS to the spleen function of mice. In particular, EAH has a strong therapeutic effect, and the spleen index of EAH is significantly different from that of the M group (P<0.001).

[0064] Based on the above description, it can be concluded that the dosage used in this experiment did not cause damage to the heart, liver, lungs, or kidneys of the mice. However, in the spleen section results, it was observed that the spleen structure of the EAH group mice gradually returned to normal, indicating that the EAH group has a significant therapeutic effect in improving acute UC in mice.

[0065] V. Colon AB-PAS staining analysis.

[0066] 1. Experimental Methods: Mouse colon tissue (1 cm posterior to the cecum) was embedded and sectioned, then dewaxed and dehydrated using gradients of xylene and ethanol at different concentrations. First, sections of appropriate thickness were immersed in 1% Alcian Blue staining solution for 5 minutes, followed by gentle rinsing with running water. Then, Schiff's reagent was used for staining for 15-30 minutes, followed by rinsing with running water, resulting in a purplish-red color. Next, the sections were oxidized with 1% periodic acid solution for approximately 5 minutes, followed by rinsing with running water to remove excess stain. Hematoxylin was then used to counterstain the sections for 30-60 seconds, followed by blue reversion with running water. The sections were then dehydrated using gradients of ethanol, cleared with xylene, and finally mounted with neutral resin. Structural changes in the mouse colorectal tissue were observed under an optical microscope, including the integrity of crypt structures, the number and distribution of goblet cells, and the degree of inflammatory infiltration. Images were collected using a slide scanner and viewed and analyzed using CaseViewer software.

[0067] 2. Experimental results: such as Figure 7 As shown, according to the AB-PAS staining results, the colon and rectum of mice in the Ctrl group had normal morphology, appeared blue after AB-PAS staining, and contained a large number of goblet cells. Compared with the Ctrl group, the tissue sections of mice in the M group showed a lighter color after staining, with very few goblet cells, almost invisible, indicating that the intestinal mucosal barrier of mice in the M group was severely damaged. Among the drug-treated groups, the number of goblet cells in the P and EA groups increased significantly after staining, gradually approaching the strong positive reaction of the Ctrl group. This indicates that the P and EA groups can significantly improve DSS-induced acute UC, secrete sufficient mucin, and form an intestinal mucosal barrier to protect epithelial cells. In conclusion, the P and EA groups have a significant advantage in improving acute UC in mice; conversely, the other groups have weaker therapeutic effects.

[0068] VI. Expression of related proteins in the colorectal region of mice with acute UC.

[0069] 1. Experimental Methods: Protein samples were prepared from colonic tissue of mice with acute UC. First, 30 mg of mouse colonic and rectal tissue was weighed and placed in a 2 mL centrifuge tube. Then, lysis buffer (RIPA tissue lysis buffer: phosphatase inhibitor: PMSF = 100:1:1) was added to each centrifuge tube, and the tissue was thoroughly homogenized using a multi-sample tissue homogenizer. After lysis for 30 min, the tissue was centrifuged at 12000 rpm for 20 min at 4°C. The supernatant was collected and transferred to pre-chilled EP tubes. Next, a working solution (A:B = 50:1) was prepared for BCA protein quantification. An appropriate amount of protein supernatant was added, and the tubes were incubated at 37°C for 30 min. The absorbance of the protein samples was measured using a microplate reader, and the values ​​were calculated based on the results. The remaining samples were added to 4× protein loading buffer and heated in a metal bath at 100°C for 10 min to completely denature the protein samples. The samples were then stored at -20°C.

[0070] The target protein was separated using SDS-PAGE protein gel electrophoresis. PVDF membranes were pre-activated with methanol and transferred using a wet transfer method. After transfer, the membrane was blocked by immersing it in 5% skim milk powder for 1 hour, followed by washing three times with PBST for 10 minutes each time. The transferred PVDF membrane was then completely immersed in primary antibody solution and incubated overnight at 4°C, followed by washing three times with PBST for 10 minutes each time. It was then incubated with secondary antibody at room temperature for 1 hour, followed by washing three times with PBST for 10 minutes each time. Finally, the bands were placed flat in a chemiluminescence imaging system, and ECL ultrasensitive luminescent solution was evenly added above the bands for exposure and development to obtain clear images. The saved files were imported into ImageJ for grayscale analysis, using β-actin as an internal control protein to calculate the relative expression level of the target protein.

