CN110755594B - Application of double-ginger stomachache prescription in preparation of medicine for treating inflammatory bowel disease - Google Patents
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Abstract
The invention discloses an application of a double-ginger stomachache prescription in preparing a medicament for treating inflammatory bowel diseases, wherein the double-ginger stomachache prescription is an extract obtained by mixing and extracting turmeric, purple ginger, delavay stephania root, grassleaf sweelflag rhizome and sow thistle seeds according to the weight ratio of 1:1:1:1: 1. Tests prove that the double-ginger stomachache prescription can obviously improve the mouse colon inflammation induced by the dextran sodium sulfate, and is shown in that the disease activity index of the mouse is obviously reduced, and the reduction of the mouse colon length caused by model building is obviously improved; the activity of MPO in serum is obviously reduced; obviously reduces the content of proinflammatory factors IL-1 beta, IL-6 and IL-1 beta in colon tissues, has obvious activity of resisting ulcerative colitis, and has good application prospect in preparing medicaments for treating inflammatory bowel diseases.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to application of a double-ginger stomachache treating preparation in preparation of a medicine for treating inflammatory bowel diseases.
Background
The prescription standard number WS-11105 (ZD-1105) -2002 for stomachache of ginger is recorded in the spleen and stomach booklet in the department of internal medicine in the compilation of national standards of Chinese patent medicines. Is prepared from 200g of turmeric, 200g of purple ginger, 200g of delavay stephania root, 200g of grassleaf sweelflag rhizome and 200g of Chinese ixeris seed as raw material medicaments, and has the effects of regulating qi, relieving pain, harmonizing stomach and lowering adverse qi.
The formula of Shuangjiang stomachache is used for treating epigastric fullness and distending pain, belching and acid regurgitation and chronic superficial gastritis with the symptoms caused by qi stagnation of middle energizer, but the medical application of the formula is not fully discovered and utilized.
Disclosure of Invention
The invention aims to provide application of a double-ginger stomachache treating preparation in preparation of a medicine for treating inflammatory bowel diseases.
The formula of the double-ginger stomachache is an extract obtained by mixing and extracting turmeric, purple ginger, delavay stephania root, grassleaf sweelflag rhizome and sow thistle according to the weight ratio of 1:1:1:1: 1.
The preparation method of the double-ginger stomachache treating prescription comprises the following steps: weighing turmeric, purple ginger, stephania delavayi, rhizoma acori graminei and sow thistle according to the weight ratio of 1:1:1:1:1, adding 4-5L of ethanol into every 500g of the total weight of the medicinal materials, soaking, heating and refluxing, filtering, refluxing and extracting filter residues for 2-3 times by using ethanol, combining filtrates, drying after reduced pressure concentration, and grinding to obtain the double-ginger stomachache treating prescription.
The concentration of ethanol was 75%.
The drying method is vacuum reduced pressure drying.
Application of SHUANGJIANGWEITONG preparation in preparing medicine for treating ulcerative colitis is provided.
Application of a formula of 'Shuangjiangweitong' in preparing a medicament for treating mouse colonic inflammation induced by dextran sodium sulfate.
The double-ginger stomachache prescription comprises a treatment effective dose of the double-ginger stomachache prescription and a pharmaceutically acceptable carrier.
The medicine is pill, capsule, tablet, powder, granule, injection, compound preparation or other oral dosage forms.
The dose of the formula for treating stomachache with ginger is 2-3 times per day, and 2.5-5 g for each time.
The dose of the formula for treating stomachache with ginger is 3 times a day, and 3 g of the formula is used for each time.
The invention has the advantages that:
tests prove that the double-ginger stomachache prescription can obviously improve the mouse colon inflammation induced by the dextran sodium sulfate, and is shown in that the disease activity index of the mouse is obviously reduced, and the reduction of the mouse colon length caused by model building is obviously improved; the activity of MPO in serum is obviously reduced; obviously reduces the content of proinflammatory factors IL-1 beta, IL-6 and IL-1 beta in colon tissues, relieves the inflammatory reaction of colon parts, has obvious anti-ulcerative colitis activity, and has good application prospect in preparing medicaments for treating inflammatory bowel diseases.
Detailed Description
The present invention is further illustrated but not limited in any way by the following examples, and any modifications made thereto are intended to fall within the scope of the present invention.
