Application of gypenoside in preparation of medicine for preventing and treating enterotoxigenic escherichia coli diarrhea disease

Abstract

The invention discloses application of gypenoside in preparation of a medicine for preventing and treating enterotoxigenic escherichia coli diarrhea, and belongs to the technical field of medicines. According to the present invention, the gypenoside GP can provide significant prevention and treatment effects on diarrhea diseases caused by enterotoxigenic Escherichia coli (ETEC), particularly the diarrhea diseases caused by multi-drug resistance ETEC, and further can provide significant prevention and treatment effects on the diarrhea diseases caused by the enterotoxigenic Escherichia coli (ETEC), and further can provide significant prevention and treatment effects on the diarrhea diseases caused by the multi-drug resistance ETEC, and further can provide significant prevention and treatment effects on the diarrhea diseases caused by the multi-drug resistance ETEC, animal experiments prove that the GP can effectively relieve diarrhea caused by multi-drug-resistance ETEC. Therefore, development and application of the GP in preparation of the medicine for preventing and treating the ETEC diarrhea disease can provide a new strategy for prevention and control of the disease, dependence on traditional antibiotics can be reduced, the problem of drug resistance faced by clinical strains can be relieved, and the GP has important significance in optimization of public health safety and clinical medication strategies.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to the application of gypenosides in the preparation of medicines for preventing and treating enterotoxigenic Escherichia coli diarrhea. Background Art

[0002] Enterotoxigenic Escherichia coli Escherichia coli ETEC (E. coli) is the main bacterial pathogen that causes diarrhea in infants and young animals. Its pathogenic mechanism mainly depends on the binding of adhesin fimbriae to the surface receptors of intestinal epithelial cells and the subsequent production of enterotoxins. Once ETEC enters the body, it firmly adheres to the intestinal epithelial cells through adhesins and then secretes a large amount of enterotoxins. These enterotoxins disrupt the normal metabolic process of the intestine, affect the normal absorption and secretion function of the host intestinal cells of electrolytes and water, and ultimately lead to diarrhea. Antibiotics have long been the main means of preventing and treating ETEC infections. However, frequent and improper use of antibiotics will accelerate the generation and accumulation of ETEC resistance genes, thereby promoting the emergence of multidrug-resistant strains. These resistant strains are no longer sensitive to commonly used antibiotics, making the treatment of ETEC infections more difficult and complicated ([1] LAIRD TJ, ABRAHAM S, JORDAN D, et al. Porcine enterotoxigenic Escherichia coli : Antimicrobial resistance and development of microbial-based alternative control strategies[J]. Vet Microbiol , 2021, 258:109117. [2] NTAKIYISUMBA E, LEE S, WON G. Evidence-based approaches for determining effective target antigens to develop vaccines against post-weaning diarrhea caused by enterotoxigenic Escherichia coli in pigs: Systematic review and network meta-analysis[J]. Animals (Basel) , 2022, 12(16):2136.). Therefore, the development of new drugs that can effectively control ETEC infection, especially for drug-resistant strains, has become an important issue that needs to be solved urgently.

[0003] Gypenoside (GP) is a saponin from Gynostemma pentaphyllum ( Gynostemma pentaphyllum (Thunb.) Makino) is a major active ingredient of Gynostemma pentaphyllum, and its chemical structure is characterized by a dammarane-type skeleton of a tetracyclic triterpene. GP has been shown to have a wide range of pharmacological activities, including anti-tumor, anti-inflammatory, hypoglycemic, and neuroprotective (Fan Dongdong, Kuang Yanhui, Xiang Shixie, et al. Research Progress on Chemical Components and Pharmacological Activities of Gynostemma Pentaphyllum [J]. Chinese Journal of Pharmaceutical Sciences. 2017, 52(5):342-352.). It is worth noting that GP, as a plant-derived drug, has unique advantages such as good safety, abundant natural resources, low economic cost, and low resistance to bacterial resistance. However, to date, there have been no reports on the role of GP in preventing or treating diarrhea caused by ETEC. Summary of the invention

[0004] The purpose of the present invention is to solve the above-mentioned drug resistance problem in clinical practice and provide the use of GP in drugs for preventing and treating diarrhea caused by ETEC, especially multi-drug resistant ETEC.

[0005] To achieve the above-mentioned purpose of the invention, the technical solution of the present invention is as follows: In one aspect, the present invention provides the use of gypenosides in the preparation of a medicament for preventing and treating enterotoxigenic Escherichia coli diarrhea.

[0006] The GP of the present invention has a purity of greater than 95%. The GP is dissolved in PBS to prepare a GP solution of a desired concentration, and is stored in a refrigerator at 4°C for later use.

