WO2020187003A1 - 金芪降糖口服制剂在制备治疗溃疡性结肠炎的药物中的应用 - Google Patents

金芪降糖口服制剂在制备治疗溃疡性结肠炎的药物中的应用 Download PDF

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WO2020187003A1
WO2020187003A1 PCT/CN2020/077204 CN2020077204W WO2020187003A1 WO 2020187003 A1 WO2020187003 A1 WO 2020187003A1 CN 2020077204 W CN2020077204 W CN 2020077204W WO 2020187003 A1 WO2020187003 A1 WO 2020187003A1
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oral preparation
colon
jinqi jiangtang
preparation
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English (en)
French (fr)
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王治平
王一飞
吴子聪
邓剑平
赵振岭
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广东药科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
    • A61K36/718—Coptis (goldthread)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

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  • the invention relates to a new use of a traditional Chinese medicine compound preparation, in particular to the application of Jinqi Jiangtang oral preparation in the preparation of a medicine for treating ulcerative colitis.
  • Ulcerative colitis is a disease caused by the interaction of genetic background and environmental factors. It presents a chronic inflammatory reaction state with continuous lesions that can involve different parts of the rectum and colon. The specific cause is not clear It is clinically characterized by alternation of onset, remission and relapse, and is a common intractable disease of the digestive system. It is estimated that the prevalence of ulcerative colitis in China is 11.6 per 100,000, and there is no epidemiological data for a large sample of people. According to statistics from Nanchang City, patients with ulcerative colitis accounted for 1.37% of the total colonoscopy population.
  • patients with ulcerative colitis accounted for 3.27% of the total number of inpatients in the digestive department.
  • the clinical manifestations of the patients are: diarrhea, mucus and pus-blooded stools, 3 to 4 times a day in mild cases, dozens of times a day or diarrhea and constipation in severe cases; patients with mild abdominal pain have no abdominal pain or only abdominal discomfort.
  • abdominal pain which are pains in the left lower abdomen or lower abdomen, involving total abdominal pain, and the regularity of relief after defecation; constipation, constipation, constipation, defecation once in 4 to 5 days, stools are like sheep feces, even if you do not take laxatives Stool; other symptoms: abdominal distension, weight loss, fatigue, bowel ringing, insomnia, dreaminess, fear of cold and other symptoms.
  • Ulative colitis 15% to 30% of them have the disease. It seems that the occurrence of colitis is related to genetic factors.
  • the current methods of treating colitis mainly take western medicine and enema as the main methods.
  • western medicine is prone to relapse and has relatively large side effects.
  • the enema is effective in a short time, but it is easy to cause imbalance of the intestinal flora. Therefore, searching for Chinese medicines with good therapeutic effects on ulcerative colitis is of great significance for discovering new therapeutic drugs for ulcerative colitis.
  • Jinqi Jiangtang oral preparation is a marketed traditional Chinese medicine preparation.
  • the marketed dosage forms include tablets, capsules and granules.
  • the main function is to clear away heat and replenish qi. It is used for the syndrome of qi deficiency and internal heat in diminishing thirst. , Shortness of breath and fatigue, etc., for mild and moderate type 2 diabetes, see the above syndromes.
  • the purpose of the present invention is to provide the application of Jinqi Jiangtang oral preparation in the preparation of drugs for treating ulcerative colitis.
  • the Jinqi hypoglycemic oral preparation of the present invention is made from the following traditional Chinese medicine raw materials: coptis, astragalus and honeysuckle.
  • the Jinqi hypoglycemic oral preparation is made from the following raw materials in parts by weight: 2-20 parts of Coptidis, 3-30 parts of Astragalus, and 12-120 parts of honeysuckle.
  • the Jinqi Jiangtang oral preparations are Jinqi Jiangtang tablets, Jinqi Jiangtang granules or Jinqi Jiangtang capsules, which are all marketed traditional Chinese medicine preparations, and may also be Jinqi Jiangtang formula granules.
  • the Jinqi Jiangtang oral preparation can significantly reduce the DAI index of mice with ulcerative colitis, alleviate the shortening of the colon to varying degrees, and can significantly inhibit the MDA content in the colon tissue of the mice, and increase H 2 O 2 content in mouse colon tissue, and can significantly inhibit the three inflammatory factors (IL-1 ⁇ , IL-6 and TNF- ⁇ ) in mouse colon tissue, and have varying degrees of therapeutic effect on ulcerative colitis .
  • the present invention has the following beneficial effects:
  • the present invention provides a new use of Jinqi Jiangtang oral preparation, and provides a new drug choice for ulcerative colitis, and the Jinqi Jiangtang oral preparation has no toxicity report in animal experiments and clinical use, and has a drug source With rich, safe and non-toxic characteristics, it can be further developed as anti-inflammatory drugs, with clinical application prospects and high economic value.
  • Figure 1 shows the daily weight loss rate DAI (A) of each group of mice in Example 1, the daily DAI (B) of each group of mice, the colon length statistics (C), and the representative colon physical map (D).