Application of a formula of 'Shuangjiangweitong' in preparing a medicament for treating inflammatory bowel disease.
The formula of the double-ginger stomachache is an extract obtained by mixing and extracting turmeric, purple ginger, delavay stephania root, grassleaf sweelflag rhizome and sow thistle according to the weight ratio of 1:1:1:1: 1.
The preparation method of the double-ginger stomachache treating prescription comprises the following steps: weighing turmeric, purple ginger, stephania delavayi, rhizoma acori graminei and sow thistle according to the weight ratio of 1:1:1:1:1, adding 4-5L of ethanol into every 500g of the total weight of the medicinal materials, soaking, heating and refluxing, filtering, refluxing and extracting filter residues for 2-3 times by using ethanol, combining filtrates, drying after reduced pressure concentration, and grinding to obtain the double-ginger stomachache treating prescription.
The concentration of ethanol was 75%.
The drying method is vacuum reduced pressure drying.
Application of SHUANGJIANGWEITONG preparation in preparing medicine for treating ulcerative colitis is provided.
Application of a formula of 'Shuangjiangweitong' in preparing a medicament for treating mouse colonic inflammation induced by dextran sodium sulfate.
The double-ginger stomachache prescription comprises a treatment effective dose of the double-ginger stomachache prescription and a pharmaceutically acceptable carrier.
The medicine is pill, capsule, tablet, powder, granule, injection, compound preparation or other oral dosage forms.
The dose of the formula for treating stomachache with ginger is 2-3 times per day, and 2.5-5 g for each time.
The dose of the formula for treating stomachache with ginger is 3 times a day, and 3 g of the formula is used for each time.
Example 1 preparation of a formula for stomachache
Weighing 100 g (total 500 g) of turmeric, purple ginger, stephania root, grassleaf sweelflag rhizome and sow thistle seed, putting the weighed materials into a 5L round-bottom flask, adding 5L 75% ethanol, soaking for 1 h, respectively using 5L, 4L and 4L 75% ethanol, refluxing and extracting once in water bath for 1.5 h, 1 h and 0.5 h respectively, combining three filtrates, recovering ethanol under reduced pressure until no alcohol smell exists, putting the mixed filtrate into a vacuum reduced-pressure drying oven, drying and grinding to obtain an extract, wherein the extraction rate is 43% for later use.
Example 2
Concentrating the extract obtained in example 1, evaporating to dryness under reduced pressure, pulverizing, sieving to obtain dry extract powder, and encapsulating to obtain capsule.
Example 3
The extract obtained in the embodiment 1 is taken, concentrated, evaporated to dryness under reduced pressure, crushed and sieved to obtain dry paste powder. Mixing the above dry extract powder with starch and dextrin, granulating with ethanol as wetting agent, adding magnesium stearate, and making into tablet.
Example 4: experimental study on treatment of ulcerative colitis caused by dextran sodium sulfate by using double-ginger stomachache prescription
1 materials of the experiment
1.1 Main Instrument
HH-6 constant temperature water bath, national electric appliances Limited; 282-2 desk low speed centrifuge, Shanghai medical instruments group, Inc.; desktop high speed refrigerated centrifuge HR/T16M, Hainan Hexi instrumentation, Inc.; BS-124S optical reading analytical balance, Beijing Sartorius balance, Inc.; one hundred thousand model TB-25 electronic balance, beijing danver instruments ltd; vortex oscillator, LAB-BLOGEN QUASI TECHNOLOGY VTX-E; water purification machines (Milli-Q Academic A10 ultra pure water system), Millipore USA; ultra low temperature (-80 ℃) refrigerator, Saimer Feishale science and technology (China) Co., Ltd; enzyme-labeled analyzers (Molecular Devices), Molecular Devices, U.S. Molecular instruments; ultrasonic cleaning machine, Ningbo Xinzhi Biotech GmbH.
