阅读说明：本技术 对羟基苯甲酸钠在制备治疗或预防炎症性肠病药物中的应用 (Application of sodium p-hydroxybenzoate in preparing medicine for treating or preventing inflammatory bowel disease ) 是由 王宇晖 段小群 徐笑天 郑董璇 张溪杨 魏承琼 于 2019-12-09 设计创作，主要内容包括：本发明公开了对羟基苯甲酸钠在制备治疗或预防炎症性肠病药物中的应用。在剂量明显低于美沙拉嗪的情况下,对羟基苯甲酸钠可明显抑制葡聚糖硫酸钠诱导的肠炎模型小鼠疾病活动指数评分的升高,改善结肠炎模型小鼠结肠长度缩短,降低小鼠结肠MPO活力,减轻结肠炎模型结肠组织炎性细胞浸润。本发明还公开了一种治疗或预防炎症性肠病的药物,该药物以对羟基苯甲酸钠为有效成分,含有治疗上有效剂量的对羟基苯甲酸钠。(The invention discloses an application of sodium p-hydroxybenzoate in preparing a medicine for treating or preventing inflammatory bowel disease. Under the condition that the dosage is obviously lower than that of mesalazine, the sodium p-hydroxybenzoate can obviously inhibit the increase of disease activity index scores of enteritis model mice induced by dextran sodium sulfate, improve the reduction of colon length of colitis model mice, reduce the MPO activity of the colon of mice and reduce the infiltration of colitis model colon tissue inflammatory cells. The invention also discloses a medicament for treating or preventing inflammatory bowel diseases, which takes sodium p-hydroxybenzoate as an effective component and contains the sodium p-hydroxybenzoate with a therapeutically effective dose.)

1. Application of sodium p-hydroxybenzoate in preparing medicine for treating or preventing inflammatory bowel disease is provided.

2. The use according to claim 1, characterized in that said inflammatory bowel disease is ulcerative colitis.

3. Use according to claim 1 or 2, characterized in that said inflammatory bowel disease is DSS-induced colitis.

4. A medicament for treating or preventing inflammatory bowel disease is characterized by comprising sodium p-hydroxybenzoate as an active ingredient.

5. The medicament of claim 4, wherein the medicament is in a pharmaceutically acceptable dosage form.

6. The medicament according to claim 4, wherein the dosage form is capsule, tablet, granule, suppository, sustained release agent.

7. A medicament according to claim 4, characterized in that said inflammatory bowel disease is ulcerative colitis.

8. The medicament according to claim 4 or 7, characterized in that said inflammatory bowel disease is DSS-induced colitis.

Technical Field

The invention relates to application of sodium p-hydroxybenzoate, in particular to application of sodium p-hydroxybenzoate in preparation of a medicine for treating or preventing inflammatory bowel disease.

Background

Inflammatory Bowel Disease (IBD) is an idiopathic inflammatory bowel disease mainly affecting the ileum, rectum and colon, and clinically manifested as diarrhea, abdominal pain and even bloody stool. Inflammatory bowel disease includes Ulcerative Colitis (UC) and Crohn's Disease (CD). Ulcerative colitis is a continuous inflammation of the mucosal layer and submucosa of the colon, and the disease usually affects the rectum first and gradually spreads to the whole colon; crohn's disease is a non-continuous, full-thickness inflammation that affects the entire digestive tract, the most commonly affected areas being the terminal ileum, colon and perianal. UC, a typical inflammatory bowel disease, is better in young and strong years of 30-40 years old, has no sex difference, is manifested by clinical symptoms such as diarrhea, abdominal pain, mucopurulent bloody stool and the like, is complicated by multiple intestinal perforation, polyp, ulcer and the like, has a long course of disease and a high cancer rate, and seriously affects the life quality and survival of patients. Epidemiological studies have shown that UC incidence in european regions is 505/100000 high, in canadian regions 248/100000 and in the united states 214/100000. With the change of life style and diet rules, the incidence of UC in Asia and middle east is increasing year by year.