  • Figure 2 is a representative colon H&E stained section (A) of each group of mice in Example 1, and a histogram of colon pathology score (B);
  • Figure 3 is a bar graph of H 2 O 2 content (A) and MDA content (B) in the colon tissue of each group of mice in Example 1;
  • Figure 4 is a bar graph showing the protein content of inflammatory factors IL-1 ⁇ (A), IL-6 (B) and TNF- ⁇ (C) in the colon tissue of each group of mice in Example 1;
  • Figure 5 is a bar graph showing the relative gene expression levels of IL-1 ⁇ (A) and IL-6 (B) in the colon tissue of each group of mice in Example 1;
  • Figure 6 shows the daily DAI diagram (A), colon length statistics (B) and representative colon physical diagram (C) of each group of mice in Example 2;
  • Figure 7 is a bar graph of the spleen index of mice in each group in Example 2.
  • Figure 8 is a representative colon H&E stained section of each group of mice in Example 2 (A), and a histogram of colon pathology score (B);
  • Figure 9 is a bar graph showing the H 2 O 2 content (A) and MPO content (B) in the colon tissue of each group of mice in Example 2.
  • Example 1 Animal experiment (acute model) of Jinqi Jiangtang oral preparation for treating ulcerative colitis:
  • mice 6-8 weeks old, male, SPF grade, Balb/c mice.
  • the mice were randomly divided into: blank group (Control), model group (Model), positive drug group (SASP), Jinqijiangtang oral preparation low-dose group (JQ(L)) and Jinqijiangtang oral preparation high-dose group Group (JQ(H)). There are 6 in each group, 30 in total. In the SPF laboratory adaptive feeding for one week.
  • the dosage of Jinqi Jiangtang oral preparation high-dose group was 1.0g/kg/d, and the dosage of Jinqi Jiangtang oral preparation low-dose group was 0.5g/kg/d.
  • the dosage was 0.6g/kg/d, the dosage was gavage, and the dosage was 0.2ml/10g.
  • the blank group and the model group were given an equal volume of distilled water every day. The drug was administered at the same time on the day of modeling. 4.
  • Weight loss Calculated by the percentage of weight loss, no weight loss is 0 points, weight loss 1%-5% is 1 point, weight loss 6%-10% is 2 points, and weight loss 11%-15% is 3 points. Weight loss greater than 15% is 4 points.
  • Stool viscosity 0 for normal, 2 points for loose stool, 4 points for diarrhea.
  • Stool bleeding 0 points for normal, 1 point for occult bleeding, 3 points for obvious bleeding.
  • DAI (weight score + stool trait score + bleeding score)/3, the results are shown in Table 3 and Table 4:
  • mice were sacrificed on the first day after the last dose.
  • the abdomen of the mouse was opened, the abdominal cavity was exposed, and the entire colon from 2 cm from the anus to the cecum was cut off.
  • Fresh tissues are fixed with fixative for more than 24 hours. Take the tissue out of the fixative solution and place it in a fume hood to trim the target site tissue with a scalpel. Put the trimmed tissue and the corresponding label in the dehydration box.
  • Dewatering and soaking wax Put the dehydration box into the hanging basket and dehydrate with gradient alcohol in the dehydrator. 75% alcohol 4h-85% alcohol 2h-90% alcohol 2h-95% alcohol 1h-anhydrous ethanol I 30min-anhydrous ethanol II 30min-alcohol benzene 5-10min-xylene I5-10min-xylene II 5-10min -65°Melting Paraffin I 1h-65°Melting Paraffin II 1h-65°Melting Paraffin III 1h.
  • Embedding Embed the wax-soaked tissue in the embedding machine. First put the melted wax into the embedding frame, and before the wax solidifies, take the tissue out of the dehydration box and put it into the embedding frame according to the requirements of the embedding surface and attach the corresponding label. After cooling on a -20°freezing table, remove the wax block from the embedding frame and trim the wax block after the wax solidifies.
  • Sectioning Put the trimmed wax block in a paraffin microtome for sectioning, with a thickness of 4 ⁇ m. Float the slices on the spreader's 40°C warm water to flatten the tissue, lift the tissue with a glass slide, and bake the slices in a 60°C oven. After the water-baked dry wax is baked, take it out and store at room temperature for later use.
  • Paraffin sections are deparaffinized to water: sequentially put the sections into xylene I 20 min-xylene II 20 min-anhydrous ethanol I 5 min-anhydrous ethanol II 5 min-75% alcohol for 5 min, and wash with tap water.
  • Hematoxylin staining Stain the slices in hematoxylin staining solution for 3-5 minutes, wash with tap water, differentiate in differentiation solution, wash with tap water, return blue solution to blue, and rinse with running water.
  • Eosin staining the sections were sequentially dehydrated with 85% and 95% gradient alcohol for 5 minutes, and stained in eosin staining solution for 5 minutes.
  • Histopathology score was based on the Cooper inflammation grading standard. At 200 times magnification, each specimen is in the pathological section of the same colon segment, and 8 fields of view are randomly selected. Observe and score each field of view. Take these 8 average values as the histopathological score value of this specimen, the specific scoring method is as follows:
  • the mucosal layer is moderately inflamed, and 2/3 of the crypts at the base disappear;
  • FIG. 2(A) The histopathological results of representative colonic mucosa of mice in each group are shown in Figure 2(A). It can be seen from the figure that the colon mucosa tissue of the blank group is intact, the crypt structure is intact, and there is no edema in the submucosa. The mucosal epithelium of mice in the model group was largely damaged, shedding, crypt structure disappeared, ulcers were formed, and a large number of inflammatory cell infiltrations were seen in the submucosa. Sulfasalazine and Jinqi Jiangtang oral preparations can alleviate the above pathological features. It reduces the shedding of epithelial tissue and also reduces the infiltration of inflammatory cells.
