Application of maslinic acid or derivative thereof in preparation of product for treating clostridium perfringens infection

Abstract

The invention relates to the technical field of biological medicine, and discloses application of maslinic acid or a derivative thereof in preparation of a product for treating clostridium perfringens infection. In-vitro experiments and in-vivo infection models verify that maslinic acid has a good inhibition effect on clostridium perfringens and has a good treatment effect on mammal clostridium perfringens intestinal infection and poultry necrotic enteritis, and a new way and thought are provided for treatment of clostridium perfringens infection; important clinical significance and application value are realized.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to the use of maslinic acid or its derivatives in the preparation of products for treating Clostridium perfringens infection. Background Art

[0002] Clostridium perfringens is a major and common zoonotic pathogen, causing a variety of serious systemic infections and intestinal diseases in both humans and animals, including gas gangrene, food poisoning, enterocolitis, and necrotizing enterocolitis. This not only causes significant economic losses to the aquaculture industry but also severely endangers public health and safety for both humans and animals. In clinical treatment, C. perfringens is increasingly resistant to antibiotics, with multidrug resistance becoming a prominent phenomenon, severely impacting the effectiveness of antibiotics in clinical treatment. Furthermore, excessive antibiotic use in aquaculture not only promotes the development of antibiotic resistance in pathogens but also leads to antibiotic residues in aquaculture products, posing a serious threat to human health. Therefore, promoting antibiotic-free aquaculture is the future of healthy aquaculture. Therefore, developing new, cost-effective anti-infection strategies to partially reduce antibiotic use is crucial for the prevention and control of C. perfringens infections.

[0003] Maslinic acid, a pentacyclic triterpenoid compound primarily extracted from olives, exhibits multiple pharmacological activities, including anti-inflammatory, antioxidant, anti-tumor, and neuroprotective properties. It is also highly safe and has low toxicity. A mature synthetic route for maslinic acid is currently available, enabling its commercial supply. However, to date, maslinic acid has not been used in the treatment of Clostridium perfringens infections. Summary of the Invention

[0004] The purpose of this application is to provide the use of hawthorn acid in the preparation of a product for treating Clostridium perfringens infection. Through research, this application has found that hawthorn acid has a good inhibitory effect on Clostridium perfringens, and through in vitro experiments and in vivo infection models, it has been verified that hawthorn acid can be used to treat Clostridium perfringens infection.

[0005] In order to achieve the above-mentioned invention objectives, the technical solutions adopted in this application are:

[0006] In a first aspect, the present application provides the use of maslinic acid or its derivatives in the preparation of products for treating and / or improving Clostridium perfringens infection.

[0007] In an optional embodiment, the use includes use in preparing a product for treating and / or improving diseases caused by Clostridium perfringens infection and its complications.

[0008] In an optional embodiment, the symptoms of the disease caused by Clostridium perfringens infection include at least one of abdominal distension, abdominal pain, diarrhea, bloody stools, fever, nausea and vomiting, tissue pain and tissue necrosis;

[0009] And / or, the complications of the disease caused by the Clostridium perfringens infection include at least one of severe dehydration, hypoproteinemia, shock, toxic megacolon, intestinal obstruction, intestinal perforation, systemic inflammatory response syndrome and sepsis.

[0010] In an optional embodiment, the treatment and / or improvement of Clostridium perfringens infection comprises:

[0011] reducing the abundance of Clostridium perfringens in the intestine;

[0012] and / or, improving the disease activity index of colitis caused by Clostridium perfringens infection;

[0013] and / or, improving colon tissue damage caused by Clostridium perfringens infection;

[0014] and / or, improving decreased weight gain caused by Clostridium perfringens infection.

[0015] In an optional embodiment, the use includes use in preparing a product for treating and / or improving intestinal infection of mammals caused by Clostridium perfringens and / or necrotic enteritis in poultry caused by Clostridium perfringens infection.

[0016] In an optional embodiment, the product includes at least one of a medicine, a health product and a feed additive.

[0017] In an optional embodiment, the medicine comprises an effective amount of maslinic acid or a derivative thereof, and a pharmaceutically acceptable carrier.