Currently, drug therapy and surgical resection are the main methods for controlling UC, and the following 5 types of commonly used therapeutic drugs are available: (1) aminosalicylates, such as sulfasalazine and mesalamine; (2) glucocorticoids, such as dexamethasone and beclomethasone dipropionate; (3) immunosuppressants such as 6-mercaptopurine and cyclosporine; (4) biologics such as the TNF-alpha inhibitor infliximab; (5) anti-infective drugs such as metronidazole and ciprofloxacin. The medicines mainly relieve local inflammation of colon and prevent disease progression by inhibiting abnormal immune response, but have the defects of unstable curative effect, more adverse reactions, poor tolerance, incapability of completely improving all symptoms, unsuitability for long-term administration, high price and the like. Therefore, the finding of the anti-UC medicament with definite curative effect, less adverse reaction and controllable quality has important value.

The inventor finds that the p-hydroxybenzoic acid can effectively relieve symptoms of enteritis model mice induced by Dextran Sodium Sulfate (DSS) under the condition that the dose of the p-hydroxybenzoic acid is lower than that of mesalazine in earlier experiments, but finds that the p-hydroxybenzoic acid has obvious relieving effect on UC symptoms but has side effect on livers when the p-hydroxybenzoic acid is administrated to the model mice by gastric lavage in follow-up researches.

Disclosure of Invention

The inventor researches sodium p-hydroxybenzoate, and discovers that the sodium p-hydroxybenzoate can obviously inhibit the increase of Disease Activity Index (DAI) scores of enteritis model mice induced by dextran sodium sulfate, improve the reduction of colon length of colitis model mice, reduce the MPO activity of the colon of mice and reduce the inflammatory cell infiltration of colon tissues of colitis model mice through a large number of experiments under the condition that the dosage is obviously lower than that of mesalazine. The inventor further finds that the sodium p-hydroxybenzoate has a better relieving effect on inflammatory bowel diseases than p-hydroxybenzoate, and has no obvious side effect. Meanwhile, the pH value of the sodium hydroxybenzoate is 7.3, the pH value of the p-hydroxybenzoic acid is about 3, the alkaline sodium hydroxybenzoate is more easily absorbed in the gastrointestinal tract of a human body according to the principle of ion barrier, and the acidic substance can obviously stimulate the intestinal tract, so that the disease condition is obviously aggravated after the acidic substance is repeatedly infected and influenced especially for patients with ulcerative colitis.

The purpose of the invention is realized by the following technical scheme:

application of sodium p-hydroxybenzoate in preparing medicine for treating or preventing inflammatory bowel disease is provided.

Another object of the present invention is to provide a medicament for treating or preventing inflammatory bowel disease, which comprises sodium p-hydroxybenzoate as an active ingredient in a therapeutically effective amount.

The dosage form of the medicament can be a pharmaceutically acceptable dosage form; the preparation forms are capsules, tablets, granules, suppositories, sustained-release agents and the like.

Preferably, the inflammatory bowel disease is ulcerative colitis.

Preferably, the inflammatory bowel disease is DSS-induced colitis.

Drawings

FIG. 1 is a graph showing the effect of sodium p-hydroxybenzoate on the body weight of mice with UC induced by DSS; wherein normal group, DSS group, HBSS (10mg/kg) group, HBSS (20mg/kg) group, HHA (20mg/kg) group, and Mesalazine (200mg/kg) group, respectively, represent the normal group, DSS group, and HBSS (10mg/kg) group, and HBSS (20mg/kg) group, and sodium paraben group, respectively, and HHA (20mg/kg) group, and Mesalazine group, respectively; in comparison to the normal group, # # indicates p < 0.01; compared to the model group, denotes p <0.05, denotes p < 0.01.

FIG. 2 is a graph showing the effect of sodium p-hydroxybenzoate on DAI scores in DSS-induced UC mice; in comparison to the normal group, # # indicates p < 0.01; compared to the model group, denotes p <0.05, denotes p < 0.01.

FIG. 3 is a graph showing the effect of sodium p-hydroxybenzoate on colon length of DSS-induced UC mice; in comparison to the normal group, # # indicates p < 0.01; compared to the model group, denotes p <0.05, denotes p < 0.01.

FIG. 4 is a graph showing the effect of sodium p-hydroxybenzoate on MPO activity in DSS-induced UC mice; in comparison to the normal group, # # indicates p < 0.01; compared to the model group, denotes p <0.05, denotes p < 0.01.