  • the pathological scores of the colonic mucosa of mice in each group are shown in Table 6 and Figure 2 (B).
  • the model group had the highest pathological score, which was significantly different from the blank group (P ⁇ 0.01).
  • the pathological scores of each treatment group were reduced, indicating that each administration group has a certain degree of therapeutic effect on ulcerative colitis, and it is statistically significant (P ⁇ 0.01).
  • MDA MDA-binding protein
  • the molar concentration of MDA is calculated according to the standard curve. After calculating the MDA content in the sample solution, the MDA content in the initial sample is expressed by the protein content per unit weight: ⁇ mol/mg protein.
  • the background MDA of the blank group is about 100ng/mg protein.
  • the MDA in the model group was significantly higher, with a significant difference (P ⁇ 0.05).
  • the positive drug sulfasalazine group was reduced, but there was no significant difference (P>0.05), while the MDA in the colon tissue of mice in the Jinqi Jiangtang oral preparation group was reduced Significantly inhibited, and statistically very significant (P ⁇ 0.01).
  • Hydrogen peroxide concentration (mM) 22.94X A240, so as to calculate the actual concentration of hydrogen peroxide standard provided by the kit; melt the hydrogen peroxide detection reagent on ice or ice water bath; add 50 micrometers to the detection hole Liter of sample or standard; add 100 microliters of hydrogen peroxide detection reagent to each well; gently shake or beat to mix, incubate at 37°C for 30 minutes, and immediately determine A540; calculate the amount of hydrogen peroxide in the sample according to the standard curve concentration.
  • the background hydrogen peroxide content of the blank group is about 600ng/mg protein.
  • H 2 O 2 in the model group was reduced, but there was no significant difference (P>0.05).
  • the positive drug sulfasalazine group increased, but there was no significant difference (P>0.05), while the Jinqi Jiangtang oral preparation group had an increase in the colon tissue of mice H 2 O 2 increased significantly, and there was a statistically significant difference (P ⁇ 0.05).
  • Wash Buffer Dilute the Wash Buffer (20 ⁇ ) 20 times with distilled water to obtain Wash Buffer (1 ⁇ ); dilute the standard in a 1.5 mL EP tube, and use Sample Diluent to dilute the sample to 1000, 500, 250, 125, 62.5, 31.25, 15.6, 0pg/mL; add 100 ⁇ L of diluted standard to the standard wells; add 100 ⁇ L Sample Diluent to the blank wells; add 100 ⁇ L of sample to the sample wells, paste the sealing membrane, and incubate at 37°C for 90min; wash the plate: 350 ⁇ L Wash Buffer/well, wash 3 times; prepare the biotinylated antibody working solution, dilute the concentrated biotinylated antibody (30X) 30 times with the biotinylated antibody diluent; add 100 ⁇ L biotinylated antibody working solution to each well.
  • Sample Real-timePcR detection dilute the cDNA obtained in the previous step, and then use the primers of the target gene and the internal reference gene to amplify respectively.
  • the primer sequence is shown in Table 12, and the PCR reaction solution is prepared according to the components in Table 11 (on ice)
  • the amplification program is: 95°C pre-denaturation 30s, 95°C denaturation 5s, annealing 60°C, 10s, a total of 40 cycles, extension 95°C, 15s, 60°C, 1min, 95°C, 15s.
  • mice 6-8 weeks old, male, SPF grade, Balb/c mice.
  • the mice were randomly divided into: blank group (Control), model group (Model), positive drug group (SASP) and Jinqi Jiangtang oral preparation (JQ). There are 6 in each group, 24 in total.
  • the chronic ulcerative colitis model is divided into three cycles, the model group, the positive drug group and the Jinqi Jiangtang oral preparation administration group in the first cycle, from the first day to the sixth day, 2% DSS is used instead of drinking water.
  • Mice were given ordinary drinking water from 7 to 14 days; in the second cycle, 2% DSS was used instead of drinking water to give mice from total 15 days to total 21 days, and mice were given from total 22 days to 23 days.
  • With ordinary drinking water in the third cycle, the mice were given 2% DSS instead of drinking water from the 31st day to the 25th day, and ordinary drinking water was given from the 36th day to the 43rd day.
  • the blank group Always give regular drinking water.
  • the dosage of Jinqi Jiangtang oral preparation group is 1.0g/kg/d
  • the dosage of positive drug mesalazine group is 0.6g/kg/d
  • the administration method is intragastric administration
  • the administration volume is 0.2m L/10g, once a day from the 6th day of modeling, to the 43rd day.
  • the blank group and the model group were given an equal volume of distilled water every day.
  • DAI disease activity index
  • mice were sacrificed on the first day after the last dose.
  • the abdomen of the mouse was opened, the abdominal cavity was exposed, and the entire colon from 2 cm from the anus to the cecum was cut off.
  • FIG. 8(A) The histopathological results of representative colonic mucosa of mice in each group are shown in Figure 8(A). It can be seen from the figure that the colon mucosa tissue of the blank group is intact, the crypt structure is intact, and there is no edema in the submucosa. The mucosal epithelium of mice in the model group was largely damaged, shedding, crypt structure disappeared, ulcers were formed, and a large number of inflammatory cell infiltrations were seen in the submucosa. Sulfasalazine and Jinqi Jiangtang oral preparations can alleviate the above pathological features. It reduces the shedding of epithelial tissue and also reduces the infiltration of inflammatory cells.
  • the pathological scores of the colonic mucosa of mice in each group are shown in Table 17 and Figure 8 (B).
