Use of extracts of amomum villosum in the preparation of a medicament for treating ulcerative colitis

Abstract

The application discloses application of Jatrophulang fruit or stem extract in preparation of a medicine for treating ulcerative colitis. The Jatrophulang fruit or stem extract is an ethanol extract of Jatrophulang fruit or stem. The Jatrophulang fruit or stem extract provided by the application can significantly improve colitis inflammation of a mouse induced by dextran sulfate sodium, reduce a disease activity index of the mouse, obviously improve a shortened colon length of the mouse caused by modeling, significantly improve a survival rate of the mouse with ulcerative colitis, and significantly reduce inflammatory cell infiltration of colon tissue, and has a significant anti-ulcerative colitis activity, and the effect is equivalent to that of a positive medicine, i.e., sulfasalazine, and has a good application prospect in preparation of a medicine for treating inflammatory bowel disease.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to application of a cardamom extract in preparing a medicine for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory disease primarily affecting the colonic mucosa, with an incompletely elucidated etiology. It can occur anywhere in the colorectal system, but is most common in the rectum and sigmoid colon. The main pathological changes of UC are: ① diffuse, continuous mucosal inflammation; ② mucosal ulceration; ③ crypt abscesses; ④ pseudopolyps; and ⑤ specific cellular changes, such as Paneth cell hyperplasia and goblet cell reduction. Clinical manifestations include abdominal pain, diarrhea, mucus, and bloody stools. Symptoms vary in severity, alternating between remissions and flares. Patients may present with only colonic symptoms or with concurrent systemic symptoms. UC is characterized by recurrent exacerbations and is prone to complications such as toxic colon dilatation, intestinal perforation, lower gastrointestinal bleeding, fistulas, rectal and colonic strangulation, and colorectal cancer. UC carries a high risk of disability and a high risk of malignant transformation, and is classified by the World Health Organization as one of the most intractable diseases in modern medicine. The global prevalence of ulcerative colitis is estimated to be 5 million cases in 2023. In Europe and the United States, the incidence remains high and is on the rise. In recent years, with changes in diet, lifestyle, and environment, as well as continuous improvements in diagnostic technology, the incidence of ulcerative colitis in my country has increased year by year, gradually becoming one of the most common and difficult diseases in gastroenterology.

[0003] Currently, the treatment of UC focuses on inducing and maintaining disease remission. The main therapeutic agents include: ① aminosalicylic acids; ② glucocorticoids; ③ immunosuppressants and biologics. Clinical data show that some UC patients are unable to effectively control UC progression with existing medications, and up to 15% ultimately require surgical resection. Furthermore, these medications pose significant side effects with long-term use, such as relapse after withdrawal of glucocorticoids and osteoporosis. Traditional Chinese medicine (TCM) offers a unique understanding of UC, boasting extensive diagnostic and treatment experience and a wealth of theoretical, methodological, and prescription-based therapies. In TCM, ulcerative colitis can be categorized as large intestine damp-heat syndrome, spleen deficiency and dampness obstruction syndrome, liver depression and spleen deficiency syndrome, and mixed cold-heat syndrome. TCM treatments, such as oral TCM formulas, offer superior clinical efficacy to Western medications, boosting patients' immunity, delaying relapse time, and reducing recurrence rates with minimal side effects. Therefore, identifying effective, well-defined therapies for the prevention and treatment of UC from traditional Chinese medicines and ethnic medicines is a promising approach to addressing the current challenges in UC treatment.

[0004] Nine-winged cardamom Amomum maximum Roxb. is a member of the Zingiberaceae family (Zingiberaceae) AmomumCardamom is a medicinal plant with glabrous, oblong or elliptical leaves and nine distinct wings on the pericarp, hence the name "Nine-winged Cardamom." It is found in tropical regions of Asia and Oceania, with over 150 species, primarily found in Yunnan, southern Tibet, Guangdong, and Guangxi. There are 24 species and two varieties in my country. Its fruit has medicinal properties, stimulating appetite, aiding digestion, promoting qi circulation, and relieving pain.