[0007] Preferably, the enterotoxigenic Escherichia coli diarrheal disease is caused by enterotoxigenic Escherichia coli.

[0008] The ETEC described in the present invention is isolated from the intestinal contents of diarrhea piglets and stored in the pig virus disease research group of the Institute of Animal Husbandry and Veterinary Medicine of Zhejiang Academy of Agricultural Sciences. The strain has virulence factors such as enterotoxin STb, edema disease causative virus Stx2e, non-adhesin AIDA (adhesin involved in diffuse adhesion) and adhesin F18, and is resistant to antibiotics such as cefotaxime, cephalothin, penicillin, kanamycin, streptomycin, erythromycin, tetracycline, trimethoprim-sulfamethoxazole and ciprofloxacin, and belongs to multi-drug resistant ETEC.

[0009] In some embodiments of the present invention, the dosage form of the drug is liquid, powder injection, tablet, capsule, powder, pill, paste, granule or dressing.

[0010] Preferably, the drug is in the form of a liquid, and when used, the dosage of the drug is 100-400 mg / kg.

[0011] The present invention induces a mouse diarrhea model by intraperitoneal injection of multidrug-resistant ETEC, and observes the severity of diarrhea, mortality, changes in blood routine indicators, morphological changes in intestinal tissue, and changes in bacterial load in mice. The present invention's research shows that GP has good preventive and therapeutic effects and no adverse reactions.

[0012] On the other hand, the present invention also provides a medicine for preventing and treating enterotoxigenic Escherichia coli diarrhea, wherein the effective ingredient of the medicine contains gypenosides.

[0013] Preferably, the enterotoxigenic Escherichia coli diarrheal disease is caused by enterotoxigenic Escherichia coli.

[0014] Furthermore, the enterotoxigenic Escherichia coli is a multidrug-resistant enterotoxigenic Escherichia coli; the multidrug-resistant enterotoxigenic Escherichia coli shows resistance to cefotaxime, cephalothin, penicillin, kanamycin, streptomycin, erythromycin, tetracycline, trimethoprim-sulfamethoxazole and ciprofloxacin.

[0015] The dosage form of the medicine of the present invention is liquid, powder injection, tablet, capsule, powder, pill, paste, granule or dressing.

[0016] Preferably, the drug is in the form of a liquid, and when used, the dosage of the drug is 100-400 mg / kg.

[0017] Beneficial effects of the present invention: In the present invention, GP has a significant preventive and therapeutic effect on diarrhea caused by ETEC, especially multidrug-resistant ETEC. Animal experiments have shown that GP can effectively reduce diarrhea caused by multidrug-resistant ETEC. Therefore, the development and application of GP in the preparation of drugs for the prevention and treatment of ETEC diarrhea can provide a new strategy for the prevention and control of this disease, which can not only reduce the dependence on traditional antibiotics, but also help alleviate the drug resistance problems faced by clinical strains, which is of great significance to public health safety and the optimization of clinical drug use strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Figure 3 is a graph showing the PCR amplification results of the virulence gene of the isolated ETEC strain; wherein, Figure 3 is the isolated ETEC strain, i.e., the target strain; Figure 2 Representative visual images of GP alleviating diarrhea symptoms induced by multidrug-resistant ETEC in mice; Figure 3Schematic diagram of the results of GP reducing the mortality of mice caused by multidrug-resistant ETEC; Figure 4 The results of GP improving blood routine indicators are shown in the figure; A is the white blood cell level, B is the neutrophil count, C is the lymphocyte level, D is the monocyte percentage, and E is the eosinophil count; ns means p>0.05, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001; Figure 5 Representative scanning electron micrographs of GP improving intestinal tissue lesions; Figure 6 Schematic diagram of the results of GP reducing bacterial load in mouse tissues; A is the bacterial load in the liver, B is the bacterial load in the spleen, and C is the bacterial load in the feces; **** p<0.0001. DETAILED DESCRIPTION

[0019] The test reagents used in the following examples, unless otherwise specified, are conventional reagents available from commercial companies; the experimental methods, unless otherwise specified, are conventional methods.

[0020] In the following embodiments, without departing from the spirit of the present invention, those skilled in the art may make many modifications to the present invention in combination with known techniques, and such modifications also fall within the scope of protection of the present invention.

[0021] The present invention is described in detail below in conjunction with experimental examples and accompanying drawings: The ETEC described in the present invention was isolated from the intestinal contents of diarrheal piglets and stored in the Pig Virus Disease Research Group of the Institute of Animal Husbandry and Veterinary Medicine of Zhejiang Academy of Agricultural Sciences. The strain has virulence factors such as enterotoxin STb, edema disease-causing toxin Stx2e, non-adhesin AIDA and adhesin F18 (PCR results are shown in Figure 1 , primer sequences are shown in Table 1), and they showed resistance to cefotaxime, cephalothin, penicillin, kanamycin, streptomycin, erythromycin, tetracycline, trimethoprim-sulfamethoxazole and ciprofloxacin antibiotics, belonging to multidrug-resistant ETEC. The drug sensitivity results are shown in Table 2.

