阅读说明：本技术 高良姜素在制备治疗鸡坏死性肠炎药物中的用途 (Application of galangin in preparation of medicine for treating necrotic enteritis of chicken ) 是由 吕强华 陈停停 邓旭明 王建锋 宋泽宇 邱家章 周永林 徐蕾 王琳 邓彦宏 于 2021-09-08 设计创作，主要内容包括：本发明涉及高良姜素在治疗鸡坏死性肠炎中的医用用途,并通过产气荚膜梭菌滑动运动抑制试验、产气荚膜梭菌生长曲线、生物被膜形成抑制试验、产气荚膜梭菌粘附至Caco-2细胞试验和雏鸡坏死性肠炎模型免疫器官指数和回肠菌落定植试验验证高良姜素对产气荚膜梭菌感染所致的鸡坏死性肠炎具有良好的保护作用。由于传统抗生素的滥用和细菌耐药性的不断加强,高良姜素具有成本低廉,无耐药性、药物残留和治愈率高的特点。因此,高良姜素用于治疗鸡坏死性肠炎可以增加使用药物的选择性,具有广阔的应用前景,对新药开发具有重要意义。(The invention relates to medical application of galangin in treating necrotizing enterocolitis of chickens, and the galangin is proved to have good protection effect on necrotizing enterocolitis of chickens caused by clostridium perfringens infection through a clostridium perfringens sliding motion inhibition test, a clostridium perfringens growth curve, a biofilm formation inhibition test, a clostridium perfringens adhesion to Caco-2 cell test, a chicken necrotizing enterocolitis model immune organ index and ileum colony colonization test. Due to abuse of traditional antibiotics and continuous reinforcement of bacterial drug resistance, galangin has the characteristics of low cost, no drug resistance, no drug residue and high cure rate. Therefore, the galangin used for treating the necrotic enteritis of the chickens can increase the selectivity of the used medicine, has wide application prospect and has important significance for the development of new medicines.)

1. The galangin can be used for treating necrotic enteritis in chicken.

2. The use of claim 1, wherein the galangin is in any pharmaceutically acceptable dosage form with galangin as an active ingredient.

3. The use of claim 1, wherein the necrotic enteritis in chickens is caused by bacteria.

4. The use of claim 1, wherein the bacterial necrotic enteritis is caused by Clostridium perfringens.

Technical Field

The invention relates to application of galangin in treatment of necrotic enteritis in chickens, relates to application of galangin in preparation of medicines for treating necrotic enteritis in chickens, and belongs to the technical field of medical pharmacy.

Background

Clostridium perfringens (Clostridium perfringens) is a gram-positive, spore-forming, strictly anaerobic and special capsule-forming Clostridium, one of the main members of Clostridium, widely exists in intestinal tracts of human and animals, and is the main pathogen causing sudden death of domestic poultry in recent years. The bacterial infection can cause various diseases of human and animals, mainly causing gas gangrene and food poisoning of human. In poultry, clostridium perfringens is a key pathogen causing necrotic enteritis, severely compromising the development of poultry farming. The flora belongs to typical conditioned pathogenic bacteria, clostridium perfringens normally inhabits the cecum of chickens and does not cause diseases, but clostridium perfringens is propagated in large quantity and generates a large amount of toxin under the condition that the internal environment of small intestine is changed (hypoxia) or the intestinal tract is damaged (coccidian and mycotoxin) under the condition that organisms are stressed or diseases occur. Clostridium perfringens produces a variety of toxins, and clostridium perfringens can be classified into five toxin types a-E according to the most predominant 4 exotoxins (alpha, beta, epsilon, and iota), with poultry primarily infected with type a. After poultry is infected, necrotic enteritis is mainly caused, lesions are mainly concentrated on small intestine parts, and the lesions are marked that the small intestine is obviously enlarged to 2-3 times of the normal small intestine, the intestinal canal is shortened, the surface of the intestinal canal is gray black, the intestinal wall is thinned, gray white or yellow white clothes-like exudates are fully filled in the intestinal cavity, and the film is seriously necrotized by cellulose. Statistically, the economic loss caused by necrotic enteritis outbreaks is as high as 20 billion dollars worldwide, seriously jeopardizing the development of poultry farming.