[0005] Currently, there are no research reports on the effect of the extract of Amomum villosum in treating ulcerative colitis. The present invention aims to provide the use of the ethanol extract of Amomum villosum in preparing a drug for treating UC. Summary of the Invention

[0006] The present invention aims to provide an application of a cardamom extract in preparing a medicine for treating ulcerative colitis.

[0007] The object of the present invention is achieved by using a cardamom extract in preparing a drug for treating ulcerative colitis, wherein the cardamom extract is an ethanol extract of the fruit or stem of the cardamom;

[0008] The specific preparation method is as follows: the dried fruits or stems of Amomum villosum are crushed into coarse powder, the powder is passed through a 20-mesh sieve, 90-95% ethanol is added to every 100 g of the fruit coarse powder, and the powder is soaked for 10-28 hours, and then heated under reflux at 80-90° C. for 0.5-1.5 hours. After filtering, the residue is further extracted with 90-95% ethanol under reflux for 2-3 times, the filtrates are combined, concentrated under reduced pressure, and then dried to obtain the Amomum villosum extract.

[0009] The dosage of the cardamom extract for mice is 100-200 mg / kg.

[0010] The cardamom extract provided by the present invention can significantly improve the colon inflammation of mice induced by sodium dextran sulfate, which is manifested by significantly reducing the disease activity index of mice, significantly improving the shortening of the colon length of mice caused by modeling, and significantly improving the survival rate of mice with ulcerative colitis; significantly reducing the inflammatory cell infiltration of colon tissue, and having significant anti-ulcerative colitis activity, the effect is comparable to that of the positive drug sulfasalazine, and has good application prospects in the preparation of drugs for treating inflammatory bowel disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Effects of extracts from the fruits and stems of Cardamom oleifera on DAI in UC model mice (n=8) (compared with the normal group, # : p <0.05, ## : p <0.01; compared with the model group, * : p <0.05, ** : p<0.01);

[0012] Figure 2 Effects of the extracts of cardamom fruit and stem on the colon length of UC model mice (n=8) (compared with the normal group, ## : p <0.01; compared with the model group, * : p <0.05);

[0013] Figure 3 Effects of extracts from the fruits and stems of Amomum villosum on the survival rate of UC model mice (n=9);

[0014] Figure 4 Effect of extracts from the fruits and stems of Cardamom nematodes on colon histopathology of UC model mice (H&E staining)

[0015] the impact of;

[0016] Figure 5 This study investigates the effects of extracts from the fruits and stems of Amomum villosum on colon histopathological scores in UC model mice (n=8). DETAILED DESCRIPTION

[0017] The present invention is further described below with reference to the embodiments, but the present invention is not limited in any way. Any changes or substitutions made based on the technical teachings of the present invention fall within the scope of protection of the present invention.

[0018] The present invention provides an application of a cardamom extract in preparing a drug for treating ulcerative colitis, wherein the cardamom extract is an ethanol extract of the fruit or stem of the cardamom;

[0019] The specific preparation method is as follows: the dried fruits or stems of Amomum villosum are crushed into coarse powder, the powder is passed through a 20-mesh sieve, 90-95% ethanol is added to every 100 g of the fruit coarse powder, and the powder is soaked for 10-28 hours, and then heated under reflux at 80-90° C. for 0.5-1.5 hours. After filtering, the residue is further extracted with 90-95% ethanol under reflux for 2-3 times, the filtrates are combined, concentrated under reduced pressure, and then dried to obtain the Amomum villosum extract.

[0020] The dosage of the cardamom extract for mice is 100-200 mg / kg.

[0021] The medicine for treating ulcerative colitis is prepared by taking a therapeutically effective amount of the cardamom extract as a main active ingredient and adding pharmaceutically acceptable excipients or carriers.

[0022] The dosage form of the medicine for treating ulcerative colitis is pills, capsules, tablets, powders, granules, injections or compound preparations.