  • the model group had the highest pathological score, which was significantly different from the blank group (P ⁇ 0.01).
  • the pathological scores of each treatment group were reduced, indicating that each administration group has a certain degree of therapeutic effect on ulcerative colitis, and it is statistically significant (P ⁇ 0.01).
  • the MPO activity of the model group was significantly higher than that of the blank group (P ⁇ 0.01).
  • MPO can recruit neutrophils and cause colonic tissue damage, while the positive drugs SASP and Jinqin reduce blood sugar
  • the tablet has a significant inhibitory effect on the increase of MPO activity induced by DSS (P ⁇ 0.01), which is consistent with the colonic pathological observation results.

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Abstract

金芪降糖口服制剂在制备治疗溃疡性结肠炎的药物中的应用。金芪降糖口服制剂在动物实验和临床应用中未见毒性报告，具有药源丰富、安全无毒的特征，可进一步开发为用于抗炎药物，具有临床应用前景和经济利用价值。

Description

金芪降糖口服制剂在制备治疗溃疡性结肠炎的药物中的应用 技术领域
本发明涉及一种中药复方制剂的新用途，具体涉及金芪降糖口服制剂在制备治疗溃疡性结肠炎的药物中的应用。
背景技术
溃疡性结肠炎(ulcerative colitis)是一种由遗传背景与环境因素相互作用而产生的疾病，呈慢性的炎性反应状态，病变呈连续性，可累及直肠、结肠的不同部位，具体病因尚未明确，临床以发作、缓解和复发交替为特点，是常见的消化系统疑难病。据推测中国溃疡性结肠炎患病率为11.6/10万，目前尚无大样本人群的流行病学资料。据南昌市统计，溃疡性结肠炎患者占结肠镜检查总人群的1.37％，宁夏自治区统计2014年溃疡性结肠炎患者占全年消化科住院总人数的3.27％。患者的临床表现为：腹泻黏液便及脓血便，轻者每天3～4次，重者数十次或腹泻与便秘交替出现；腹痛轻度患者无腹痛或仅有腹部不适。一般有轻度至中度腹痛，系左下腹或下腹阵痛，涉及全腹痛，排便后缓解的规律；便秘大便秘结4～5日排便一次，粪便如羊屎样，甚则不吃泻药不能通便；其他症状腹胀、消瘦、乏力、肠鸣、失眠、多梦、怕冷等症。溃疡性结肠炎患者的直系亲属中，15％～30％的人发病，由此看来结肠炎的发生于遗传因素有一定的关系。
目前治疗结肠炎的方法主要以西药、灌肠等手段为主要手段，但是西药容易复发，且副作用较大，灌肠短时间内见效，但容易导致肠内菌群失调。因此，寻找对溃疡性结肠炎具有良好治疗作用的中药，对于发现溃疡性结肠炎的新型治疗药物具有重要意义。
金芪降糖口服制剂为上市中药制剂，上市剂型包括片剂、胶囊剂及颗粒剂，功能主要为清热益气，用于消渴病气虚内热证，症见口渴喜饮，易饥多食，气短乏力等，用于轻、中型二型糖尿病见上述证候者。目前尚无将金芪降糖口服制剂用于治疗溃疡性结肠炎的研究报道。
发明内容
鉴于此，本发明的目的在于提供金芪降糖口服制剂在制备治疗溃疡性结肠炎的药物中的应用。
本发明所述的金芪降糖口服制剂，由下列中药原料制作而成：黄连、黄芪、金银花。
作为优选，所述的金芪降糖口服制剂，按重量份数计，由如下原料制成：黄连2-20份、黄芪3-30份、金银花12-120份。
在本发明中，所述金芪降糖口服制剂为金芪降糖片、金芪降糖颗粒或金芪降糖胶囊，均为已上市中药制剂，也可以为金芪降糖配方颗粒。
本发明经药理研究发现，金芪降糖口服制剂能显著降低溃疡性结肠炎小鼠DAI指数、对结肠缩短均有不同程度的缓解，且能显著抑制小鼠结肠组织中的MDA含量、提高小鼠结肠组织中的H 2O 2含量，并能显著抑制小鼠结肠组织中的三种炎症因子(IL-1β、IL-6和TNF-α)，对溃疡性结肠炎有不同程度的治疗作用。
本发明与现有技术相比，具有如下有益效果：
本发明提供了金芪降糖口服制剂的新用途，为溃疡性结肠炎提供了一种新的药物选择，且金芪降糖口服制剂在动物实验和临床使用中未见毒性报告，具有药源丰富、安全无毒的特征，可进一步开发为用于抗炎药物，具有临床应用前景和很高的经济利用价值。
附图说明
