Golden flower and fruit extract for treating ulcerative colitis

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a golden flower and fruit extract for treating ulcerative colitis. According to the traditional Chinese medicine composition, natural medicine components are extracted from golden flower and fruit, the components can enhance the shielding guarantee effect of colonic mucosa, reduce inflammation gene expression, prevent colon shortening and improve the curative effect of ulcerative colitis, and the traditional Chinese medicine composition has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to an extract of golden flower fruit for treating ulcerative colitis. Background Art

[0002] Chronic non-specific ulcerative colitis (hereinafter referred to as ulcerative colitis (UC)) is a chronic non-specific inflammation mainly involving the rectal and colonic mucosa and submucosa, with abdominal pain, diarrhea, mucous bloody stools, and tenesmus as the main clinical manifestations. The disease has a protracted course and can last for more than ten years or even decades, and there is also a possibility of canceration. This disease belongs to the categories of "intestinal bi" "diarrhea" "dysentery" in traditional Chinese medicine. Its etiology is complex, the course of disease is long, and it is difficult to cure. At present, the curative effect of chemical drugs is limited, and the commonly used drugs are aminosalicylates. Sometimes hormones or immunosuppressants need to be used. The drug side effects are large, and the compliance of patients taking drugs is poor, resulting in the protracted course of the disease and seriously affecting the quality of life of patients. In recent years, due to various reasons, the incidence and the number of patients with ulcerative colitis have increased significantly. However, so far, the varieties of drugs for treating ulcerative colitis are limited, the curative effect is not exact, some patients need to add drugs such as hormones and immunosuppressants for treatment, and the treatment cycle is long, the treatment cost is high, and the treatment compliance of patients is poor, which seriously affects the clinical prognosis of patients with ulcerative colitis.

[0003] Sulfasalazine is a sulfonamide antibacterial drug, an oral sulfonamide that is not easily absorbed, and is used for inflammatory bowel disease, namely Crohn's disease and ulcerative colitis. After being absorbed, sulfasalazine is decomposed into 5-aminosalicylic acid and sulfapyridine under the action of intestinal microorganisms. 5-Aminosalicylic acid complexes with the connective tissue of the intestinal wall and stays in the intestinal wall tissue for a long time to play an antibacterial, anti-inflammatory and immunosuppressive role.

[0004] Golden flower fruit is Dioscorea cirrhosa Lour. of the genus Dioscorea in the family Dioscoreaceae. According to "Xishuangbanna Dai Medicine Records" and "Chinese Materia Medica · Dai Medicine Volume", golden flower fruit, also known as "bao le", is cool in nature, astringent in taste, promoting blood circulation and nourishing blood, and astringing and consolidating. It is used for functional uterine bleeding, postpartum hemorrhage, hemoptysis, hematemesis, hematochezia, hematuria, diarrhea; for external use in treating burns.

[0005] The main clinical manifestations of non-specific ulcerative colitis in traditional Chinese medicine are diarrhea, mucopurulent bloody stools accompanied by abdominal pain, tenesmus and other symptoms in traditional Chinese medicine diseases such as dysentery and damp-heat dysentery. Hudi Enteric Capsule is a widely used traditional Chinese medicine formula preparation on the current market for the treatment of non-specific ulcerative colitis. The prescription consists of eight herbs, namely Zhushaqi, Polygonum cuspidatum, Hedyotis diffusa, Patrinia villosa, Limonium bicolor, Sanguisorba officinalis (charcoal), Bletilla striata, and Glycyrrhiza uralensis. Its functions and indications are to clear heat, promote diuresis, and cool the blood. It is used for the syndrome of damp-heat accumulation in non-specific UC, with symptoms such as abdominal pain, dysentery with purulent and bloody stools, and tenesmus. The current expert consensus treatment principle for Hudi Enteric Capsule in the treatment of ulcerative colitis at the grass-roots level is to clear heat, promote diuresis, and cool the blood.

[0006] The functions and indications of Jinhua Fruit are to clear fire and detoxify, cool the blood and stop bleeding, and astringe the intestine and stop diarrhea. The traditional medicinal experience is to treat dysentery, diarrhea, abdominal pain and burns. Hudi Enteric Capsule is a compound preparation, and the prescription contains Bletilla striata and Glycyrrhiza uralensis. During the medication process, the medication risks of "the eighteen antagonisms and nineteen contraindications" of Bletilla striata and Glycyrrhiza uralensis need to be considered. Jinhua Fruit can be a single preparation, and Jinhua Fruit has a history of being edible and has better safety. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the present invention provides an extract of Jinhua Fruit for the treatment of ulcerative colitis.