Type IV pili (TFP) is a mobile organ present on the bacterial cell membrane that plays a key role in host cell colonization and invasion, biofilm formation and gliding movement, which are critical for clostridium perfringens infection. The adhesion and colonization of bacteria in intestinal tracts can be interfered by targeting the TFP function, so that the aim of treating clostridium perfringens infection is fulfilled. At present, the first choice method for treating necrotizing enteritis in chicks caused by clostridium perfringens is to add antibiotics into feed, but the long-term use and abuse of the antibiotics often cause the generation of bacterial resistance. Therefore, the development of safe and effective antibiotic substitutes is urgently needed to solve the technical problem, and the natural compound has the advantages of wide source, small toxic and side effect, low cost, low drug residue, wide safety range, difficult induction of drug resistance and the like, and is an important resource for screening the antibiotic substitutes.

Galangin (Patchouli alcohol), chemically known as 3, 5, 7-trihydroxyflavone, is a flavonoid extracted and separated from dried rhizome of Alpinia officinarum Linn of Zingiberaceae. Modern medical research shows that galangin has the functions of resisting tumor, bacteria, oxidation, inflammation, etc. In recent years, a large number of literatures report pharmacological activities of galangin. However, no research on galangin for treating necrotizing enterocolitis of chicken caused by clostridium perfringens exists at home and abroad until now.

Disclosure of Invention

The invention provides a medical application of galangin in treating necrotic enteritis in chickens, and the galangin has a good treatment effect on necrotic enteritis in chickens caused by clostridium perfringens.

According to the invention, the clostridium perfringens sliding motion inhibition test, the biofilm formation inhibition test, the growth curve test and the clostridium perfringens adhesion to Caco-2 cell test prove that galangin obviously inhibits the activity and the function of a clostridium perfringens type IV pilus system (TFP) and has no bactericidal or antibacterial activity on clostridium perfringens. The chicken clostridium perfringens necrotic enteritis model shows that galangin has good protective effect on clostridium perfringens necrotic enteritis.

The molecular formula of galangin is C₁₅H₁₀O₅And the molecular weight is 270.23. The chemical structural formula of galangin is as follows:

drawings

FIG. 1: galangin inhibits the gliding movement of clostridium perfringens on semi-solid BHI plates;

FIG. 2: galangin does not inhibit the growth of clostridium perfringens in an effective concentration range;

FIG. 3: galangin inhibits the formation of clostridium perfringens biofilm;

FIG. 4: galangin inhibits the adhesion of clostridium perfringens to Caco-2 cells;

FIG. 5: galangin increases immune organ index of infected broiler chicken;

FIG. 6: galangin reduces colonization of infected broiler chicken ileum.

Detailed Description

Example 1

The galangin is used in treating necrotic enteritis in chicken, and is prepared into any pharmaceutically acceptable dosage form with galangin as active component.

Example 2

The galangin is used for treating necrotic enteritis of chicken caused by bacteria.

Example 3

The galangin is used for treating necrotic enteritis of chicken caused by clostridium perfringens.

1. Clostridium perfringens glide movement inhibition assay

Clostridium perfringens standard strain ATCC13124 was anaerobically cultured in 2ml of liquid BHI medium at 37 ℃ for 12 hours. Mu.l of this culture was inoculated into 2ml of liquid BHI and anaerobic culture was continued at 37 ℃ for 5 hours. After the culture, 1ml of the culture was taken, centrifuged at 12000rpm for 5min and the supernatant was discarded, and the cells were resuspended in 500. mu.l of BHI liquid medium to prepare a bacterial suspension.

And respectively dripping 5 mu l of the bacterial suspension on 0.7 percent agar BHI plates containing galangin with different final concentrations (4-32 mu g/ml), carrying out anaerobic culture at 37 ℃ for 72h, and counting the sliding movement result of bacteria.

The results show that: at a concentration of 16 μ g/ml, galangin significantly inhibited the gliding motility of clostridium perfringens ATCC13124 (see fig. 1).

2. Growth curve of clostridium perfringens

Clostridium perfringens was anaerobically cultured in 2ml of liquid BHI medium at 37 ℃ for 12 h. The next day, the cells were inoculated in 200ml of fresh BHI medium and cultured until OD was reached_600nmWhen the volume is 0.3, the mixture is divided into 6 conical flasks, and each flask contains 20ml of the mixture. Adding galangin to the final concentration of 4, 8, 16, 32 and 64 μ g/ml, respectively, adding DMSO control group, standing at 37 deg.C for anaerobic culture, and measuring bacterial culture OD at intervals of 1 hr_600nmValues were recorded until the bacteria grew to plateau and growth curves were plotted. The results show that: within the effective concentration range (4-64 mu g/ml), galangin does not affect the growth of clostridium perfringens (see figure 2).