[0071] 2. Experimental results: such as Figure 8 As shown, compared with the Ctrl group, the inflammatory factors iNOS and IL-1β in the colon of mice in the M group were significantly upregulated, showing a significant positive reaction. This indicates that the overexpression of iNOS and IL-1β proteins aggravated the inflammatory damage, intestinal mucosal erosion, and ulceration of the colorectal tissue in mice, demonstrating the successful establishment of a DSS-induced acute UC model. Compared with the M group, the expression levels of iNOS and IL-1β proteins in the P, TEH, and EAH groups were significantly decreased, and the number of iNOS-positive cells in the colorectal mucosa of mice with UC gradually approached that of the Ctrl group, indicating that they could significantly alleviate colorectal mucosal damage in mice, protect the intestinal epithelial barrier function, and improve the inflammatory response of DSS-induced acute UC. In this experiment, the results of the EAH group were found to be more significant in improving acute UC in mice, showing potential clinical application value.

[0072] like Figure 9As shown, compared with the Ctrl group, the expression levels of IL-6 and TNF-α proteins in the colon and rectum of mice in the M group were significantly upregulated, indicating a severe degree of inflammation. The changes in IL-6 and TNF-α protein levels promoted the activation of other inflammatory cells, causing tissue damage and a more widespread inflammatory response, successfully establishing a DSS-induced acute UC model. Compared with the M group, the expression levels of IL-6 and TNF-α proteins in the colon and rectum of mice in the P, TEH, TEL, and EAH groups were significantly decreased, indicating that the treatment groups alleviated the DSS-induced acute UC inflammatory response. The protein expression levels in the EAH group were close to those in the Ctrl group, and its improvement effect was superior to other treatment groups, significantly reducing the inflammatory response in mice and demonstrating a highly effective therapeutic effect.

[0073] Analysis of the expression levels of iNOS, IL-1β, IL-6, and TNF-α proteins in the colorectal region of acute UC revealed that the EAH group had the most significant therapeutic effect, significantly reducing the expression levels of iNOS, IL-1β, IL-6, and TNF-α proteins, significantly inhibiting the inflammatory response, and effectively improving the symptoms of acute UC in mice. Other treatment groups showed poorer therapeutic effects.

[0074] like Figure 10 As shown in A, compared with the Ctrl group, the expression levels of ZO-1 and Occludin tight junction proteins in the colorectal region of mice in the M group were significantly downregulated, indicating that the expression and function of tight junction proteins were inhibited, leading to severe damage to the intestinal barrier and further exacerbating the inflammatory response. Compared with the M group, the expression levels of ZO-1 and Occludin proteins in the P, TE, and EA groups were significantly upregulated, indicating that they protect the integrity of tight junctions between intestinal epithelial cells, reduce intercellular permeability, hinder the invasion of inflammatory mediators, and maintain the intestinal mucosal barrier function of mice. Combining the statistical results of B and C in 10, a significant difference was found between the EA and H groups (P < 0.001), indicating a strong advantage in maintaining the intestinal mucosal barrier function of mice.

[0075] Analysis of the expression levels of ZO-1 and Occludin tight junction proteins in the colorectal region of acute UC revealed that the EAH group had the best therapeutic effect, significantly increasing the expression levels of ZO-1 and Occludin proteins and effectively protecting the intestinal barrier function of mice. This further demonstrates that the EAH group has a significant advantage in improving acute UC in mice.

[0076] In the above experimental results, it has been determined that the EA group is the most effective component of turmeric slices, significantly reducing the expression levels of inflammatory factors (iNOS, IL-1β, IL-6, TNF-α) and significantly increasing the expression levels of tight junction proteins (ZO-1, Occludin), effectively reducing inflammatory cell infiltration and maintaining intestinal functional integrity. It shows outstanding advantages in treating acute UC in mice, especially the EAH group. Therefore, we further selected the Ctrl, M, P, EAH, and EAL groups to continue our research, exploring the expression levels of apoptosis proteins in the colorectal region of mice with acute UC, and understanding the potential mechanism of action of the EA component in the treatment of acute UC.