图1为实施例1中各组小鼠每天体重减轻率DAI(A)，各组小鼠每天DAI(B)，结肠长度统计(C)，代表性结肠实物图(D)。
图2为实施例1中各组小鼠代表性结肠H&E染色切片图(A)，及结肠病理评分(B)柱形图；
图3为实施例1中各组小鼠结肠组织中H 2O 2含量(A)和MDA含量(B)柱形图；
图4为实施例1中各组小鼠结肠组织中炎症因子IL-1β(A)，IL-6(B)和TNF-α(C)蛋白含量柱形图；
图5为实施例1中各组小鼠结肠组织中IL-1β(A)，IL-6(B)相对基因表达量柱形图；
图6为实施例2中各组小鼠每天DAI图(A)，结肠长度统计(B)以及代表性结肠实物图(C)；
图7为实施例2中各组小鼠脾脏指数柱形图；
图8为实施例2中各组小鼠代表性结肠H&E染色切片图(A)，及结肠病理评分柱形图(B)；
图9为实施例2中各组小鼠结肠组织中H 2O 2含量(A)和MPO含量(B)柱形图。
具体实施方式
下面通过具体实施方式来进一步说明本发明，以下实施例为本发明具体的实施方式，但本发明的实施方式并不受下述实施例的限制。
实施例1：金芪降糖口服制剂治疗溃疡性结肠炎的动物实验(急性模型)：
一、主要仪器及试剂(见表1和表2)：
表1主要仪器
Figure PCTCN2020077204-appb-000001
Figure PCTCN2020077204-appb-000002
表2主要试剂及药物
Figure PCTCN2020077204-appb-000003
Figure PCTCN2020077204-appb-000004
二、动物实验方法
1、分组，造模及给药
6-8周龄，雄性，SPF级，Balb/c小鼠。将小鼠随机分为：空白组(Control)、模型组(Model)、阳性药组(SASP)、金芪降糖口服制剂低剂量组(JQ(L))及金芪降糖口服制剂高剂量组(JQ(H))。每组6只，共30只。在SPF级实验室适应性喂养一周。
2、溃疡性结肠炎模型的建立
配制浓度为4％的DSS水溶液。模型组自由饮用4％DSS水溶液。空白组自由饮用蒸馏水。常规饮食不变，连续饮用7天。
3、动物实验给药
3.1动物实验给药准备
取金芪降糖片(药物组成：黄连103g，黄芪154g，金银花618g，规格：0.42g/片)，称取2.5g，在20mL蒸馏水中分散，超声30min，使用容量瓶定容至50mL。
3.2实验给药
金芪降糖口服制剂高剂量组给药剂量为1.0g/kg/d，金芪降糖口服制剂低剂量组给药剂量为0.5g/kg/d。阳性药美沙拉嗪组，给药剂量为0.6g/kg/d，给药方式为灌胃给药，给药体积为0.2m L/10g。空白组和模型组每天灌胃等体积的蒸馏水。造模当天同时开始给药。4、疾病活动指数(the disease activity index，DAI)评分
4.1大便隐血检测方法
按照隐血检测试剂盒说明书进行检测。
4.2 DAI评分
体重减少：按减少体重的百分比计算，无体重减轻为0分，体重减轻1％-5％为1分，体重减轻6％-10％为2分，体重减轻11％-15％为3分，体重减轻大于15％为4分。
大便黏稠度：正常为0，松散便2分，腹泻4分。
大便出血：正常0分，隐血出血1分，显性出血3分。
分别就体重、大便粘稠度、大便出血进行评分。DAI值即这三项的平均值。DAI＝(体重评分+大便性状评分+出血评分)/3，结果见表3和表4所示：
表3DAI评分标准
Figure PCTCN2020077204-appb-000005
表4各组小鼠DAI指数(x±S)(n＝6)
Figure PCTCN2020077204-appb-000006
见表4与图1(A)，与空白组相比，模型组DAI指数在第二天就开始上升，并在第三天明显上升，且存在显著性差异(P<0.01)。与模型组相比，阳性药和金芪降糖口服制剂能降低溃疡性结肠炎小鼠DAI指数，而金芪降糖口服制剂则和模型组相比存在显著性差异。说明阳性药和金芪降糖口服制剂能对溃疡性结肠炎有不同程度的治疗作用。
5、取材
于最后一次给药后的第一天处死小鼠。剖开小鼠腹部，暴露腹腔，剪去肛门上2cm至盲肠的整段结肠。将整段结肠拍照并测量长度。沿肠系膜边缘纵向剖开，用生理盐水冲洗肠内容物并测量结肠重量。将整段结肠一分为二，一半放入10％福尔马林缓冲液中固定备用，另一半迅速放入液氮中，置于-80℃保存。
表5各组小鼠结肠长度(x±S)(n＝6)
Figure PCTCN2020077204-appb-000007
各组小鼠代表性的结肠外观图见图1(D)。从表5以及图1(C)中可以看出，模型组小鼠结肠明显缩短，与空白组相比，存在极显著性差异(P<0.01)。与模型组相比，金芪降糖口服制剂组对结肠缩短均有不同程度的缓解，且具有统计学意义(P<0.05)。
6、结肠病理观察
6.1石蜡切片制备方法
1.取材：新鲜组织用固定液固定24h以上。将组织从固定液取出在通风橱内用手术刀将目的部位组织修平整，将修切好的组织和对应的标签放于脱水盒内。
2.脱水浸蜡：将脱水盒放进吊篮里于脱水机内依次梯度酒精进行脱水。75％酒精4h-85％酒精2h-90％酒精2h-95％酒精1h-无水乙醇I 30min-无水乙醇II 30min-醇苯5-10min-二甲苯I5-10min-二甲苯II 5-10min-65°融化石蜡I 1h-65°融化石蜡II 1h-65°融化石蜡III 1h。