[0008] An extract of Jinhua Fruit for the treatment of ulcerative colitis is prepared by the following method: After crushing the Jinhua Fruit medicinal material, weigh the Jinhua Fruit sample and put it into a round-bottom flask, add a methanol aqueous solution, heat to boiling, keep slightly boiling for a period of time, then let it cool and filter, collect the filtrate, and then repeat the extraction of the residue twice according to the above method. Finally, combine the filtrates of the three times and concentrate to obtain the extract of Jinhua Fruit, and then perform freeze-drying to obtain the extract of Jinhua Fruit.

[0009] Preferably, the methanol aqueous solution is a 90% methanol aqueous solution

[0010] Preferably, the mass-volume ratio of the Jinhua Fruit sample to the methanol aqueous solution is 1:5.

[0011] The application of the extract of Jinhua Fruit provided by the present invention in the preparation of a drug for the treatment of ulcerative colitis.

[0012] The present invention provides a pharmaceutical composition for the treatment of ulcerative colitis, which includes the above-mentioned Jinhua Fruit as a drug active ingredient and pharmaceutically acceptable carriers and excipients.

[0013] The mass percentage of the drug active ingredient in the whole drug is 10% - 40%.

[0014] The pharmaceutical composition can be prepared into tablets, capsules, pills or oral liquids.

[0015] The said pharmaceutical composition can be prepared into various conventional dosage forms by conventional preparation methods.

[0016] Advantages of the present invention: Natural pharmaceutical ingredients are extracted from golden flower fruits, which can strengthen the protective function of the colonic mucosa, reduce the expression of inflammatory genes, prevent colon shortening, and improve the curative effect of ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 qPCR detection

[0018] Figure 2 Body weight loss rate and DAI, ***P < 0.001 vs Control; **P < 0.01 vs Control; *P < 0.05 vs Control; ### P < 0.001 vs Model; ## P < 0.01 vs Model

[0019] Figure 3 Anal bleeding

[0020] Figure 4 Colon length, ***P < 0.001 vs Control; ### P < 0.001 vs Model; ## P < 0.01 vs Model

[0021] Figure 5 Colonic mucosa, ***P < 0.001 vs Control; ### P < 0.001 vs Model; ## P < 0.01 vs Model; # P < 0.05 vs Model

[0022] Figure 6 HE staining DETAILED DESCRIPTION OF THE INVENTION

[0023] The following examples can enable those skilled in the art of this specialty to understand the present invention more comprehensively, but do not limit the present invention within the scope of the described examples.

[0024] MAIN REAGENTS AND INSTRUMENTS

[0025]

[0026]

[0027] EXPERIMENTAL METHODS

[0028] Extraction process of golden flower fruits

[0029] After pulverizing the Jinhua guo medicinal material, weigh 100 g of the Jinhua guo sample and place it in a 1000 ml round-bottom flask. Add 500 ml of 90% methanol aqueous solution, add boiling chips, heat to boiling, then maintain gentle boiling for 1 h, allow to cool, filter, collect the filtrate. Then repeat the extraction of the residue twice according to the above method. Finally, combine the filtrates from the three extractions and concentrate to obtain the Jinhua guo extract. After freeze-drying, 47 g of freeze-dried powder is obtained, and the yield is 47%.

[0030] Cell seeding

[0031] Collect the precipitates of Raw264.7 cells in the logarithmic growth phase, adjust the cell density to 1×10 5 cells / mL, and then add 500 μL of the cell suspension to each well in a 24-well cell culture plate, divided into a control group, an LPS group, a drug group, and an LPS + drug group. Incubate overnight in an incubator. The drug group is treated with different concentrations of Jinhua guo (50, 100, and 200 μg / mL) for 24 h, and the LPS + drug group is treated with LPS (1 μg / mL) and different concentrations of Jinhua guo (50, 100, and 200 μg / mL) in combination for 24 h. Each group is repeated in 3 wells. The grouping is as follows:

[0032] Group number Group A NC B LPS C Golden flower and fruit 50 μg / mL D Golden flower and fruit 100 μg / mL E Golden flower and fruit 200 μg / mL F Golden flower and fruit 50 μg / mL G Golden flower and fruit 100 μg / mL H Golden flower and fruit 200 μg / mL

[0033] RNA extraction

[0034] Prepare the sample and add 0.3 mL of Trizol lysis solution;

[0035] Add 0.2 mL of chloroform, shake vigorously for 15 s, and let stand at room temperature for 5 min;

[0036] Centrifuge at 12000×g for 15 min at 4℃. The sample is divided into three layers: the bottom layer is a yellow organic phase, the upper layer is a colorless aqueous phase, and an intermediate layer;