[0077] Inflammatory factors accumulate in large quantities on the colorectal mucosa of mice with acute ulcerative colitis (UC), triggering a series of inflammatory cascade reactions with other pro-inflammatory cytokines. Reactive oxygen species (ROS) generated during inflammation damage cellular DNA, lipids, and proteins, significantly increasing apoptosis, leading to damage to intestinal mucosal epithelial cells, increased intestinal permeability, and allowing various harmful substances to enter the intestine, exacerbating inflammatory symptoms. The mechanism of apoptosis is also a hot research topic in inflammatory response studies. As a programmed cell death process, apoptosis, regulated through endogenous and exogenous apoptosis signaling pathways, can eliminate excess cells, maintain normal tissue and organ function, and plays an important role in alleviating inflammation.

[0078] Pro-apoptotic proteins (Bcl-2-associated X protein, Bax) belong to the Bcl-2 family and are located on the outer mitochondrial membrane, participating in the apoptosis process. Activation of Bax protein can alter mitochondrial membrane permeability, release related apoptotic molecules, and maintain mitochondrial function. Anti-apoptotic proteins (B-cell lymphoma 2, Bcl-2) can inhibit Bax protein activity, suppressing changes in mitochondrial membrane permeability and apoptosis. The Bax / Bcl-2 ratio is a key indicator regulating apoptosis; changes in this ratio can assess the sensitivity of cells to apoptosis. A decrease in the Bax / Bcl-2 ratio indicates that apoptosis is inhibited, thereby reducing the inflammatory response. Statistical analysis revealed that the Bax / Bcl-2 ratio in group M was 2.0, while that in group EAH was 1.25, significantly lower than that in group M, indicating that EAH had the best efficacy and could significantly inhibit apoptosis.

[0079] The active site of aspartate-cysteine-specific proteinase (Caspase) contains a cysteine ​​residue, enabling it to hydrolyze its substrate, the aspartate residue. In ulcerative colitis, Caspase apoptotic proteins play a crucial role in regulating apoptosis and inflammatory responses. The Caspase family comprises more than 13 enzymes, among which Caspase 9 plays a role in apoptosis signal transduction. Activation of the precursor Caspase 9 protein allows it to cleave specific aspartate residues on target proteins, catalytically converting them into the active form, Cleaved Caspase 9, which further activates downstream effector Caspases, enhancing apoptosis signaling.

[0080] Cleaved poly(ADP-ribose) polymerase 1 (Cleaved PARP 1) is a marker protein of apoptosis. In ulcerative colitis, PARP 1 is activated due to oxidative stress in intestinal epithelial cells. Caspase proteases cleave inactive PARP 1 protein into Cleaved PARP 1, inducing apoptosis.

[0081] Combination Figure 11 Results A showed that, compared with the Ctrl group, the expression level of Bax protein in the M group was significantly increased, indicating enhanced apoptotic activity of mouse intestinal mucosal cells. Compared with the M group, the expression level of EAH protein was significantly downregulated in the P and EA groups, suggesting that the EAH dosage can inhibit apoptosis and thus alleviate acute UC (e.g., Figure 11 B).

[0082] Compared with the Ctrl group, the Bcl-2 protein expression level in the M group was significantly downregulated, indicating excessive apoptosis of intestinal mucosal cells, damage to the intestinal mucosal barrier, and exacerbation of the inflammatory response. Furthermore, compared with the M group, the Bcl-2 protein expression level in the drug-treated group was significantly upregulated, suggesting that drug intervention can inhibit cell apoptosis, protect the intestinal mucosal barrier, and thus improve acute UC (such as...). Figure 11 (C).

[0083] Compared with the Ctrl group, the M group showed a significant increase in the expression level of cleaved caspase 9 protein, indicating a significant increase in the number of apoptotic intestinal mucosal cells, which disrupted the integrity of the intestinal mucosal barrier, thereby increasing intestinal mucosal permeability and further exacerbating the inflammatory response. Secondly, compared with the M group, the EAH group showed a significant decrease in the expression level of cleaved caspase 9 protein, suggesting that EAH can significantly resist apoptosis and restore intestinal function in mice (e.g., ...). Figure 11 D).

[0084] Compared to the Ctrl group, the M group showed a significant increase in cleaved PARP 1 protein expression, indicating activation of the intrinsic apoptosis pathway and initiation of the apoptosis process. Secondly, compared to the M group, the P and EAH groups showed a significant decrease in cleaved PARP 1 protein expression, suggesting that they can significantly inhibit intestinal mucosal cell apoptosis and may be potential targets for the treatment of acute UC (e.g., Figure 11 E).