3. Clostridium perfringens biofilm formation inhibition assay

Performing anaerobic culture of Clostridium perfringens in 2ml liquid BHI culture medium at 37 deg.C for 12h, centrifuging at 12000rpm for 5min, discarding supernatant, washing with sterilized PBS once, then re-suspending with TSB liquid culture medium and adjusting OD of bacterial suspension_600nmMu.l of the bacterial suspension was placed in a 24-well plate, galangin was added to final concentrations of 2. mu.g/ml, 4. mu.g/ml, 8. mu.g/ml and 16. mu.g/ml, respectively, a DMSO control group and a TSB medium control group were added, 3 replicates of each group were added, and the 24-well plate was placed in anaerobic culture at 30 ℃ for 120 h. After the incubation, the supernatant was discarded, washed twice with sterile PBS, dried at 37 ℃ for 1h, and added 400. mu.l of 1% crystalsStanding the purple dye solution at room temperature, incubating for 45min, discarding the crystal purple dye solution, and washing with sterile PBS twice. Adding 33% acetic acid into each well, blowing, uniformly mixing, adding 100 μ l of each well into a 96-well plate, measuring the absorbance at 570nm by using an enzyme-labeling instrument, setting the galangin group which is not added to be 100%, and calculating the biofilm formation rate according to the crystal violet absorbance.

The results show that: galangin significantly inhibited the formation of clostridium perfringens biofilms at concentrations of 16 μ g/ml (see fig. 3).

4. Inhibition of clostridium perfringens adhesion to Caco-2 cells by galangin

Adjusting the concentration to 3X 10 with DMEM medium without serum and antibiotics⁵cells/ml, Caco-2 cells were seeded in 24-well plates, 1ml of cell suspension was added per well, placed at 37 ℃ and 5% CO₂The incubator was used for overnight culture. Performing anaerobic culture of Clostridium perfringens in 2ml liquid BHI culture medium at 37 deg.C for 12h, respectively incubating with galangin (0, 2, 4, 8 and 16 μ g/ml) at different concentrations for 4h, and adjusting bacterial suspension concentration to 6 × 10 with DMEM culture medium without serum and antibiotics⁷CFUs, cells were infected at MOI 200 for 1 h. And (3) washing bacteria which are not adhered to cells by using sterilized PBS (phosphate buffer solution), washing 3 times per hole, adding 1ml of TritonX-100 containing 0.2 percent, repeatedly blowing and beating for 50 times, placing in a horizontal shaking table, shaking for 10min, coating on a BHI (baby hamster kidney) plate by a multiple dilution method, carrying out anaerobic culture at 37 ℃ overnight, and counting bacterial colonies.

The results show that: galangin significantly inhibited clostridium perfringens adhesion to Caco-2 cells within an effective concentration range (see fig. 4).

Treatment effect of 5 galangin on necrotic enteritis of clostridium perfringens of broiler chickens

5.1 establishment of necrotic enteritis model of clostridium perfringens of broiler chickens

23-day-old male Alisma orientalis (Arbor Acre) broiler chickens, gavaged for four consecutive days with 0.5ml of Clostridium perfringens ATCC13124 bacterial suspension (concentration 1X 10)⁹CFUs), a necrotic enteritis model of the chicks was established. 5.2 therapeutic action of galangin on necrotizing enterocolitis of Clostridium perfringens of broiler chickens

According to the method, a clostridium perfringens necrotic enteritis model is established, 50mg/kg galangin is given to a treatment group once a day for five days, and a blank control group and an infection control group are arranged at the same time.

5.3 immune organ index

Weighing the broilers 30 days old after the administration, performing complete autopsy, weighing spleen, thymus and bursa of Fabricius of each group of broilers, and counting and analyzing immune organ indexes of the broilers in each group. Immune organ index is immune organ weight (g)/broiler weight (g).

5.4 ileal colony colonization count

Sterile collecting the above groups of broiler ileum, preparing 1% PBS tissue grinding homogenate, diluting and coating on BHI blood plate in multiple times, anaerobic culturing at 37 deg.C overnight, and counting colony colonization number of ileum of each group (see figure 5).

The results show that: compared with an infected control group, the galangin treatment obviously reduces the colony colonization number of clostridium perfringens infected broiler ileum, and obviously improves the immune organ index of infected broiler chickens. In conclusion, galangin has a good protective effect on necrotic enteritis caused by clostridium perfringens infection of broiler chickens.