[0037] Transfer the aqueous phase to a new tube and precipitate the RNA in the aqueous phase with isopropanol. Add 0.5 mL of isopropanol for every 1 mL of Trizol used and let stand at room temperature for 5 min;

[0038] Centrifuge at 12000×g for 10 min at 4℃. After centrifugation, a gelatinous precipitate appears on the side and bottom of the tube. Remove the supernatant;

[0039] Wash the RNA precipitate with 0.5 mL of 75% ethanol. Centrifuge at no more than 7500×g for 5 min at 4℃ and discard the supernatant. (Repeat twice)

[0040] Let stand at room temperature to dry, then add 50 μL of DEPC water and store at -80℃;

[0041] RT-qPCR

[0042] Place the reverse-transcribed cDNA on ice for later use. At the same time, keep the Taq Pro Universal SYBR qPCR Master Mix on ice and prepare the reaction system as follows. The reaction system is 20 μL:

[0043] Name Volume TaqPro Universal SYBR qPCR Master Mix 10 μL *Primers(F) 0.4 μL *Primers(R) 0.4 μL cDNA 2 μL <![CDATA[ddH2O]]> 7.2 μL

[0044] Reaction program:

[0045]

[0046] *Primer is designed as shown in the following table:

[0047]

[0048]

[0049] Animal experiment

[0050] Thirty-six healthy male C57BL / 6 mice aged 6 - 8 weeks (purchased from Yangzhou University Animal Experiment Center) were housed under conditions of a 12-hour light-dark cycle, 18 - 26 °C, and 40% - 70% relative humidity. After one week of adaptive feeding, they were randomly divided into six groups (n = 6), namely the control group (Control), the model group (Model), the groups treated with 50 mg / kg, 100 mg / kg, and 200 mg / kg of Jinhua Guo, and the group treated with sulfasalazine (100 mg / kg). The mice in the control group drank normal water for 7 days, and the mice in the model group freely drank a 2.5% DSS solution (weigh 2.5 g of DSS powder and dissolve it thoroughly in 100 mL of mouse drinking water) for 7 days. The DSS solution was freshly prepared and changed every other day. On the 8th day, an intraperitoneal injection was given, and on the 14th day, colon tissues were taken for subsequent experiments. The grouping is as follows:

[0051] Group number Group A Control B Model C Golden flower and fruit 50 mg / kg D Golden flower and fruit 100 mg / kg E Golden flower and fruit 200 mg / kg F Sulfasalazine G H

[0052] Disease activity index (DAI) scores and anal conditions of mice in each group

[0053] Record the changes in mouse body weight, bloody stools, and stool viscosity every day, and score each item. The average of the three scores is the DAI score. The DAI score reflects the degree of colon tissue damage; the higher the score, the more severe the colon tissue damage. The specific criteria for DAI scoring include weight loss (score 0 for stable weight; score 1 for 1 - 5% weight loss; score 2 for 5 - 10% weight loss; score 3 for 10 - 15% weight loss; score 4 for weight loss exceeding 15%), (score 0 for normal; score 2 for loose stools; score 4 for diarrhea), and bloody stools (score 0 for negative; score 2 for occult blood; score 4 for visible bleeding). After treatment, check the anal area condition of the mice and take images.

[0054] Colon length and Colon Mucosal Damage Index (CMDI)

[0055] During the evaluation of colon tissue and related pathology, the mouse was dissected along the midline of the abdomen from the anus to the cecum to excise the colon for length measurement. Subsequently, the colon tissue was opened, and the degree of mucosal damage was visually evaluated and quantified according to the CMDI scoring system. The CMDI score is described by the following scale: 0 indicates no mucosal damage; 1 indicates a smooth mucosal surface that shows mild congestion and edema without erosion or ulceration; 2 indicates mucosa with congestion, edema, roughness, granularity, erosion, or adhesions; 3 indicates that the mucosal surface shows necrosis, ulceration, significant congestion and edema, the ulcer size is less than 1.0 cm, accompanied by intestinal wall necrosis, inflammation, or hyperplasia; 4 indicates cases where the largest ulcer exceeds 1.0 cm or the full-thickness wall necrosis and inflammation are very severe.

[0056] HE staining

[0057] The mouse colon tissue was fixed with 4% paraformaldehyde solution to denature and coagulate the proteins in the colon tissue; the mouse colon tissue was taken out of the 4% paraformaldehyde fixative and the fixative was rinsed off with running water; then dehydration was carried out successively with alcohol from low concentration to high concentration, and the colon tissue was put into xylene for clearing; subsequently, the tissue was immersed in paraffin, and the paraffin-impregnated tissue block was vertically embedded overnight; the embedded paraffin was cut into 5-μm thin wax sections with a microtome; it was immersed in a 42°C water bath for spreading, then laid flat on a glass slide and dried in an oven; dewaxed with xylene, and the tissue section was hydrated with alcohol from high concentration to low concentration to distilled water; stained with hematoxylin and eosin respectively, and dehydrated; after dehydration, it was sealed with neutral gum; after sealing, it was observed and photographed under a high-power optical microscope.