[0018] In an optional embodiment, the dosage form of the drug includes at least one of a pill, a capsule, a tablet, a powder or a granule;

[0019] And / or, the medicine uses maslinic acid or its derivatives as the only active ingredient.

[0020] In a second aspect, the present application provides the use of maslinic acid or its derivatives in the preparation of products for inhibiting and / or killing Clostridium perfringens.

[0021] In an optional embodiment, the inhibiting and / or killing of Clostridium perfringens comprises:

[0022] Inhibits the growth and viability of Clostridium perfringens;

[0023] And / or, the product includes at least one of an inhibitor, a disinfectant, or a bactericide.

[0024] Among them, the chemical structural formula of maslinic acid involved in this application is:

[0025]

[0026] Based on the above technical solution, this application has at least the following beneficial effects:

[0027] Maslinic acid, a pentacyclic triterpenoid compound extracted from olives, is highly safe and lacks drug resistance. This application, using in vitro and in vivo infection models, demonstrates for the first time that maslinic acid has a potent inhibitory effect on Clostridium perfringens. It also demonstrates promising therapeutic effects against C. perfringens intestinal infections in mammals and necrotic enteritis in poultry. Therefore, this application provides a new approach and strategy for the treatment of C. perfringens infections, with significant clinical significance and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0029] Figure 1 This is a graph showing the growth inhibition of Clostridium perfringens by maslinic acid in Example 1 of the present application;

[0030] Figure 2 This is a graph showing the results of a colony spot plate experiment of maslinic acid inhibiting Clostridium perfringens in Example 1 of the present application;

[0031] Figure 3 This is a fluorescence image of live bacteria / dead bacteria staining showing that maslinic acid inhibits the activity of Clostridium perfringens in Example 1 of the present application;

[0032] Figure 4 This is a graph showing the effect of maslinic acid on the abundance of Clostridium perfringens in the cecal contents of mice infected with Clostridium perfringens in the intestine in Example 2 of the present application;

[0033] Figure 5 This is a graph showing the effect of maslinic acid on the colitis disease activity index (DAI) score of mice infected with Clostridium perfringens in the intestine in Example 2 of the present application;

[0034] Figure 6 This is a graph showing the effect of maslinic acid on colon histological damage in mice infected with Clostridium perfringens in Example 2 of the present application (histopathological hematoxylin-eosin staining);

[0035] Figure 7 This is a graph showing the effect of maslinic acid on body weight changes in broiler chickens with necrotic enteritis caused by Clostridium perfringens infection in Example 3 of the present application;

[0036] Figure 8 This is a graph showing the effect of maslinic acid on the abundance of Clostridium perfringens in the ileum contents of broiler chickens with necrotic enteritis caused by Clostridium perfringens infection in Example 3 of the present application. DETAILED DESCRIPTION

[0037] To further illustrate the technical means and results employed by this application to achieve the intended invention objectives, the following describes in detail the specific implementation methods, technical solutions, and features of this application using preferred embodiments. The specific features, structures, or characteristics of the various embodiments described below may be combined in any suitable manner.

[0038] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0039] The present application is further described in detail below with reference to specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0040] Example 1 In vitro antibacterial experiment

[0041] 1. Experimental Materials

[0042] Maslinic acid was purchased from MedChemExpress with a purity of ≥98.0%; Brain Heart Infusion Broth (BHI) and Tryptose Soya Agar (TSA) were purchased from Qingdao Haibo Biotechnology Co., Ltd.; defibrinated sheep blood was purchased from Beijing Solebold Technology Co., Ltd.; and the LIVE / DEAD BacLight Bacterial Viability kit was purchased from Molecular Probes.

[0043] 2. Preparation of main reagents

[0044] (1) Preparation of maslinic acid solution: Weigh maslinic acid, prepare the solution to the corresponding concentration with dimethyl sulfoxide (DMSO), perform gradient dilution, and store at -80°C in the dark.

[0045] (2) Preparation of brain heart infusion broth: Weigh 19.25 g of BHI powder, dissolve in 500 mL of distilled water, autoclave at 121°C for 15 min, and store at 4°C.

[0046] (3) Preparation of soybean casein agar plates: Weigh 5 g of TSA powder and dissolve it in 100 mL of distilled water. Autoclave at 121°C for 15 min. After the culture medium cools to 50°C, add defibrinated sheep blood and store at 4°C after coagulation.