[0022] Table 1 Primer sequences

[0023] Table 2 Antibiotic susceptibility results

[0024] Among them, "R" stands for resistant, which means that the antibiotic is ineffective or has very poor effect on the pathogen; "S" stands for sensitive, which means that the drug is effective against the pathogen; and "I" stands for intermediate, which means that the pathogen is moderately sensitive to the antibiotic.

[0025] Example 1 Preparation of enterotoxigenic E. coli diarrheal disease animal model: Multidrug-resistant ETEC strains were inoculated into LB liquid medium and cultured in a 37°C constant temperature incubator with shaking until OD 600nm =0.9, and the bacterial solution concentration was calculated using the plate count method. The bacterial solution was centrifuged and resuspended in PBS to adjust the bacterial solution concentration to 1×10 9 Balb / c mice (3 weeks old) were intraperitoneally injected with 0.5 mL of bacterial solution (5×10 8 CFU / each).

[0026] The grouping is shown in Table 3: Table 3

[0027] From day 1 to day 7 of the experiment, mice in the control group and ETEC model group were gavaged with 0.5 mL of normal saline every day, and mice in the GP treatment group were gavaged with the same volume of GP solution every day, with doses of 400, 200, and 100 mg / kg, respectively. Two hours after gavage on day 7 of the experiment, mice in the ETEC model group and GP treatment group were intraperitoneally injected with 0.5 mL of 1×10 9 CFU / mL ETEC bacterial solution, and the control group mice were intraperitoneally injected with 0.5 mL normal saline. The diarrhea of ​​the mice was observed, and the death time of the mice was continuously observed and recorded for 7 days, and the survival curve of the mice was drawn.

[0028] The experimental results are as follows: Depend on Figure 2 It can be seen that the multidrug-resistant ETEC strain caused severe diarrhea symptoms in mice after infection, manifested as loose and unformed feces, while the diarrhea symptoms of mice pretreated with GP were significantly alleviated, indicating that GP has a preventive and therapeutic effect on diarrhea symptoms in mice caused by ETEC infection.

[0029] like Figure 3As shown in the results, the survival rate of mice in the ETEC model group was only 5% 24 hours after infection, and all died after 168 hours. In contrast, the survival rates of mice pretreated with 400, 200, and 100 mg / kg GP increased to 55%, 60%, and 75% 24 hours after infection, respectively, and the survival rates were maintained at 40%, 35%, and 55% at 168 hours, respectively. The results showed that GP pretreatment can significantly improve the survival rate of mice against ETEC infection, and 100 mg / kg GP concentration has the best effect. Therefore, 100 mg / kg GP was selected as the test concentration in subsequent experiments.

[0030] Example 2

[0031] The preparation of the enterotoxigenic Escherichia coli diarrheal disease animal model is the same as that in Example 1.

[0032] The grouping is shown in Table 4: Table 4

[0033] From day 1 to day 7 of the experiment, mice in the control group and ETEC model group were gavaged with 0.5 mL of normal saline every day, and mice in the GP treatment group were gavaged with the same volume of GP solution every day, with a dose of 100 mg / kg. Two hours after gavage on day 7 of the experiment, mice in the ETEC model group and GP treatment group were intraperitoneally injected with 0.2 mL of 1×10 9 CFU / mL ETEC bacterial solution, and the control group mice were intraperitoneally injected with 0.2 mL of normal saline. 24 h after mice were infected with ETEC, the mice were killed and blood was collected for the detection of blood routine indicators. The abdominal cavity of the mice was opened under sterile conditions, and the liver, spleen and intestinal contents (i.e., feces) were taken out for the determination of bacterial load. In addition, the duodenum, jejunum, ileum and colon tissues were separated and fixed in 2.5% glutaraldehyde to observe the ultrastructure of intestinal tissue.

[0034] Routine blood test: The collected blood was placed in an EDTA anticoagulant tube, and the total white blood cell count, neutrophil percentage, lymphocyte percentage, monocyte percentage and eosinophil percentage were tested and analyzed using a veterinary fully automatic routine blood cell analyzer.

[0035] Determination of bacterial load: The liver, spleen and intestinal contents of mice were collected aseptically and weighed, and sterile PBS was added at a ratio of 1:10 for grinding. The homogenate was diluted 10 times and evenly spread on MacConkey plates. After incubation at 37°C for 12 h, the number of colonies on the plates was counted.