1.2 major drugs and kits
Sulfasalazine enteric coated tablets (0.25 g, Shanghai Yinyi balance pharmaceutical Co., Ltd., lot number: 0917018); 4% paraformaldehyde (Wuhan Severer Biotech Co., Ltd., lot number: 183440); occult blood test box (Nanjing institute of bioengineering, lot number: 20161228); mouse interleukin 6 (IL-6) ELISA kit (Hangzhou Union Biotechnology, Inc., lot number: A20690151), interleukin-1 beta (IL-1 beta) ELISA kit (Hangzhou Union Biotechnology, Inc., lot number: A201B 81053), tumor necrosis factor-alpha (TNF-alpha) ELISA kit (Hangzhou Union Biotechnology, Inc., lot number: A28281212), and Myeloperoxidase (MPO) activity detection kit (Nanjing Jiancheng City bioengineering research institute, lot number: 20180728). Dextran sulfate sodium salt (DSS; MW: 36000-. Other chemical reagents were purchased from Kunming Hao technologies, Inc.
1.3 Experimental animals
Liaoning Changsheng Biotechnology GmbH, license number: SCXK (Liao) 2015-0001. Experimental mouse feed and padding purchased from sieke scenda biotechnology limited, hannan, license number: 2016-0002. After the experimental animal is fed into the animal feeding room, the experimental animal is adaptively fed for 1 week under the laboratory condition. The experimental method and conditions are approved by the ethical committee of the animal experiment center of Yunnan Chinese medicine university, and the animal ethical examination document number is as follows: r-06201934.
1.4 Main reagent preparation
Weighing 20 g of CMC-Na in a 1% CMC-Na solution, adding 2000 mL of distilled water, uniformly mixing to fully swell, standing overnight, and uniformly stirring to obtain the CMC-Na composite material.
Weighing 30 g of DSS in a 3% DSS solution, adding 1000 mL of distilled water, and uniformly mixing to fully dissolve the DSS. Storing at 4 deg.C for use.
Sulfasalazine suspension: taking 3 sulfasalazine enteric-coated tablets (0.25 g) and carefully scraping off the enteric-coated tablets by using a scalpel, fully grinding the tablets, and adding 0.5% CMC-Na solution to ensure that the final volume is 25 mL to obtain sulfasalazine suspension (0.03 g/mL) for later use. The intragastric volume is 0.1 mL/10 g, and the administration dosage is 0.3 g/kg.
Preparing a ginger stomachache sample: as in example 1.
The double-ginger stomachache prescription is prepared from liquid medicines in a high-dose group: taking a proper amount of the extract of the double-ginger stomachache prescription, and preparing a suspension of 0.204 g (crude drug)/mL by using a 1% CMC-Na solution. The gavage volume was 0.1 mL/10 g, and the administration dose was 2.04 g (crude drug)/kg.
The double-ginger stomachache prescription is prepared from liquid medicines in a low-dose group: taking a proper amount of the extract of the double-ginger stomachache prescription, and preparing a suspension of 0.068 g (crude drug)/mL by using a 1% CMC-Na solution. The gavage volume was 0.1 mL/10 g, and the administration dose was 0.68 g (crude drug)/kg.
2 method of experiment
2.1 animal grouping, modeling and administration
Animals were randomly divided into a normal group, a model group, a positive drug group (sulfasalazine, 0.3 g/kg), a dual-ginger stomachache prescription high dose group (2.04 g/kg) and a dual-ginger stomachache prescription low dose group (0.68 g/kg). The day of the start of the experiment was noted as day 0. Except for the normal group, the other groups were free to drink 3% DSS solution from day 0 of the start of the experiment, and when mice began to develop significant hematochezia (about 4 to 6 days), the DSS was stopped and replaced with pure water until day 10 of the experiment. The 3% DSS solution was replaced every 2 days. Each group was administered 1 time per day for 11 consecutive days starting on day 0 of the experiment. Both the normal and model groups were given equal volumes of 1% CMC-Na solution.
2.2 mouse Disease Activity Index (DAI) score
From the modeling day to the end of the experiment, the mental state, the eating condition, the hair regularity and glossiness, the weight, the stool property and the occult blood condition of the mice are observed every day for 11 days, and the weight, the stool property and the occult blood condition are selected and graded according to literature standards. The percentage of weight loss of animals (weight not changed to 0, 1% -5% to 1 point, 5% -10% to 2 points, 10% -20% to 3 points, more than 20% to 4 points), stool characteristics (normally 0 points, loose stool 2 points, water sample diarrhea 4 points), and stool bleeding [ normally 0 points, occult blood positivity 2 points, dominant bleeding (i.e., visible to the naked eye) 4 points ], wherein the occult blood condition of the stools was examined according to occult blood test kit instructions. Adding the above scores to obtain the Disease Activity Index (DAI) of mouse.