[0058] Experimental results

[0059] qPCR detection of RNA expression level

[0060] After extracting cell RNA and reverse transcription, qPCR detection was carried out, and the results are as Figure 1 shown. Using the combined treatment of Jinhua Fruit and LPS, the expression of inflammatory genes il-1, il-6, and il-10 was significantly inhibited at all three concentrations, and among them, tnf-a showed the most obvious inhibitory effect at LPS + 50 μg / mL.

[0061] Body weight loss rate and DAI

[0062] The body weight, bloody stool, and fecal consistency of the mice were observed and recorded every day, and the results are as Figure 2 shown. There were significant differences in the body weight loss rate and DAI index among the Jinhua Fruit 200 mg / kg, Jinhua Fruit 100 mg / kg, and sulfasalazine groups compared with the Model group.

[0063] Anal bleeding

[0064] As Figure 3 shown, compared with the control group, obvious peripheral bloody stools were observed in the model group, slight bloody stools in the Jinhua fruit 50mg / kg group, and no bloody stools in the Jinhua fruit 200mg / kg, Jinhua fruit 100mg / kg and sulfasalazine groups, showing some recovery.

[0065] Colon length

[0066] As Figure 4 shown, by measuring the colon length, the average colon length of the Control group was about 6.1 cm, that of the Model group was about 4.1 cm, that of the Jinhua fruit 200mg / kg group was about 5.6 cm, that of the Jinhua fruit 100mg / kg group was about 4.88 cm, that of the Jinhua fruit 50mg / kg group was about 4.32 cm, and that of the sulfasalazine group was about 5.87 cm. Compared with the Control group, there were significant differences in the Model, Jinhua fruit 100mg / kg, and Jinhua fruit 50mg / kg groups. Compared with the Model group, there were significant differences in the Jinhua fruit 200mg / kg, Jinhua fruit 100mg / kg, and sulfasalazine groups.

[0067] Colonic mucosa

[0068] As Figure 5 shown, there were significant differences between the Model group and the Control group. Compared with the Model group, there were significant differences in the Jinhua fruit 200mg / kg, Jinhua fruit 100mg / kg, Jinhua fruit 50mg / kg, and sulfasalazine groups.

[0069] HE

[0070] The results of HE staining were as Figure 6 shown. In the Control group, the crypt structure was clear and a large number of goblet cells were visible. Compared with the Control group, in the Model group, the crypt structure was lost, the epithelial cells were arranged disorderly, a large number of inflammatory cells infiltrated the basal layer, and the goblet cells were significantly reduced. Compared with the Model group, in the Jinhua fruit 200mg / kg, Jinhua fruit 100mg / kg, and sulfasalazine groups, the destruction of the colonic mucosa structure in mice was recovered to varying degrees, the inflammatory infiltration was reduced, and the number of goblet cells increased.

Claims

1. A golden flower fruit extract for treating ulcerative colitis is prepared by the following method: after pulverizing the golden flower fruit medicinal material, weigh the golden flower fruit sample and put it into a round-bottom flask, add a methanol aqueous solution, heat to boiling, keep slightly boiling for a period of time, then let it cool and filter, collect the filtrate, and then repeat the extraction of the residue twice according to the above method. Finally, combine the filtrates of the three times and concentrate to obtain a concentrated solution of the golden flower fruit extract, and then perform freeze-drying to obtain the golden flower fruit extract.

2. The extract of golden flower and fruit according to claim 1, characterized in that, The methanol aqueous solution is a 90% methanol aqueous solution.

3. The extract of golden flower and fruit according to claim 1, characterized in that, The mass-volume ratio of the golden flower fruit sample to the methanol aqueous solution is 1:

5.

4. Use of the golden flower fruit extract according to claim 1 in the preparation of a drug for treating ulcerative colitis.

5. A pharmaceutical composition for treating ulcerative colitis, comprising the golden flower fruit as a pharmaceutically active ingredient and a pharmaceutically acceptable carrier and excipient.

6. The pharmaceutical composition according to claim 5, wherein The mass percentage of the pharmaceutically active ingredient in the whole drug is 10% to 40%.

7. The pharmaceutical composition according to claim 5, wherein The pharmaceutical composition can be prepared into tablets, capsules, pills or oral liquids.

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