[0023] Example 1

[0024] Weigh 100 g each of the fruits and stems of Cardamom chinensis, crush them into coarse powder, pass through a 20-mesh sieve, and place them in a 2-L round-bottom flask. Add 1 L of 95% ethanol, soak for 12 h, and reflux extract in an 85°C water bath for 1 h. After filtering, the filter residue is reflux extracted with 95% ethanol 2 to 3 times. The filtrate is combined, and the ethanol is recovered under reduced pressure until there is no alcohol taste. Place it in a vacuum drying oven, dry it, and grind it into powder to obtain the extracts of the fruits and stems of Cardamom chinensis.

[0025] The extracts of the fruit and stem of the cardamom fructus are respectively obtained, concentrated, evaporated to dryness under reduced pressure, crushed and sieved to obtain dry paste powder, and filled into capsules to obtain capsules.

[0026] The extracts of the fruit and stem of the cardamom are respectively concentrated, evaporated to dryness under reduced pressure, crushed and sieved to obtain a dry paste powder. An appropriate amount of the dry paste powder is mixed with equal amounts of starch and dextrin, and ethanol of appropriate concentration is used as a wetting agent. Granulation is carried out by conventional methods, and an appropriate amount of magnesium stearate is added and mixed to prepare tablets.

[0027] Example 2

[0028] Weigh 100 g each of the fruits and stems of Cardamom chinensis, crush them into coarse powder, pass through a 20-mesh sieve, and place them in a 2-L round-bottom flask. Add 1 L of 90% ethanol, soak for 24 h, and reflux extract in an 80°C water bath for 1.5 h. After filtration, the filter residue is reflux extracted with 90% ethanol three times. The filtrates are combined, and the ethanol is recovered under reduced pressure until there is no alcohol taste. Place the mixture in a vacuum drying oven, dry it, and grind it into powder to obtain the extracts of the fruits and stems of Cardamom chinensis.

[0029] Example 3

[0030] Weigh 100 g each of the fruits and stems of Cardamom chinensis, crush them into coarse powder, pass through a 20-mesh sieve, and place them in a 2-L round-bottom flask. Add 1 L of 95% ethanol, soak for 24 h, and reflux extract in a 90°C water bath for 0.5 h. After filtration, the filter residue is reflux extracted twice with 95% ethanol. The filtrates are combined, and the ethanol is recovered under reduced pressure until there is no alcohol taste. The extracts are placed in a vacuum drying oven, dried, and ground into powder to obtain the extracts of the fruits and stems of Cardamom chinensis.

[0031] Experimental Example 1 Detection of the Anti-ulcerative Colitis Activity of Cardamom Extract Induced by Dextran Sulfate Sodium

[0032] 1. Experimental Materials

[0033] 1.1 Main instruments

[0034] HH-6 constant temperature water bath, Guohua Electric Co., Ltd.; 282-2 benchtop low-speed centrifuge, Shanghai Medical Devices Group Co., Ltd.; benchtop high-speed refrigerated centrifuge HR / T16M, Hunan Hexi Instrument Equipment Co., Ltd.; BS-124S ​​optical reading analytical balance, Beijing Sartorius Balance Co., Ltd.; TB-25 1 / 100,000 electronic balance, Beijing Denver Instrument Co., Ltd.; vortex shaker, LAB-BLOGEN VTX-E; water purifier (Milli-Q Academic A10 ultrapure water system), Millipore Corporation, USA; ultra-low temperature (-80°C) freezer, Thermo Fisher Scientific (China) Co., Ltd.; ultrasonic cleaning machine, Ningbo Xinzhi Biotechnology Co., Ltd.

[0035] 1.2 Main drugs and test kits

[0036] Sulfasalazine enteric-coated tablets (0.25 g, Shanghai Xinyi Tianping Pharmaceutical Co., Ltd., batch number: 0917018); 4% paraformaldehyde (Wuhan Saiwei Biotechnology Co., Ltd.); occult blood test kit (Nanjing Jiancheng Bioengineering Institute); dextran sulfate sodium salt (DSS; MW: 36000-50000; MP Biomedicals). All other chemical reagents were purchased from Kunming Zehao Technology Co., Ltd.