[0085] Comprehensive analysis of the expression levels of apoptosis proteins Bax, Bcl-2, Cleaved Caspase 9, and Cleaved PARP 1 in the colorectal region of acute UC revealed that, compared with other groups, the Bax / Bcl-2 ratio was decreased in the EAH group, and the levels of Cleaved Caspase 9 and Cleaved PARP 1 proteins were significantly reduced. This indicates that EAH can effectively inhibit cell apoptosis and reduce inflammatory response, suggesting that the EAH group has a significant therapeutic effect in improving acute UC in mice.

[0086] In summary, EA is the key pharmacologically active component of turmeric slices. This component can significantly reduce the rate of weight loss, increased DAI score, shortened colorectal length, and massive infiltration of inflammatory cells in intestinal mucosa induced by DSS in mice, significantly increase the number of goblet cells, and restore intestinal barrier function. Simultaneously, no toxic effects on the heart, liver, lungs, or kidneys were found at experimental doses of 300 and 150 mg / kg in mice, and the 300 mg / kg dose significantly inhibited splenomegaly in mice. This experiment has confirmed that the dosage of EA component is safe and effective. Western blotting results indicate that EA component can significantly downregulate the expression of inflammatory factors (iNOS, Il-1β, IL6, TNF-α) and related apoptosis proteins (Bax, Cleaved Csapase 9, Cleaved PARP 1) in the colorectal region, and upregulate the expression levels of tight junction proteins (ZO-1, Occludin) and the anti-apoptotic protein Bcl-2. EAH component showed even more effective therapeutic effects and has advantages in improving acute UC. EAH significantly inhibited the invasion of inflammatory factors and the number of apoptotic cells in the colorectal mucosa, cleared harmful substances such as bacteria and toxins from the intestine, reduced intestinal permeability, repaired the integrity of the intestinal mucosal barrier, and effectively alleviated the inflammatory response induced by the modeling drug. ELISA kit detection showed a decrease in the concentration of inflammatory factors in the serum of mice in the EAH group, indicating that the EAH group had a good anti-inflammatory effect, consistent with the results of Western blotting experiments. Combined with the metabolite analysis results in Example 1, it is clear that the EA component is the core component of turmeric tablets in improving DSS-induced UC in mice. Next, further investigation can be conducted to explore the monomeric components of the EA component and study the potential efficacy of this component in improving acute UC, providing a basis for the clinical development of novel therapeutic drugs. In in vivo experiments, the EA component has been screened as a key drug for improving acute UC.

[0087] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0088] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0089] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of ethyl acetate extract of turmeric in the preparation of drugs for treating acute ulcerative colitis, characterized in that, The preparation method of the ethyl acetate extract of turmeric slices is as follows: After turmeric slices are chopped, they are soaked overnight in 10 to 20 times their weight of a 60% to 80% ethanol solution. The extracts are then refluxed at 80 to 90°C for 2 to 4 times, each time for 1 to 3 hours. The extracts are combined and concentrated until no alcohol odor is detected. The extracts are then dried to constant weight to obtain the total extract. The total extract is reconstituted with water and extracted 2 to 4 times with ethyl acetate at an equal volume ratio. The extracts are combined and dried to constant weight to obtain the ethyl acetate extract of turmeric slices.

2. The application according to claim 1, characterized in that, The drug is used to relieve colonic and rectal shortening caused by acute ulcerative colitis.

3. The application according to claim 1, characterized in that, The drug is used to increase the expression of tight junction proteins ZO-1 and Occludin in the colon of patients with acute ulcerative colitis, thereby reducing intestinal barrier damage.

4. The application according to claim 1, characterized in that, The drug is used to inhibit the expression of inflammatory factors iNOS, IL-1β, IL-6 and TNF-α in the lesion colon of acute ulcerative colitis.

5. The application according to claim 1, characterized in that, The content of ethyl acetate extract of turmeric in the drug is 0.1wt% to 99wt%.

6. The application according to claim 1, characterized in that, The drug is made from the ethyl acetate extract of turmeric tablets and pharmaceutically acceptable excipients.

7. The application according to claim 6, characterized in that, The excipients include any one or more of fillers, stabilizers, diluents, and adjuvants.

8. The application according to claim 7, characterized in that, The diluent is either water or physiological saline.

Images