3.包埋：将浸好蜡的组织于包埋机内进行包埋。先将融化的蜡放入包埋框，待蜡凝固之前将组织从脱水盒内取出按照包埋面的要求放入包埋框并贴上对应的标签。于-20°冻台冷却，蜡凝固后将蜡块从包埋框中取出并修整蜡块。
切片：将修整好的蜡块置于石蜡切片机切片，厚4μm。切片漂浮于摊片机40℃温水上将组织展平，载玻片将组织捞起，60℃烘箱内烤片。水烤干蜡烤化后取出常温保存备用。
石蜡切片脱蜡至水：依次将切片放入二甲苯Ⅰ20min-二甲苯Ⅱ20min-无水乙醇Ⅰ5min-无水乙醇Ⅱ5min-75％酒精5min，自来水洗。
4.苏木素染色：切片入苏木素染液染3-5min，自来水洗，分化液分化，自来水洗，返蓝液返蓝，流水冲洗。
5.伊红染色：切片依次入85％、95％的梯度酒精脱水各5min，入伊红染液中染色5min。
6.脱水封片：切片依次放入无水乙醇I 5min-无水乙醇II 5min-无水乙醇Ⅲ5min-二甲Ⅰ5min-二甲苯Ⅱ5min透明，中性树胶封片。
7.显微镜镜检，图像采集分析。
6.2组织病理学评分
组织病理学评分依据Cooper炎症分级标准进行组织病理学评分。在放大200倍下，每个 标本在同一结肠区段的病理切片区域里，随机选取8个视野。对每个视野进行观察和评分。将这8个平均值作为这个标本的组织病理学评分值，具体评分方法如下：
0正常肠黏膜；
1黏膜层轻度炎症和水肿，基底部1/3隐窝消失；
2黏膜层中度炎症，基底部2/3隐窝消失；
3黏膜层中度炎症，隐窝完全消失，但上皮层尚完整；
4黏膜层、黏膜下层、肌层重度炎症，隐窝和上皮层消失。
各组小鼠代表性的结肠黏膜组织病理结果图见图2(A)。从图中可以发现，空白组小鼠结肠黏膜组织完好，隐窝结构完整，黏膜下层未见水肿。模型组小鼠黏膜上皮大量破损，脱落，隐窝结构消失，有溃疡形成，黏膜下层可见大量炎性细胞浸润。柳氮磺吡啶及金芪降糖口服制剂能缓解以上病理特征。减少上皮组织脱落，也可以减轻炎性细胞浸润情况。
各组小鼠结肠黏膜病理评分见表6及图2(B)。各组中，模型组病理评分最高，与空白组相比存在极显著性差异(P<0.01)。与模型组相比，各治疗组病理评分都有所降低，表明各给药组对溃疡性结肠炎都有一定程度的治疗作用，且有统计学意义(P<0.01)。
表6各组小鼠结肠黏膜病理评分(x±S)(n＝6)
Figure PCTCN2020077204-appb-000008
7、结肠组织MDA检测
取约0.5cm长度结肠使用200μL生理盐水进行匀浆或裂解；4℃，14000rpm离心5min，取上清；使用用BCA蛋白浓度测定试剂盒测定蛋白浓度，以便于后续计算单位蛋白重量结肠组织MDA含量；取适量标准品用生理盐水稀释至1、2、5、10、20、50μM，用于后续制作标准曲线；在离心管或其它适当容器内加入0.1ml生理盐水作为空白对照，加入0.1ml上述不同浓度标准品用于制作标准曲线，加入0.1ml样品用于测定；随后加入0.2ml MDA检测工作液；100℃或沸水浴加热15分钟；水浴冷却至室温，1000g室温离心10分钟。取200微升上清加入到96孔板中，随后用酶标仪在540nm测定吸光度。根据标准曲线计算获得MDA的摩尔浓度，计算出样品溶液中的MDA含量后，通过单位重量的蛋白含量来表示最初样品中的MDA含量：μmol/mg 蛋白。
表7各组小鼠结肠MDA含量(x±S)(n＝6)
Figure PCTCN2020077204-appb-000009
从表7和图3(B)中可以看出，空白组MDA本底约为100ng/mg蛋白。与空白组相比，模型组中MDA显著增高，有显著性差异(P<0.05)。在给药治疗后，与模型组相比，阳性药柳氮磺吡啶组有所降低，但没有显著性差异(P>0.05)，而金芪降糖口服制剂组小鼠结肠组织中的MDA被显著抑制，且统计学上非常显著(P<0.01)。
8、结肠组织过氧化氢检测
取约0.5cm长度结肠使用200μL生理盐水进行匀浆或裂解；4℃，14000rpm离心5min，取上清；使用用BCA蛋白浓度测定试剂盒测定蛋白浓度，以便于后续计算单位蛋白重量结肠组织MDA含量；取适量标准品用生理盐水稀释至1、2、5、10、20、50μM，用于后续制作标准曲线；把浓度约为1M的过氧化氢用水稀释100倍，使过氧化氢的浓度约为10mM，测定A240。过氧化氢浓度(mM)＝22.94X A240，从而计算出试剂盒提供的过氧化氢标准品的实际浓度；把过氧化氢检测试剂在冰上或冰水浴上融解；在检测孔内加入50微升样品或标准品；在每个孔内加入100微升过氧化氢检测试剂；轻轻振荡或敲打混匀，37℃孵育30分钟，立即测定A540；根据标准曲线计算出样品中过氧化氢的浓度。
表8各组小鼠结肠H 2O 2含量(x±S)(n＝6)
Figure PCTCN2020077204-appb-000010