[0037] 1.3 Experimental Animals

[0038] Liaoning Changsheng Biotechnology Co., Ltd., License No. SCXK(Liao)2015-0001. Mouse feed and bedding were purchased from Hunan Slake Jingda Biotechnology Co., Ltd., License No. 2016-0002. After the experimental animals were housed in the animal housing, they were fed under laboratory conditions for one week.

[0039] 1.4 Preparation of main reagents

[0040] 1% CMC-Na solution: weigh 20 g of CMC-Na, add 2000 mL of distilled water, mix well, allow it to swell fully, leave it overnight, and stir evenly to obtain.

[0041] To prepare a 3% DSS solution, weigh 30 g of DSS and add 1000 mL of distilled water. Mix thoroughly and dissolve thoroughly. Store at 4°C until ready for use.

[0042] Sulfasalazine suspension: Take three sulfasalazine enteric-coated tablets (0.25 g), carefully scrape off the enteric coating with a scalpel, and thoroughly grind. Add 0.5% CMC-Na solution to a final volume of 25 mL to obtain a sulfasalazine suspension (0.03 g / mL) for later use. The oral gavage volume is 0.1 mL / 10 g, for a dose of 0.3 g / kg.

[0043] Preparation of the cardamom extract sample: as shown in Example 1.

[0044] For the extract from the fruit of Amomum villosum: Take an appropriate amount of the extract from the fruit of Amomum villosum prepared in Example 1 and prepare a 200 mg / mL suspension in 1% CMC-Na solution. The gavage volume was 0.1 mL / 10 g, and the dosage was 200 mg / kg.

[0045] For the extract from the stems and branches of Amomum villosum: Take an appropriate amount of the Amomum villosum stem and branches extract prepared in Example 1 and prepare a 200 mg / mL suspension with 1% CMC-Na solution. The gavage volume is 0.1 mL / 10 g, and the dosage is 200 mg / kg.

[0046] 2. Experimental methods

[0047] 2.1 Animal grouping, modeling, and drug administration

[0048] Animals were randomly divided into a normal group, a model group, a positive drug group (sulfasalazine, 0.3 g / kg), a cardamom fruit extract group (200 mg / kg), and a cardamom stem and twig extract group (200 mg / kg). The day of the experiment was designated day 0. All groups, except the normal group, were given free access to 3% DSS solution starting on day 0. When mice began to experience noticeable hematochezia (approximately 4-6 days), DSS was discontinued and replaced with pure water until day 10. The 3% DSS solution was replaced every 2 days. Drugs were administered to each group once daily starting on day 0 for 11 consecutive days. Both the normal and model groups received an equal volume of 1% CMC-Na solution.

[0049] 2.2 Disease Activity Index (DAI) Scoring in Mice

[0050] From the day of model establishment until the end of the experiment, for a total of 11 days, mice were observed daily for their mental state, food intake, hair neatness and glossiness, body weight, stool characteristics, and occult blood. Weight, stool characteristics, and occult blood were scored according to literature standards. The scores were based on percentage weight loss (no change in weight = 0, 1%-5% = 1, 5%-10% = 2, 10%-20% = 3, and >20% = 4), stool characteristics (normal = 0, loose stools = 2, and watery diarrhea = 4), and fecal bleeding (normal = 0, positive occult blood = 2, overt bleeding (i.e., visible bleeding) = 4, and death = 12). Occult blood was detected using an occult blood test kit according to the instructions. The sum of these scores was used to calculate the disease activity index (DAI).

[0051] 2.3 Survival rate

[0052] From the day of modeling to the end of the experiment, a total of 11 days, the survival rate of mice in each group was observed and recorded every day.