[0047] 3. Recovery and culture of Clostridium perfringens

[0048] Clostridium perfringens (ATCC 13124) was purchased from the American Type Culture Collection. C. perfringens was inoculated onto soy casein agar plates containing 10% defibrinated sheep blood and resuscitated under anaerobic conditions at 37°C for 24 hours. The resuscitated plates were subjected to biological characterization and biochemical analysis. Authenticated and contaminant-free C. perfringens were serially subcultured on brain heart infusion broth and soy casein agar plates under the aforementioned culture conditions to ensure bacterial viability.

[0049] 4. Experimental methods

[0050] (1) Growth curve of maslinic acid inhibiting Clostridium perfringens ATCC13124

[0051] Pick a single colony from the TSA agar plate and transfer it to BHI medium for overnight culture. The next day, dilute the bacterial culture with BHI medium to a concentration of 1 × 10 6 CFU / mL, and then add maslinic acid solution with gradient concentrations to make the final concentration of maslinic acid 0, 3, 4, and 5 μM, respectively. Then, incubate the culture at 37°C for anaerobically, and measure the absorbance of the bacterial solution at OD600nm every 1 hour until the bacterial growth reaches the plateau phase. Record the absorbance of each treatment group at the corresponding time point and draw a growth curve.

[0052] (2) Maslinic acid inhibits the colony formation of Clostridium perfringens ATCC13124

[0053] Take the overnight culture of Clostridium perfringens and dilute the bacterial culture with BHI medium to a concentration of 1×10 6 CFU / mL, and then add gradient concentrations of maslinic acid solution to make the final concentration of maslinic acid 0, 3, 4, and 5 μM, respectively. After static anaerobic culture at 37°C for 6 hours, each group of bacterial solution is diluted in multiple ratios, and 5 μL is spotted on TSA agar plates containing 10% defibrinated sheep blood. The plates are statically cultured anaerobically at 37°C for 20 hours, and the colonies of Clostridium perfringens on the plates are photographed and recorded.

[0054] (3) Live / dead bacteria staining experiment of hawthorn acid inhibiting the activity of Clostridium perfringens ATCC13124

[0055] Take the overnight culture of Clostridium perfringens and dilute the bacterial culture with BHI medium to a concentration of 1×10 6 CFU / mL, and then add maslinic acid solution with gradient concentration to make the final concentration of maslinic acid 0, 3, 4, and 5 μM, respectively. After static anaerobic culture at 37°C for 6 h, the cells were stained with a bacterial viability assay kit (LIVE / DEAD BacLight Bacterial Viability kit) and photographed with a fluorescence microscope.

[0056] 5. Experimental results

[0057] (1) Growth curve of maslinic acid inhibiting Clostridium perfringens ATCC13124

[0058] The results are as follows Figure 1 As shown, maslinic acid can inhibit the growth of Clostridium perfringens. At a working concentration of 4 μM, maslinic acid can significantly prolong the logarithmic phase of Clostridium perfringens growth and increase the time it takes to enter the stable growth phase. At a working concentration of 5 μM, maslinic acid can completely inhibit the growth of Clostridium perfringens.

[0059] (2) Results of the colony spot test of hawthorn acid against Clostridium perfringens ATCC13124

[0060] The results are as follows Figure 2 As shown, the colony formation of Clostridium perfringens in the maslinic acid-treated group was reduced and correlated with the maslinic acid concentration.

[0061] (3) Fluorescence staining of live / dead bacteria showing the inhibition of hawthorn acid on the activity of Clostridium perfringens ATCC13124

[0062] The results are as follows Figure 3 As shown in the figure, maslinic acid can inhibit the activity of Clostridium perfringens. As the concentration of maslinic acid increases, the number of dead bacteria in Clostridium perfringens increases (red) and the number of live bacteria decreases (green).

[0063] Example 2 Treatment of Clostridium perfringens intestinal infection in mice using hawthorn acid

[0064] 1. Experimental Materials and Methods

[0065] (1) Experimental animals: SPF-grade C57BL / 6 mice (female, weighing 18-22 g) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0066] (2) Preparation of Maslinic Acid Dosage Suspension: Maslinic acid was dissolved in dimethyl sulfoxide (DMSO). The resulting solution was stored at -80°C. Corn oil was added before each use to make the final concentration of maslinic acid 20 mg / kg, where the concentration of dimethyl sulfoxide (DMSO) was 3%. The suspension was thoroughly shaken and mixed.