从表8和图3(A)中可以看出，空白组过氧化氢含量本底约为600ng/mg蛋白。与空白组相比，模型组中H 2O 2有所降低，但没有显著性差异(P>0.05)。在给药治疗后，与模型组相比，阳性药柳氮磺吡啶组有所升高，但也没有显著性差异(P>0.05)，而金芪降糖口服制剂组小鼠结肠组织中的H 2O 2则显著升高，且统计学上有显著性差异(P<0.05)。
9、ELISA检测
检测结肠组织中炎症因子IL-1β、IL-6和TNF-α表达水平。按照ELISA试剂盒说明书操作如下：
用蒸溜水将Wash Buffer(20×)稀释20倍，得到Wash Buffer(1×)；在1.5mL EP管中梯度稀释标准品，用Sample Diluent将样本稀释成1000、500、250、125、62.5、31.25、15.6、0pg/mL；向标准孔中加入100μL稀释后标准品；向空白孔中加入100μLSample Diluent；向样本孔中加入100μL样品，贴封板膜，37℃孵育90min；洗板：350μL Wash Buffer/孔，洗3次；准备生物素化抗体工作液，用生物素化抗体稀释液将浓缩生物素化抗体(30X)稀释30倍；每孔加100μL生物素化抗体工作液。贴封板膜，37℃孵育60min；洗板：350μL Wash Buffer/孔，洗3次；准备酶结合物工作液，用酶结合物稀释液将浓缩酶结合物工作液(30X)稀释30倍；每孔加100μL酶结合物工作液。贴封板膜，37℃孵育60min；洗板：350μL Wash Buffer/孔，洗3次；每孔加100μL TMB显色液，37℃避光孵育15min；每孔加100μL终止液；使用酶标仪，在450nm波长处测吸光值；结果判断：每个标准品和样品的OD值应减去空白孔的OD值，以标准品浓度作横坐标，OD值作纵坐标，绘制标准曲线，通过样品的OD值可在标准曲线上计算出其浓度。
表9各组小鼠结肠中IL-1β、IL-6和TNF-α含量(x±S)(n＝6)
Figure PCTCN2020077204-appb-000011
从表9和图4中可以看出，与空白组相比，模型组中三种炎症因子IL-1β、IL-6和TNF-α含量显著增高，且都有显著性差异(P<0.05)。在给药治疗后，与模型组相比，阳性药柳氮磺吡啶组有所降低，但都没有显著性差异(P>0.05)，而金芪降糖口服制剂组小鼠结肠组织中的三种炎症因子都被显著抑制，且都存在显著性差异(P<0.01)。
10、Real-timePCR检测
检测结肠中中IL-1β，IL-6、TNF-α、IFN-β和TERT mRNA表达水平，具体操作步骤如下：
取约0.5mL长度结肠组织，置于1.5mL离心管中，加入1mL Trizol，使用匀浆仪进行冰上匀浆，室温放置5min；4℃，14000rpm离心5min，取上清，Trizon补充至1mL；加入0.2mL 氯仿，剧烈震荡30s，室温放置5min；4℃，14000rpm离心15min，取上层水相400uL至新1.5mL离心管中；向离心管中加入1倍体积异丙醇，混匀室温放置5min；4℃，14000rpm离心15min，小心吸走上清，保留沉淀；加入75％乙醇1mL，震荡30s；4℃，7500rpm离心5min；小心吸走上清，保留沉淀；加入20μL DEPC水，使用掌式离心机离心30s，将所得到的溶液置于-80℃保存；逆转录合成cDNA，使用微量紫外分光光度计测定RNA浓度。按照PrimeScript TM RT Master Mix试剂盒说明书进行操作，按表10组份配制逆转录反应液，轻柔混匀后进行反转录反应，条件如下:37℃15min(反转录应)；85℃5s(反转录酶的失活应)；4℃。
表10逆转录反应液配制方法
Figure PCTCN2020077204-appb-000012
11、样品Real-timePcR检测，将上一步得到的cDNA稀释，然后用目的基因和内参基因的引物分别进行扩增，引物序列见表12，按表11组份配制PCR反应液(冰上进行)，扩增程序为：95℃预变性30s，95℃变性5s，退火60℃，10s，共40个循环，延伸95℃，15s，60℃，1min，95℃，15s。
表11 RT-qPCR反应液配制方法
Figure PCTCN2020077204-appb-000013
表12引物序列
Figure PCTCN2020077204-appb-000014
使用BioRad CFX96 Real-Time PCR Detection Systems进行检测，结果见表13。
表13各组小鼠结肠中IL-1β及IL-6相对表达量(x±S)(n＝6)(以模型组为100％)
Figure PCTCN2020077204-appb-000015
从表13和图5中可以看出，与空白组相比，模型组中两种炎症因子IL-1β和IL-6表达显著增高，且都有显著性差异(P<0.05)。在给药治疗后，与模型组相比，阳性药柳氮磺吡啶组有所降低，但都没有显著性差异(P>0.05)，而金芪降糖口服制剂组小鼠结肠组织中的两种炎症因子表达都被显著抑制，且都存在显著性差异(P<0.05)。
实施例2金芪降糖口服制剂治疗溃疡性结肠炎的动物实验(慢性模型)：
一、主要仪器及试剂同实施例1。
二、动物实验方法
1、分组，造模及给药