[0053] 2.4 Colon length

[0054] On the 11th day, the mice were killed and dissected, the entire colon was cut out and neatly placed on filter paper, and the length of the colon was measured and recorded.

[0055] 2.5 H&E staining of colon tissue

[0056] The intestinal contents were gently rinsed with saline, and the central portion of the colon (approximately 0.5–1 cm) was carefully excised and fixed overnight in 4% paraformaldehyde. 4 μm sections were dewaxed and stained with H&E. The sections were reviewed blindly under a microscope and scored according to the Sykes criteria (Takagi T, et al., Dig Dis Sci. 2011; 56(6): 1663–71).

[0057] 2.6 Data Statistical Analysis

[0058] The experimental data were analyzed and processed using SPSS 21.0, and the mean ± standard deviation ( Mean ± SD) was expressed and analyzed using one-way analysis of variance (ANOVA). Data were pre-tested for homogeneity of variance using the Homogeneity of Variances method. If the variances were homogeneous, the LSD test was performed, and if the variances were unequal, the Tamhane's T2 test was performed.

[0059] 3. Experimental results

[0060] 3.1 Effects on the Disease Activity Index (DAI) in Mice

[0061] While freely drinking the 3% DSS solution, mice in each model group developed physical changes such as lethargy, dulling of fur, decreased appetite, and weight loss. A small number of mice developed diarrhea and occult blood in their stools starting on the fourth day of the experiment, and most mice developed diarrhea and a small amount of visible blood in their stools by the fifth day. The 3% DSS solution in each model group was replaced with tap water. The model group mice experienced the most severe blood in their stools and diarrhea by the sixth day; by the eighth day, these symptoms had subsided.

[0062] Depend on Figure 1 As shown, compared with the normal group (C), the DAI scores of UC model mice were significantly higher than those of the model group (M). After administration of each drug group, the fruit extract (Fruits) and stem extract (Stem) of Amomum vulgare L., as well as the active drug sulfasalazine (5-ASA), all significantly reduced the DAI scores of UC model mice. After six days of administration, the fruit extract (Fruits) and stem extract (Stem) of Amomum vulgare L. were more effective in reducing DAI than the active drug sulfasalazine (5-ASA). At the end of the experiment, the fruit extract (Fruits) of Amomum vulgare L. remained more effective than sulfasalazine (5-ASA) in reducing DAI.

[0063] Conclusion: The extracts of the fruits and stems of N. chinensis can reduce the disease activity index (DAI) score of UC mice.

[0064] 3.2 Effects on the changes in mouse colon length

[0065] Depend on Figure 2 Compared with the normal control group (C, mean colon length 5.96±0.73 cm), the colon length of the UC model group (M, mean colon length 4.32±0.42 cm) was significantly shortened. After administration of each drug group, the colon length of mice treated with the fruit extract of Amomum vulgare (Fruits, mean colon length 5.06±0.53 cm), the stem extract of Amomum vulgare (Stem, mean colon length 5.18±0.40 cm), and the positive drug sulfasalazine (5-ASA, mean colon length 4.94±0.36 cm) significantly increased compared with the model group. The fruit extract and stem extract of Amomum vulgare (Stem) groups showed a more effective improvement in colon shortening in UC than the positive drug sulfasalazine (5-ASA).

[0066] It can be seen that the fruit and stem extracts of Amomum villosum can significantly improve the colon shortening phenomenon in UC, and the fruit extract is better than the stem extract.

[0067] 3.3 Effects on the survival rate and survival duration of UC model mice

[0068] Depend on Figure 3 As shown, compared with the normal group (C, survival rate 100%), the survival rate of mice in the UC model group (M) was significantly decreased, with mortality beginning on the 5th day of modeling. From the time of administration to the end of modeling, all drug groups significantly improved the survival rate of UC model mice compared with the model group (M, survival rate 77.8% on the 8th day of modeling), the fruit extract group (Fruits, survival rate 100% on the 8th day of modeling), and the positive drug sulfasalazine group (5-ASA, survival rate 85.7% on the 8th day of modeling). However, the stem and branch extract group (Stem, survival rate 77.8% on the 8th day of modeling) did not improve survival. The fruit extract group showed a superior effect on improving the survival of UC model mice compared with the positive drug sulfasalazine.