[0036] Observation of intestinal tissue ultrastructure: The duodenum, jejunum, ileum, and colon were fixed with 2.5% glutaraldehyde overnight. The fixed samples were washed and transferred to 1% OsO4 for post-fixation for 1.5 h. After washing the samples again, they were dehydrated in a series of different concentrations of ethanol gradient (30%, 50%, 70%, 90%, and three times 100%), each for 15 min, and then treated with isoamyl acetate twice, each for 15 min. Subsequently, the samples were dried using a critical point dryer, vacuum sprayed, observed under a scanning electron microscope, and photographed.

[0037] The experimental results are as follows: Depend on Figure 4 It can be seen that compared with the control group, the blood neutrophils (Neu) ( Figure 4 B in), monocytes (Mon) ( Figure 4 D) and eosinophils (Eos) ( Figure 4 E) ratio increased significantly, while the total white blood cell count (WBC) ( Figure 4 A) and lymphocytes (Lym) ( Figure 4 The proportion of C in the GP-treated group decreased significantly, indicating that ETEC infection had a certain impact on the immune system of mice. However, in the GP-treated group, the total white blood cell count and lymphocyte ratio increased significantly, the proportion of neutrophils, monocytes and eosinophils decreased significantly, and except for lymphocytes, the proportion of other cells returned to a level that was not significantly different from that of the control group, indicating that GP treatment has a restorative and improving effect on the immune function of mice.

[0038] Depend on Figure 5 It can be seen that ETEC infection mainly damaged the morphological integrity of the duodenum and jejunum of mice, which was manifested by significant damage to the villus structure, while no obvious abnormal changes were observed in the morphological structure of the ileum and colon. However, after GP pretreatment, the damage to the villus structure of the duodenum and jejunum of mice was significantly reduced, and their morphological integrity was significantly improved.

[0039] like Figure 6 As shown in the figure, compared with the control group, the number of ETEC colonies in the liver, spleen and intestinal contents of the ETEC model group mice was significantly increased, indicating that ETEC had spread and infected the mice extensively. Figure 6 A in), spleen ( Figure 6 B) and in the intestine ( Figure 6 C) The number of ETEC colonies in mice was significantly reduced, indicating that GP pretreatment can effectively inhibit the spread and infection of ETEC in mice.

[0040] In summary, GP showed good effects in preventing and treating diarrhea caused by multidrug-resistant ETEC without any adverse reactions, and has broad application prospects.

[0041] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of gypenosides in the preparation of drugs for preventing and treating enterotoxigenic Escherichia coli diarrhea.

2. The use according to claim 1, characterized in that: The enterotoxigenic Escherichia coli diarrheal disease is caused by enterotoxigenic Escherichia coli.

3. The use according to claim 2, characterized in that: The enterotoxigenic Escherichia coli is a multi-drug resistant enterotoxigenic Escherichia coli; The multi-drug resistant enterotoxigenic Escherichia coli exhibits resistance to cefotaxime, cephalothin, penicillin, kanamycin, streptomycin, erythromycin, tetracycline, trimethoprim-sulfamethoxazole and ciprofloxacin.

4. The use according to claim 1, characterized in that: The dosage form of the medicine is liquid, powder injection, tablet, capsule, powder, pill, paste, granule or dressing.

5. The use according to claim 4, characterized in that: The dosage form of the drug is liquid, and when used, the dosage of the drug is 100-400 mg / kg.

6. A drug for preventing and treating enterotoxigenic Escherichia coli diarrhea, characterized in that: The active ingredient of the medicine contains gypenoside.

7. The drug for preventing and treating enterotoxigenic Escherichia coli diarrhea according to claim 6, characterized in that: The enterotoxigenic Escherichia coli diarrheal disease is caused by enterotoxigenic Escherichia coli.

8. The drug for preventing and treating enterotoxigenic Escherichia coli diarrhea according to claim 7, characterized in that: The enterotoxigenic Escherichia coli is a multi-drug resistant enterotoxigenic Escherichia coli; The multi-drug resistant enterotoxigenic Escherichia coli exhibits resistance to cefotaxime, cephalothin, penicillin, kanamycin, streptomycin, erythromycin, tetracycline, trimethoprim-sulfamethoxazole and ciprofloxacin.

9. The drug for preventing and treating enterotoxigenic Escherichia coli diarrhea according to claim 6, characterized in that: The dosage form of the medicine is liquid, powder injection, tablet, capsule, powder, pill, paste, granule or dressing.

10. The drug for preventing and treating enterotoxigenic Escherichia coli diarrhea according to claim 9, characterized in that: The dosage form of the drug is a liquid, and when used, the dosage of the drug is 100-400 mg / kg.

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