Rutin and fangchinoline synergistic compound preparation for resisting salmonella infection and application

Abstract

The invention relates to a compound preparation for treating salmonella infection, which comprises rutin and fangchinoline, and the concentration ratio of the fangchinoline to the rutin is 1: 2. The compound preparation plays an antibacterial role through an anti-virulence strategy and a host-oriented strategy, remarkably reduces the bacteria loading amount of salmonella in tissue organs and intestinal contents of mouse and chick models, improves tissue pathological damage, and has good safety. The compound preparation disclosed by the invention provides an efficient and safe novel scheme for treating salmonella infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a compound preparation containing rutin and tetrandrine for treating salmonella infection. BACKGROUND

[0002] Salmonella is an important zoonotic pathogen that can cause gastrointestinal inflammation, sepsis and reactive arthritis in humans and animals. Salmonella can spread across species through the contamination of meat, eggs, milk and their products, posing a great threat to the livestock industry and social public health safety. In addition, the long-term use of first-line antimicrobial drugs such as fluoroquinolones, third-generation cephalosporins and carbapenems has led to the rapid emergence of multi-drug resistant salmonella, with the minimum inhibitory concentration of traditional antimicrobial drugs increasing exponentially and the clinical treatment effect declining dramatically. The development cycle of new antibiotics generally takes 10-15 years, with an average investment of more than 1 billion US dollars. The high time and economic costs have resulted in a speed of new drug development far behind the pace of bacterial drug resistance evolution, and the number of available clinical options is dwindling, with infection-related mortality rates rising year by year. Therefore, it is urgent to develop new anti-infection strategies and drugs with high efficiency and safety to break through the drug resistance bottleneck.

[0003] In recent years, anti-virulence strategies and host-directed strategies have attracted much attention as new directions in the field of anti-infection. Unlike traditional antibiotics that directly kill or inhibit bacteria, anti-virulence strategies target key pathogenic factors of pathogens. For example, the type III secretion system of Salmonella can transport effector proteins directly into host cells, driving bacterial invasion and intracellular replication. Studies have found that the absence or inhibition of the type III secretion system can significantly reduce or even completely eliminate the pathogenicity of Salmonella, indicating that the type III secretion system is one of the most promising drug targets for anti-virulence strategies. Host-directed strategies enhance the host's immune regulation capacity, such as activating the autophagy-lysosome pathway, enhancing the killing ability of macrophages, regulating the redox balance, and enhancing the integrity of the epithelial barrier, thereby improving the host's tolerance and clearance capacity for pathogens. In addition, this strategy can also be used in conjunction with existing antimicrobial drugs to expand the treatment window and reduce toxic side effects. Anti-virulence strategies and host-directed strategies approach pathogens and hosts from two perspectives, complement each other, and provide new ideas and methods for preventing and controlling Salmonella infection, showing broad application prospects.

[0004] Rutin is a flavonoid widely present in edible plants such as citrus, buckwheat and sophora. Previous studies have focused on its antioxidant, anti-inflammatory and neuroprotective activities, but the study on the anti-salmonella infection of rutin is still insufficient. Our study confirmed that rutin can significantly inhibit the expression of salmonella type III secretion system effector proteins at concentrations that do not affect bacterial growth, which is precisely in line with the concept of anti-virulence intervention. Fangchinoline is a bisbenzylisoquinoline alkaloid derived from plants of the family Lauteraceae. It has attracted attention due to its multi-target action, low toxicity and natural origin. Our and our collaborators' studies have confirmed that fangchinoline can significantly increase the number of acidic lysosomes and autophagy flux in infected macrophages by activating the AMPK-mTORC1-TFEB axis, thereby promoting the capture of salmonella by phagosomes and delivery to lysosomes for degradation, effectively controlling salmonella infection in a host-directed manner (He M, Wu H, Xu T, et al. Fangchinoline eliminates intracellular Salmonella by enhancing lysosomalfunction via the AMPK-mTORC1-TFEB axis[J]. Journal of Advanced Research,2025.)。

[0005] Based on the above findings, this study first proposes and verifies the intervention mode of "anti-virulence strategy combined with host-directed strategy", aiming to achieve efficient and safe control of salmonella infection from the "bacteria-host" double level. In the salmonella mouse infection model, fangchinoline and rutin were administered by gavage at a concentration ratio of 1:2, which significantly reduced the pathological damage of infected mice. In the 1-week-old chicken model, drug combination therapy also significantly reduced the bacterial load in the liver, spleen and cecum, and the pathological HE staining results showed that it had good therapeutic effect. In summary, the compound preparation composed of fangchinoline and rutin has dual mechanisms of host-directed clearance and anti-virulence attenuation, providing a new solution for salmonella infection that is safe, efficient and scalable. SUMMARY

[0006] The fangchinoline and rutin described in the application are purchased from Chengdu Ruifenshi Biological Technology Co., Ltd. The fangchinoline described in the application has a CAS accession number of 436-77-1, a molecular formula of C 37 H 40 N2O6, and a molecular weight of 608.72 g / mol. Rutin has a CAS accession number of 153-18-4, a molecular formula of C 27 H 30 O 16 , and a molecular weight of 610.52 g / mol.