FIG. 5 is a graph showing the effect of sodium p-hydroxybenzoate on the colon histopathology scores of DSS-induced UC mice.

FIG. 6 shows the effect of sodium p-hydroxybenzoate on the pathomorphology of DSS-induced UC mice.

FIG. 7 is a graph showing the effect of sodium p-hydroxybenzoate on the liver index of DSS-induced UC mice.

FIG. 8 is a graph showing the effect of sodium p-hydroxybenzoate on the pathological score of liver tissues of mice with DSS-induced UC.

FIG. 9 shows the effect of sodium p-hydroxybenzoate on the liver pathomorphology of DSS-induced UC mice.

Detailed Description

The technical solution of the present invention will be further explained with reference to the specific embodiments.

In animal experiments, 2.5% Dextran Sulfate Sodium (DSS) solution is adopted to induce a C57BL/6 mouse to construct a UC model, the model mouse is administered with sodium p-hydroxybenzoate, p-hydroxybenzoic acid and a positive drug mesalamine by intragastric administration, and the improvement effect of the sodium p-hydroxybenzoate on UC is analyzed.

1. Material

1.1 Experimental animals

SPF grade C57BL/6 mice, female, 6-8 weeks old, with a weight of 20 + -2 g, purchased from Schlekschad laboratory animals GmbH, Hunan, license number: SCXK (xiang) 2016-: SYXK 2013-. Feeding in an environment with a temperature of 25 + -2 deg.C and a relative humidity of 55 + -10%, and freely taking food and drinking water. Is suitable for being used after being fed for one week.

1.2 Experimental reagents

Sodium p-hydroxybenzoate; p-hydroxybenzoic acid; mesalazine, shanghai love pharmaceutical co ltd, cat #: 151007; dextran sodium sulfate, MP Biomedicals corporation, usa, cat #: 160110; myeloperoxidase (MPO) kit, tokyo institute of bioengineering, lot number: 20170717, respectively; TRIzol reagent, Invitrogen, usa, cat #: 152104, respectively; other reagents are all commercially available analytical reagents, national drug group chemical reagents, Inc.

1.3 Experimental instruments

Ultra-low temperature refrigerators (model: MDF-U74V), Panasonic;

a full-wavelength multifunctional microplate reader (model: Epoch), Biotek;

a drug storage cabinet (model: YC-300L), Mitsubishi low temperature science and technology, Inc., of Chinese medicine;

optical reading analytical balance (model: BS110S), Beijing Sidoolis balance, Inc.;

circulating water pump (model: SHZ-III), Nanjing Keel instruments and Equipment Ltd;

ultra pure water instruments (model: Master Touch series), Shanghai and Tai instruments Ltd;

low temperature high speed Centrifuge (model: Centrifuge 5434R), eppendorf;

ultrasonic cleaning apparatus (model: KQ2200B), ultrasonic instruments Inc. of Kunshan;

An electric heating constant temperature air blast drying oven (model: GZX-9140MBE), Shanghai Boxun industry Co., Ltd., medical equipment factory;

a magnetic stirrer (model: GL-3250C), Qilinbeier;

micro inching VORTEX mixer (model: VORTEX-5), Qilinbeier.

2. Experimental methods

2.1 establishment of mouse colitis model

To investigate the inhibitory effect of sodium p-hydroxybenzoate on DSS-induced colitis in mice, mice were randomly divided into 6 groups: normal group (normal), model group (DSS), sodium p-Hydroxybenzoate (HBSS) low dose group (10mg/kg), sodium p-Hydroxybenzoate (HBSS) high dose group (20mg/kg), p-hydroxybenzoic acid (HHA) group (20mg/kg), and Mesalazine (Mesalazine) group (200mg/kg), with 8 individuals per group. The normal group freely drinks double distilled water; the remaining groups, except the normal group, were free to drink 2.5% DSS for 7 days, followed by tap water for 3 days.

The day of the start of molding was d1, and the body weight of the mouse at this time was recorded as the initial body weight. Gavage (0.1mL/10g) was started on the day of molding, once a day for 10 consecutive days.

The dextran sodium sulfate, sodium p-hydroxybenzoate, p-hydroxybenzoic acid and mesalazine are dissolved and administered by double distilled water.