[0067] (3) Animal grouping, modeling, and drug administration: Dextran sulfate sodium (DSS) was used to induce colonic injury in mice, followed by infection with Clostridium perfringens. Female C57BL / 6J mice were randomly divided into three groups based on body weight: a control group, a DSS group, a DSS + Clostridium perfringens group, and a DSS + Clostridium perfringens + maslinic acid group, with 6 mice in each group.

[0068] The mice in the control group drank distilled water, while the mice in the other groups drank 3% DSS on days 0-4 and then drank distilled water on days 4-7. On day 4, overnight cultured Clostridium perfringens was taken, centrifuged (4000 g, 15 min), and resuspended in PBS to a concentration of 1×10 7 CFU / mL to obtain a bacterial suspension. The DSS+Clostridium perfringens group and the DSS+Clostridium perfringens+maslinic acid group were gavaged with 100 μL / mouse of the bacterial suspension, while the control group and the DSS group were gavaged with an equal amount of PBS. Simultaneously, on days 4-7, the DSS+Clostridium perfringens+maslinic acid group was gavaged with the above-prepared maslinic acid suspension (20 mg / kg), while the control group, the DSS group, and the DSS+Clostridium perfringens group were gavaged with an equal amount of dimethyl sulfoxide (DMSO) + corn oil based on body weight.

[0069] The mice were weighed at the same time every day, and their stool characteristics and fecal occult blood were observed. The disease activity index (DAI) was scored (the specific scoring criteria are shown in Table 1), where DAI = weight score + stool characteristics score + fecal occult blood score.

[0070] Table 1 Disease activity index (DAI) scores of mice

[0071]

[0072] On day 7, mice were dissected and cecal contents were collected. Genomic DNA from the cecal contents was extracted using the SPINeasy DNA Pro Kit for Feces (MP Biomedicals). The relative abundance of C. perfringens in the cecal contents of the mice was determined by qPCR, using the resulting DNA as a template, bacterial 16s rDNA as an internal reference gene, and Clostridium perfringens as a target gene. Paraffin sections of the entire colon (from the distal cecum to the anus) were prepared and stained with hematoxylin-eosin for assessment of colonic histological damage.

[0073] 2. Experimental results

[0074] (1) Maslinic acid reduces the abundance of Clostridium perfringens in the cecal contents of mice infected with Clostridium perfringens

[0075] The results are as follows Figure 4 As shown, there are 4 bar graphs, which correspond from left to right to the control group, DSS group, DSS+Clostridium perfringens group and DSS+Clostridium perfringens+maslinic acid group. Figure 4 It can be seen that compared with the simple DSS group, the abundance of Clostridium perfringens in the cecal contents of mice in the DSS+Clostridium perfringens group was significantly increased, while the relative abundance of Clostridium perfringens in the cecal contents of mice in the maslinic acid administration group was significantly lower than that in the DSS+Clostridium perfringens group.

[0076] (2) Improvement of the Colitis Disease Activity Index (DAI) Score in Mice Infected with Clostridium perfringens by Maslinic Acid

[0077] The results are as follows Figure 5 As shown in the results, after DSS induction was stopped, the stool characteristics and bloody stools of mice in the DSS+Clostridium perfringens group continued to deteriorate compared with those in the DSS alone group, and the DAI score continued to increase. However, the administration of hawthorn acid could significantly improve the stool characteristics and occult blood in mice caused by Clostridium perfringens infection, and the DAI score was significantly improved.

[0078] (3) Improvement of colonic histological damage in mice infected with Clostridium perfringens by hawthorn acid

[0079] The results are as follows Figure 6 As shown, from left to right they correspond to the control group, DSS group, DSS+Clostridium perfringens group and DSS+Clostridium perfringens+maslinic acid group. Figure 6It can be seen that compared with the simple DSS group, the mice in the DSS+Clostridium perfringens group had more severe pathological characteristics of colitis, manifested as severe destruction of the crypt structure, a decrease in goblet cells and significant neutrophil infiltration, while the mice in the hawthorn acid-treated group had relatively intact intestinal mucosa, a smaller degree and range of neutrophil infiltration, and a milder degree of crypt destruction, indicating that hawthorn acid can improve the histological damage of the mouse colon caused by Clostridium perfringens infection.