2.3 sample Collection
2.3.1 serum sample Collection
On the 9 th day of the experiment, the mice were fasted for 12 hours without water, on the 10 th day, after the administration was finished for 2 hours, the beard of the mice was cut off, and the eyeballs were removed to obtain blood in a centrifuge tube. Each mouse was bled at about 1 mL, left to stand at room temperature for 15 minutes, centrifuged at 3000 rpm for 15 minutes at 4 ℃ and the supernatant was collected and stored at-80 ℃ for determination of mouse MPO.
2.3.2 tissue sample Collection
The mice were sacrificed and dissected, the entire colon was cut, placed neatly on filter paper, and the colon length was measured and recorded. The lumen contents were gently rinsed off with normal saline and carefully dissected the most medial colon about 0.5-1 cm for fixation in 4% paraformaldehyde fixative overnight. Collecting the upper colon segment and the lower colon segment, immediately storing in liquid nitrogen, and storing in a refrigerator at-80 ℃ after the experiment is finished, and using for detecting mouse pro-colonic inflammatory factors IL-6, IL-1 beta and TNF-alpha.
2.4 MPO Activity detection
15. mu.L of serum reserved for each group was collected, the procedure was completed according to the kit protocol, and the absorbance of each tube was measured at 460 nm.
MPO Activity =
2.5 detection of proinflammatory factor content in colonic tissue
2.5.1 preparation of tissue homogenate
Taking a tissue sample of the upper segment of colon, shearing 50 mg into a glass homogenizer, adding 500 mu L of normal saline, fully homogenizing, collecting tissue homogenate to obtain 10% tissue homogenate, and storing at-20 ℃.
2.5.2 ELISA detection of proinflammatory factors IL-6, IL-1 beta, TNF-alpha
All reagents were transferred to room temperature and left for 30 minutes. And preparing each working solution, standard solution and sample diluent according to the kit operation instructions, and performing measurement. And calculating the content of the corresponding cytokine in each sample according to the standard curve.
2.6 statistical analysis of data
The experimental data were analyzed using SPSS 21.0 as mean. + -. standard deviation ((S))
) Expressed, the analysis was performed using one-way analysis of variance method (ANOVA). The data were analyzed by Homogeneity of varianaces in advance, and LSD was examined for HomogeneityIf the variance is not uniform, the test of Tamhane's T2 is carried out.
3 results of the experiment
3.1 Effect on the Disease Activity Index (DAI) of mice
When each model group mouse freely drinks the 3% DSS solution, physical changes such as listlessness, hair luster darkening, food intake reduction, weight loss and the like occur, a small part of mice appear diarrhea and excrement occult blood at the beginning of the 4 th day of the experiment, and most of the mice appear diarrhea and a small amount of macroscopic hematochezia at the 5 th day of the experiment. The 3% DSS solution of each modeling group was replaced with tap water. The mice in the model group have the most serious degree of hematochezia and diarrhea by the 6 th day; by the 8 th day of the experiment, the degree of hematochezia and diarrhea of the mice in the model group is relieved.
Table 1 effect of dual ginger stomachache formulation on DSS-induced UC model mouse DAI (n = 8)
Note: in comparison with the normal group,###：p<0.001; in comparison with the set of models,*：p<0.05，**：p<0.01
as can be seen from Table 1, the DAI scores of mice with UC models can be significantly reduced in the groups with high or low doses of the formula of Shuangjiang stomachache and the positive drug sulfasalazine. After 6 days of administration, the DAI reduction effect of the double-ginger stomachache prescription high-dose group is better than that of the low-dose group and sulfasalazine. By the end of the experiment, the effect of reducing DAI by using the double-ginger stomachache prescription high-dose group is still better than that of reducing DAI by using the double-ginger stomachache prescription low-dose group, and the effect is equivalent to that of sulfasalazine.
3.2 Effect on Colon Length changes in mice
Table 2 effect of dual ginger stomachache formulation on colon length change in DSS-induced UC model mice (n = 8)
Note: in comparison with the normal group,###：p<0.001; in comparison with the set of models,*：p<0.05，**：p<0.01
as can be seen from Table 2, the colon length of the model mouse is obviously shortened compared with that of the normal group mouse, and the phenomenon can be obviously improved by the group with high and low doses of the double-ginger stomachache prescription and the positive drug sulfasalazine.