2.2 disease Activity index assessment

The mental state, hair color, stool character, activity state, hematochezia and other conditions of the mice are observed every day, the weight, diarrhea index and fecal occult blood condition of the mice are recorded, and Disease Activity Index (DAI) is calculated, wherein the DAI is (weight reduction + diarrhea index + fecal occult blood) 3. The DAI scoring criteria are detailed in table 1.

Table 1: DAI scoring criteria

And (3) occult blood detection: by adopting an o-toluidine method, a small amount of excrement is picked by a cotton swab, 0.3mL of o-toluidine glacial acetic acid solution is firstly dripped, then 0.3mL of 3% hydrogen peroxide solution is quickly dripped, and blue brown is positive within 2 min.

2.3 specimen Collection

Collecting blood from fundus venous plexus 1h after the last administration, standing at 4 deg.C for 2h, centrifuging (3500rpm,4 deg.C) for 15min, sucking upper layer serum, and subpackaging at-80 deg.C for freezing and storing. The colon was removed at a distance of 1cm from the anus, the length of the colon was measured with a ruler and photographed, the colon was washed 2 times with PBS, 0.4cm of the colon was taken from the end close to the rectum, fixed in 4% paraformaldehyde solution, and the remaining colon was frozen at-80 ℃.

2.4 colonic MPO Activity assay

Weighing 40mg of the colorectal tissue collected in 2.3, adding Phosphate Buffer Solution (PBS) to prepare 10% tissue homogenate, measuring absorbance (OD) at 460nm according to the instruction of MPO kit, and calculating according to the formula to obtain the MPO content. The calculation formula is as follows:

MPO viability units/g tissue ═ (assay tube OD value-control tube OD value) × 2/22.6 × sample size (g).

2.5 Colon histopathological examination

Taking colon tissues fixed in 4% paraformaldehyde solution (not less than 24H), neutralizing with 5% sodium sulfate solution for 36H, washing with running water overnight, dehydrating with absolute ethanol, embedding in paraffin, dewaxing conventionally, staining with hematoxylin eosin (H & E), sealing with neutral resin, and observing pathological and histological changes of intestinal wall under an optical microscope, wherein the specific scores are as follows:

(1) The severity of the intestinal wall inflammation, scored as 1, 2 and 3, represents mild, moderate and severe inflammation, respectively;

(2) the severity of the lesions, scored as 1, 2 and 3, i.e., lesions located in the mucosal layer, mucosa and submucosa and transmural lesions;

(3) the extent of crypt damage, scored as 1, 2, 3 and 4, was 1/3 crypt damage, 2/3 crypt damage, crypt loss but both surface epithelium intact and crypt surface epithelium are missing.

2.6 data analysis

All data are expressed as means ± s.e.m. and statistical differences between groups were tested using one-wayANONA and Dunnett's in the SPSS software. A p-value less than 0.05 is considered to be significantly different.

3. Results of the experiment

3.1 Effect of sodium p-hydroxybenzoate on DSS-induced UC mouse body weight

FIG. 1 is a graph showing the effect of sodium p-hydroxybenzoate on the body weight of mice with UC induced by DSS, indicating that: DSS-induced colitis mice progressively lost body weight. Compared with the normal group, the weight of the mice in the model group is obviously reduced (p is less than 0.01); compared with the model group, the weight reduction rate of mice in the low-dose group, the high-dose group, the p-hydroxybenzoic acid group and the mesalazine group of the p-hydroxybenzoic acid sodium salt is obviously lower than that of the model group (p is less than 0.01), and the weight reduction rate of mice with DSS-induced colitis in the low-dose group and the high-dose group of the p-hydroxybenzoic acid sodium salt is lower than that of the p-hydroxybenzoic acid group and the mesalazine group which is a positive drug.

3.2 Effect of sodium p-hydroxybenzoate on DSS-induced DAI score in UC mice

Figure 2 is a graph of the effect of sodium p-hydroxybenzoate on the DAI score of DSS-induced UC mice, showing that: DSS-induced colitis mice have a gradual increase in the disease activity index DAI, manifested primarily as weight loss, diarrhea and hematochezia. Compared with a normal group, the DAI score of the mice in the model group is obviously increased (p is less than 0.01); compared with the model group, the DAI of the low-dose group, the high-dose group and the mesalazine of the sodium p-hydroxybenzoate is obviously reduced (p is less than 0.01), and the inhibition effect of the low-dose group and the high-dose group of the sodium p-hydroxybenzoate on DSS induced colitis is stronger than that of the p-hydroxybenzoate and the positive drug mesalazine.