[0007] The structure is as follows:

[0008] This study constructs a Salmonella mouse and chick infection model, demonstrating that the combination of Fangjiningkailing and rutin can significantly improve the survival rate of the lethal Salmonella mouse infection model and alleviate the pathological damage of Salmonella to mice and chicks. At the mechanism level, it is further verified that Fangjiningkailing has no significant effect on the type III secretion system of Salmonella, while rutin can target the virulence factors of Salmonella and exert antibacterial activity.

[0009] The positive effects of the present application are: A treatment regimen based on the combined use of Fangjiningkailing and rutin is provided for effectively controlling Salmonella infection. This regimen significantly enhances the treatment effect after Salmonella infection by combining anti-virulence strategies and host-directed strategies, providing strong support for the development of new anti-infection strategies. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 Minimum inhibitory concentrations of rutin and Fangjiningkailing on Salmonella.

[0011] Figure 2 Effect of rutin and Fangjiningkailing on Salmonella growth.

[0012] Figure 3 Effect of rutin and Fangjiningkailing on type III secretion system effector proteins.

[0013] Figure 4 Effect of rutin and Fangjiningkailing combination on the survival rate of Salmonella mouse infection model.

[0014] Figure 5 Effect of rutin and Fangjiningkailing combination on the body weight of Salmonella mouse infection model.

[0015] Figure 6 Effect of rutin and Fangjiningkailing combination on the tissue bacterial load of Salmonella mouse infection model.

[0016] Figure 7 Effect of rutin and Fangjiningkailing combination on the pathological damage of Salmonella mouse infection model.

[0017] Figure 8 Effect of rutin and Fangjiningkailing combination on the body weight of Salmonella chick infection model.

[0018] Figure 9 Effect of rutin and Fangjiningkailing combination on the tissue bacterial load of Salmonella chick infection model.

[0019] Figure 10Effect of Rutin and Tetrandrine on Pathological Damage of Salmonella-infected Chickens DETAILED DESCRIPTION

[0020] The present application is further illustrated by the following examples, which in no way should be construed as limiting the present application. The present application should be limited only by the appended claims. Any modifications of the application made by those skilled in the art that are not in the final claims are to be considered as falling within the scope of the application.

[0021] 1. Determination of minimum inhibitory concentration According to the guidelines issued by the Clinical and Laboratory Standards Institute (CLSI), the minimum inhibitory concentration of rutin and tetrandrine on Salmonella was determined by 96-well plate method. The experimental procedure was as follows: 100 μL of LB liquid medium containing different concentrations of rutin or tetrandrine was added to each well of the 96-well plate, with the concentration ranging from 0 to 2048 μg / mL. Subsequently, 100 μL of Salmonella bacterial solution (concentration of 1 x 10 8 CFUs / mL) was added to each well. The 96-well plate was incubated at 37°C for 18 hours. After incubation, the minimum inhibitory concentration of the drug on the bacteria was determined by observing the bacterial growth. Specifically, the lowest drug concentration corresponding to the wells without Salmonella growth was the MIC value.

[0022] Conclusion: The minimum inhibitory concentration of rutin and tetrandrine on Salmonella SL1344 was greater than 1024 μg / mL. (Appendix Figure 1 ) 2. Determination of growth curve The overnight cultured Salmonella was transferred to new LB liquid medium, and the bacterial solution was adjusted to OD600nm=0.1. Subsequently, it was aliquoted into 20 mL conical flasks. Drugs were added to make the final concentrations of 0, 16, 32, and 64 μg / mL, respectively. The culture flasks were placed in a 37°C shaking incubator for incubation. The absorbance value of the culture at OD600nm was measured every 2 hours, and the growth curve was plotted. (Appendix Figure 2 ) Conclusion: Rutin and tetrandrine had no significant effect on the growth of Salmonella SL1344 within the concentration range of 0-64 μg / mL.

[0023] 3. Effect of drugs on the type III secretion system of Salmonella To induce the expression of Salmonella type III secretion system effector proteins, Salmonella was inoculated in high-salt medium containing 0.3 M NaCl and cultured overnight at 37°C, 200 rpm. The next day, the cultured bacterial solution was diluted at a ratio of 1:30 to adjust the OD600nm value to about 0.2. Then, the bacterial solution was divided into several groups, including a control group without drug and experimental groups treated with different concentrations of drugs (rutin and tetrandrine). After 4 hours of continuous culture, the bacteria were separated by centrifugation. An appropriate amount of 5x SDS loading buffer was added, and after boiling treatment, the expression of type III secretion system effector proteins was analyzed by SDS-PAGE and Western blotting, and the Western blotting images were quantitatively analyzed by ImageJ software. Figure 3 ) Conclusion: The anti-virulence drug rutin can significantly inhibit the expression of Salmonella type III secretion system virulence protein SipA, while the host-directed drug tetrandrine has no significant effect on the expression of SipA.