3.3 Effect on mouse serum MPO Activity
TABLE 3 Effect of the formula of Alpinia zerumbet on the viability of mouse serum MPO (n = 8)
Note: in comparison with the normal group,###：p<0.001; in comparison with the set of models,*：p<0.05，**：p<0.01
as can be seen from Table 3, the serum MPO activity of the model mouse is significantly higher than that of the normal mouse, and the positive drug sulfasalazine can be significantly improved in the groups with high and low doses of the double-ginger stomachache prescription.
3.4 Effect on Colon tissue proinflammatory factor content in mice
As can be seen from tables 3, 4 and 5, the contents of proinflammatory factors IL-1 beta, IL-6 and TNF-alpha in colon tissues of model mice are all obviously higher than those of normal mice. The content of IL-1 beta in colon tissues of mice can be obviously reduced by using the double-ginger stomachache prescription with high dose, low dose and positive drug sulfasalazine, and the effect of the double-ginger stomachache prescription with high dose is superior to that of the low dose and positive drug sulfasalazine. The low dose and positive dose of the double-ginger stomachache prescription can obviously reduce the content of IL-6 in colon tissues of mice, and the high dose and low dose of the double-ginger stomachache prescription can obviously reduce the content of TNF-alpha in colon tissues of the mice, which indicates that the double-ginger stomachache prescription can obviously reduce the inflammatory reaction of the colon parts of mice in a UC model.
TABLE 4 Effect of the formula of Doenjang stomachache on the content of IL-1 beta in mouse colon tissue proinflammatory factor (n = 8)
Note:in comparison with the normal group,###：p<0.001; in comparison with the set of models,*：p<0.05，**：p<0.01，***：p<0.001
TABLE 5 Effect of the formula of Doenjang stomachache on the content of proinflammatory factor IL-6 in Colon tissue of mice (n = 8)
Note: in comparison with the normal group,###：p<0.001; in comparison with the set of models,*：p<0.05，**：p<0.01
TABLE 6 Effect of SANGJIANGWEITONG formulation on TNF- α content in mouse colon tissue proinflammatory factor (n = 8)
Note: in comparison with the normal group,###：p<0.001; in comparison with the set of models, **：p<0.01，***：p<0.001
in conclusion, the double-ginger stomachache formula has obvious activity of treating inflammatory bowel diseases, and has good application prospect in preparing medicines for treating inflammatory bowel diseases, in particular to ulcerative colitis.
In the mouse test, the dosage of the formula for treating the stomach ache of the ginger is 0.68 g/kg and 2.04 g/kg. Converted into human (calculated by adult weight 60 kg) dosage of 4.5 g and 13.5 g. In consideration of the difference in body weight of human and various other pathological conditions, the recommended dose is 2-3 times daily, 2.5-5 g each time. The most common dose is 3 times daily, 3 g each time.
Claims (6)
1. The formula of the double-ginger stomachache is an extract obtained by mixing and extracting turmeric, purple ginger, delavay stephania root, grassleaf sweelflag rhizome and sow thistle seeds according to the weight proportion of 1:1:1:1:1, wherein the extract is an ethanol extract, and the concentration of ethanol is 75%.
2. The use according to claim 1, wherein said formula for treating stomachache from Zingiber officinale comprises: weighing turmeric, purple ginger, stephania delavayi, rhizoma acori graminei and sow thistle according to the weight ratio of 1:1:1:1:1, adding 4-5L of ethanol into every 500g of the total weight of the medicinal materials, soaking, heating and refluxing, filtering, refluxing and extracting filter residues for 2-3 times by using ethanol, combining filtrates, drying after reduced pressure concentration, and grinding to obtain the double-ginger stomachache treating prescription.
3. The use according to claim 2, wherein the preparation method of the formula comprises vacuum drying under reduced pressure.
4. The use according to claim 1, wherein the ulcerative colitis is colitis in mice colon inflammation induced by dextran sodium sulfate.
5. The use according to claim 1, wherein said ginger heartburn formulation comprises a pharmaceutically acceptable carrier.
6. The use according to claim 1, wherein the medicament is in the form of a pill, capsule, tablet, powder, granule or other oral dosage form.
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