6-8周龄，雄性，SPF级，Balb/c小鼠。将小鼠随机分为：空白组(Control)、模型组(Model)、阳性药组(SASP)及金芪降糖口服制剂(JQ)。每组6只，共24只。在SPF级实验室适应性喂养一周。
2、慢性溃疡性结肠炎模型的建立
慢性溃疡性结肠炎模型分为三个周期，模型组、阳性药组及金芪降糖口服制剂给药组第一个周期，从第一天至第六天使用2％DSS代替饮用水给与小鼠，第7至14天给与普通饮用水；第二个周期，从总第15天至总21天使用2％DSS代替饮用水给与小鼠，从总第22天至总23天给与普通饮用水；第三个周期，从总第31天至总第25天使用2％DSS代替饮用水给与小鼠，从总第36天至总第43天给与普通饮用水，空白组一直给与普通饮用水。
3、实验给药
金芪降糖口服制剂组给药剂量为1.0g/kg/d，阳性药美沙拉嗪组给药剂量为0.6g/kg/d，给药方式为灌胃给药，给药体积为0.2m L/10g，造模第6天开始每天给药一次，至第43天。空白组和模型组每天灌胃等体积的蒸馏水。
4、疾病活动指数(the disease activity index，DAI)评分同实施例1。
表14各组小鼠DAI指数(x±S)(n＝6)
Figure PCTCN2020077204-appb-000016
Figure PCTCN2020077204-appb-000017
见表14与图6(A)，与空白组相比，模型组DAI指数在第二天就开始上升，并在第三天明显上升，且存在显著性差异(P<0.01)。与模型组相比，阳性药和金芪降糖口服制剂能降低溃疡性结肠炎小鼠DAI指数。说明阳性药和金芪降糖口服制剂能对溃疡性结肠炎有不同程度的治疗作用。
5、取材
于最后一次给药后的第一天处死小鼠。剖开小鼠腹部，暴露腹腔，剪去肛门上2cm至盲肠的整段结肠。将整段结肠拍照并测量长度。沿肠系膜边缘纵向剖开，用生理盐水冲洗肠内容物并测量结肠重量。将整段结肠一分为二，一半放入10％福尔马林缓冲液中固定备用，另一半迅速放入液氮中，置于-80℃保存。取小鼠脾脏，立即称量重量。
表15各组小鼠结肠长度(x±S)(n＝6)
Figure PCTCN2020077204-appb-000018
表16各组小鼠脾脏指数(x±S)(n＝6)
Figure PCTCN2020077204-appb-000019
各组小鼠代表性的结肠外观图见图6(C)，从表15以及图6(B)中可以看出，模型组小鼠结肠明显缩短，与空白组相比，存在极显著性差异(P<0.01)。与模型组相比，金芪降糖口服制剂组对结肠缩短均有不同程度的缓解，且具有统计学意义(P<0.05)。各组小鼠脾脏指数见表16及图7，从表16可见模型组小鼠脾脏明显肿大(P<0.01)，而金芪降糖口服制剂对DSS 诱导的脾脏肿大有明显缓解(P<0.01)。
6、结肠病理观察
表17各组小鼠结肠黏膜病理评分(x±S)(n＝6)
Figure PCTCN2020077204-appb-000020
各组小鼠代表性的结肠黏膜组织病理结果图见图8(A)。从图中可以发现，空白组小鼠结肠黏膜组织完好，隐窝结构完整，黏膜下层未见水肿。模型组小鼠黏膜上皮大量破损，脱落，隐窝结构消失，有溃疡形成，黏膜下层可见大量炎性细胞浸润。柳氮磺吡啶及金芪降糖口服制剂能缓解以上病理特征。减少上皮组织脱落，也可以减轻炎性细胞浸润情况。
各组小鼠结肠黏膜病理评分见表17及图8(B)。各组中，模型组病理评分最高，与空白组相比存在极显著性差异(P<0.01)。与模型组相比，各治疗组病理评分都有所降低，表明各给药组对溃疡性结肠炎都有一定程度的治疗作用，且有统计学意义(P<0.01)。
7、结肠组织MPO检测
表18各组小鼠结肠MPO活性(x±S)(n＝6)
Figure PCTCN2020077204-appb-000021
根据表18和图9(B)可知，模型组小鼠MPO活性比空白组明显上升(P<0.01)，MPO可以募集中性粒细胞，引起结肠组织损伤，而阳性药SASP和金芩降糖片对DSS诱导引起的MPO活性上升有明显抑制作用(P<0.01)，和结肠病理观察结果是一致的。
8、结肠组织过氧化氢检测
表19各组小鼠结肠H 2O 2含量(x±S)(n＝6)
Figure PCTCN2020077204-appb-000022
Figure PCTCN2020077204-appb-000023
从表19和图9(A)可知，DSS会明显抑制结肠组织中过氧化氢水平(P<0.05)，过氧化氢对肠道损伤有修复和抑制有害微生物作用，金芪降糖片可以明显恢复DSS诱导的过氧化氢水平下降(P<0.05)

Claims (3)

  1. 金芪降糖口服制剂在制备治疗溃疡性结肠炎的药物中的应用。
  2. 根据权利要求1所述的金芪降糖口服制剂在制备治疗溃疡性结肠炎的药物中的应用，其特征在于，所述金芪降糖口服制剂，按重量份数计，由如下原料制成：黄连2-20份、黄芪3-30份、金银花12-120份。
  3. 根据权利要求2所述的金芪降糖口服制剂在制备治疗溃疡性结肠炎的药物中的应用，其特征在于，所述金芪降糖口服制剂为金芪降糖片、金芪降糖颗粒、金芪降糖胶囊或金芪降糖配方颗粒。
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