3.3 Effect of sodium p-hydroxybenzoate on Colon Length of DSS-induced UC mice

Fig. 3 is a graph of the effect of sodium p-hydroxybenzoate on colon length of DSS-induced UC mice, showing that: compared with the normal group, the colon length of the DSS-induced colitis mouse is obviously shortened (p is less than 0.01). Compared with the model group, the colon shortening (p is less than 0.01) of the model mouse can be protected by the sodium p-hydroxybenzoate which is administrated by gavage, and the colon shortening inhibiting capability of the sodium p-hydroxybenzoate is stronger than that of the positive drug mesalazine (p is less than 0.05). And the sodium p-hydroxybenzoate has stronger colon shortening inhibiting ability than p-hydroxybenzoic acid under the condition that the dose of the sodium p-hydroxybenzoate is half of that of the p-hydroxybenzoic acid.

3.4 Effect of sodium p-hydroxybenzoate on MPO Activity of DSS-induced UC mice

FIG. 4 is a graph of the effect of sodium p-hydroxybenzoate on MPO activity of DSS-induced UC mice, showing that: compared with the normal group, the MPO activity of the mice with DSS-induced colitis is obviously enhanced (p is less than 0.01). Compared with the model group, the MPO activity of the colon of the model mouse is obviously reduced by the administration of sodium p-hydroxybenzoate through intragastric administration (p is less than 0.01), and the MPO activity reducing capability of the colon is stronger than that of p-hydroxybenzoic acid and a positive drug mesalazine (p is less than 0.05). And in the case that the dosage of the sodium p-hydroxybenzoate is half of that of the p-hydroxybenzoate, the capability of reducing the MPO activity of the colon of a model mouse is still stronger than that of the p-hydroxybenzoate.

3.5 Effect of sodium p-hydroxybenzoate on DSS-induced UC mouse pathomorphology

As can be seen from FIGS. 5 and 6, the colon tissue of the normal group of mice has no obvious pathological change; the colon tissue lesion of the model group mice mainly affects the mucosa and the submucosa, inflammatory cell types are mainly mononuclear macrophages and neutrophils, and the partial mucosa full-layer necrosis of a severe inflammation area forms ulcer. Compared with the normal group, the model group mice have colon tissue inflammatory cell infiltration and crypt damage (p is less than 0.01). Gavage administration of sodium p-hydroxybenzoate (p < 0.01) significantly reduced the above histopathological changes compared to the model group, which was more potent in reducing histopathology than p-hydroxybenzoic acid and the positive drug mesalazine (p < 0.05).

3.6 Effect of sodium p-hydroxybenzoate on the liver index of DSS-induced UC mice

As can be seen from FIG. 7, the liver-to-body weight ratio was higher in the model group mice than in the normal group. Compared with the model group, the liver index of mice in the low-dose group, the high-dose group, the p-hydroxybenzoic acid group and the mesalazine group of the sodium p-hydroxybenzoate was slightly lower, and the liver index of the sodium p-hydroxybenzoate was lower than that of the p-hydroxybenzoate group.

3.7 Effect of sodium p-hydroxybenzoate on DSS-induced UC mouse liver H & E

As can be seen from FIGS. 8 and 9, the liver of the model group mice exhibited a small amount of lymphocyte infiltration, and the liver of the parahydroxybenzoic acid group mice exhibited fragment necrosis and lymphocyte infiltration, as compared with the normal group. The degree of lesion of liver tissue was good in the p-hydroxybenzoic acid-treated mice compared to the p-hydroxybenzoic acid group (p < 0.01).

4. Conclusion and discussion

Under the condition that the dosage is obviously lower than that of the mesalazine, the sodium p-hydroxybenzoate obviously improves the weight reduction of a DSS-induced UC model mouse, inhibits DAI (deoxyribose nucleic acid) increase, reduces the length shortening of the mouse colon caused by the DSS, reduces the MPO activity of the model mouse colon, and reduces inflammatory cell infiltration and tissue injury.