[0069] Compared with the model group (M), which began to show death on the 5th day of modeling, the fruit extract group (Fruits) of Amomum villosum did not show death during the modeling period, the stem and branch extract group (Stem) of Amomum villosum began to show death on the 7th day of modeling, and the positive drug sulfasalazine group (5-ASA) began to show death on the 7th day of modeling. These results indicate that the fruit extract, stem and branch extract, and positive drug sulfasalazine can prolong the survival time of UC mice, and the effects of the fruit extract and stem and branch extract of Amomum villosum are comparable to those of the positive drug sulfasalazine.

[0070] It can be seen that the fruit extract of Amomum villosum can significantly improve the survival rate of UC mice, and the fruit and stem extracts of Amomum villosum can prolong the survival time of UC mice.

[0071] 3.4 H&E staining of mouse colon tissue

[0072] like Figure 4 As shown, mice in the normal group (C) showed intact colonic epithelial structure, normal cell morphology, and regular arrangement of intestinal glands, without significant inflammation. Mice in the UC model group (M) exhibited extensive ulceration in the colon, with microscopic loss of epithelial cell continuity and intestinal gland structure, and extensive inflammatory cell infiltration in the mucosa and submucosa. Oral administration of extracts from the fruits and stems of Amomum vulgare (Fruits) and Amomum vulgare (Stem), as well as the positive drug sulfasalazine (5-ASA), significantly improved inflammatory cell infiltration and congestion and edema in the colonic mucosa, demonstrating a significant protective effect on the integrity of the epithelium and glands.

[0073] According to the Sykes criteria (Takagi T, et al.. Dig Dis Sci. 2011; 56(6):1663-71.), the results are as follows Figure 5As shown in the results, the fruit extract group (Fruits) and the stem extract group (Stem) of Cardamom gensis significantly reduced the Sykes standard score, that is, significantly improved the inflammatory cell infiltration and congestion and edema of the colon mucosal tissue, protected the integrity of the epithelium and glands, and the effect of the stem extract was better than that of the fruit extract and the positive drug sulfasalazine.

[0074] In summary, the extracts of the fruit and stem of Cardamom oleifera have significant activity in treating ulcerative colitis and have good application prospects in the preparation of treatments for ulcerative colitis, especially ulcerative colitis.

[0075] In the mouse experiments described herein, the dose of extracts from the fruit and stems of Cardamom oleifera was 200 mg / kg. This translates to a 30 g / day dose for a human (based on a 60 kg adult). Taking into account differences in body weight and various other pathological conditions, the recommended dose is 15-10 g two to three times daily. The most commonly used dose is 10 g three times daily.

Claims

1. Use of an extract of Cardamomum villosum as the sole active ingredient in the preparation of a drug for treating ulcerative colitis, characterized in that: The cardamom extract is an ethanol extract of the cardamom fruit, and its specific preparation method is as follows: the dried cardamom fruit is crushed into coarse powder, passed through a 20-mesh sieve, 90-95% ethanol is added to every 100g of the fruit coarse powder, and soaked for 10-28h, and then heated to 80-90°C for reflux extraction for 0.5-1.5h. After filtering, the filter residue is reflux-extracted with 90-95% ethanol for 2-3 times, the filtrate is combined, concentrated under reduced pressure, and then dried to obtain the cardamom extract.

2. The use according to claim 1, characterized in that The medicine for treating ulcerative colitis is prepared by adding a therapeutically effective amount of the cardamom extract and a pharmaceutically acceptable auxiliary material or carrier.

3. The use according to claim 2, wherein the dosage form of the drug for treating ulcerative colitis is pills, capsules, tablets, powders or granules.

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