[0024] 4. Establishment of Salmonella mouse infection model 6-8-week-old female BALB / c mice (body weight 18-20 g, purchased from Liaoning Changsheng Biological Co., Ltd.) were selected and adaptively fed for 3 days with free access to food and water. Three days before infection, the mice were pretreated with 5 g / L streptomycin water to inhibit the growth of other non-target bacteria in the chicks, thereby reducing the interference with the evaluation of Salmonella infection and ensuring the accuracy of the experimental results. The mice were orally gavaged with a lethal dose of Salmonella typhimurium (1x10 7 CFU / mouse) to construct a Salmonella enteritis infection model.

[0025] 4.1. Protection rate test The mice were randomly divided into seven groups of 10 mice each: a blank control group, an infection group, a rutin treatment group (50 mg / kg), a tetrandrine treatment group (50 mg / kg), a combination treatment group 1 (rutin and tetrandrine at a concentration ratio of 1:1), a combination treatment group 2 (rutin and tetrandrine at a concentration ratio of 1:2), and a combination treatment group 3 (rutin and tetrandrine at a concentration ratio of 2:1). After infection, the mice in each treatment group were orally administered the drugs twice a day for 4 consecutive days. The mental state and survival of the mice in each group were recorded (Appendix Figure 4 ).

[0026] Conclusion: Rutin and tetrandrine can significantly improve the survival rate of infected mice, and the effect of combination treatment group 3 is better than that of the single-drug treatment groups. In addition, the mental state of the mice in each treatment group also improved during the treatment, with increased activity and improved hair luster.

[0027] 4.2. Organ bacterial load and histopathological analysis The mice were given a sub-lethal dose of Salmonella (5 x 10 6 CFU / mouse) by gavage. The changes in body weight of the mice were monitored (Fig. 2) and the bacterial loads in the organs were determined (Fig. 3) after infection. Figure 5 Based on the results of the previous study, the Salmonella began to grow rapidly in the mice 3 days after infection. Therefore, the treatment was started 3 days after infection and lasted for 4 days. After the treatment, the mice were euthanized and the organs were collected aseptically. The organs were weighed, ground into homogenate, diluted with PBS, and then spread on LB agar plates containing 40 pg / mL streptomycin. The plates were incubated at 37 °C for 12 hours and the bacterial loads in the organs were recorded (Fig. 3). Figure 6 Some of the organs were fixed, dehydrated, embedded, and then sectioned into paraffin sections for HE staining and pathological analysis (Fig. 4). Figure 7

[0028] Conclusion: The combination of rutin and tetrandrine can significantly prevent the decrease in body weight caused by Salmonella infection in mice, reduce the bacterial load in target organs, and alleviate pathological damage. The effect of the combination therapy group is better than that of the single-drug therapy group.

[0029] 5. Establishment of a Salmonella infection model in chicks One-week-old female white-feathered broiler chickens (purchased from Jilin Chunhui Animal Husbandry Co., Ltd.) were selected and adaptively fed for 1 week with free access to food and water. Three days before infection, the chicks were pretreated with 5 g / L streptomycin water. A sub-lethal dose of chicken salmonella (1 x 10 9 CFU / chick) was given to the chicks by oral gavage to establish a Salmonella enteritis infection model. The chicks were divided into groups in the same way as the mouse infection model. After infection, the changes in body weight of the chicks were monitored (Fig. 5) and the bacterial loads in the organs were determined (Fig. 6). Figure 8 The treatment was started 3 days after infection and lasted for 4 days. After the treatment, the chicks were euthanized and the organs were collected aseptically, ground, and then used for bacterial load determination (Fig. 6). Figure 9 Some of the organs were fixed, dehydrated, embedded, and then sectioned into paraffin sections for HE staining and pathological analysis (Fig. 7). Figure 10

[0030] Conclusion: The combination of rutin and tetrandrine can significantly prevent the decrease in body weight caused by Salmonella infection in chicks, reduce the bacterial load in target organs, and alleviate pathological damage. The effect of the combination therapy group is better than that of the single-drug therapy group.​​

Claims

1. A composition for treating Salmonella infection, characterized by comprising, The compound preparation contains rutin and fangchinoline, and the concentration ratio of fangchinoline to rutin is 1:

2.

2. The complex preparation according to claim 1, characterized in that, The compound preparation exerts antibacterial effect through an antitoxic strategy and a host-directed strategy.

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