[0080] Example 3 Maslinic acid in the treatment of necrotic enteritis in broiler chickens caused by Clostridium perfringens infection

[0081] 1. Experimental Materials and Methods

[0082] (1) Experimental animals: 15-day-old AA+ broiler chickens were purchased from a large-scale chicken farm near Beijing.

[0083] (2) Preparation of Maslinic Acid Dosage Suspension: Maslinic acid was dissolved in dimethyl sulfoxide (DMSO). The resulting solution was stored at -80°C. Corn oil was added before each use to make the final concentration of maslinic acid 20 mg / kg, where the concentration of dimethyl sulfoxide (DMSO) was 3%. The suspension was thoroughly shaken and mixed.

[0084] (3) Animal grouping, modeling, and drug administration: 36 healthy broilers aged 15 days were selected and randomly divided into a control group, a Clostridium perfringens infection group, and a Clostridium perfringens infection + maslinic acid group, with 12 chickens in each group. Overnight cultured Clostridium perfringens was taken, the bacterial culture was centrifuged (4000 g, 15 min), and then resuspended in PBS to a concentration of 1×10 9 CFU / mL of bacterial suspension. On days 1-7, the Clostridium perfringens infection group and the Clostridium perfringens infection + maslinic acid group were gavaged with 1 ml / each of the above bacterial suspension, while the control group was gavaged with an equal amount of PBS. Simultaneously, on days 1-7, the Clostridium perfringens infection + maslinic acid group was also gavaged with the above-prepared maslinic acid administration suspension (20 mg / kg), while the control group and the Clostridium perfringens infection group were gavaged with an equal amount of dimethyl sulfoxide (DMSO) + corn oil based on body weight. The rats were weighed on day 1 and day 8 and recorded as initial and final body weights, respectively, to calculate weight gain. On day 8, all broilers were killed, the small intestine was dissected, and the ileum contents were obtained. Genomic DNA from the ileum contents was extracted using the SPINeasy DNA Pro Kit for Feces (MP Biomedicals). The relative abundance of Clostridium perfringens in the ileum contents of broiler chickens was detected by qPCR using the obtained DNA as a template, bacterial 16s rDNA as an internal reference gene, and Clostridium perfringens as the target gene.

[0085] 2. Experimental results

[0086] (1) Maslinic acid reduces the abundance of Clostridium perfringens in the ileum of broiler chickens with necrotic enteritis caused by Clostridium perfringens infection

[0087] The results are as follows Figure 7 As shown, from left to right they correspond to the control group, Clostridium perfringens infection group and Clostridium perfringens infection + maslinic acid group. Figure 7 It can be seen that compared with the control group, the abundance of Clostridium perfringens in the ileal contents of broiler chickens in the Clostridium perfringens infection group was significantly increased, and the administration of hawthorn acid significantly downregulated the relative abundance of Clostridium perfringens in the ileal contents of broiler chickens.

[0088] (2) Maslinic acid improves weight changes in broiler chickens with necrotic enteritis caused by Clostridium perfringens infection

[0089] The results are as follows Figure 8 As shown, from left to right they correspond to the control group, Clostridium perfringens infection group and Clostridium perfringens infection + maslinic acid group. Figure 8 It can be seen that compared with the control group, Clostridium perfringens infection caused a decrease in broiler weight gain, and the administration of maslinic acid significantly improved the decrease in broiler weight gain caused by Clostridium perfringens infection.

[0090] The above description is merely a preferred embodiment of the present application; however, the scope of protection of the present application is not limited thereto. Any person skilled in the art who, within the technical scope disclosed in the present application, makes equivalent substitutions or modifications based on the technical solutions and improved concepts of the present application shall be covered by the scope of protection of the present application.

Claims

1. Use of maslinic acid or its derivatives in preparing products for treating and / or improving Clostridium perfringens infection.

2. The use according to claim 1, characterized in that The application includes application in preparing products for treating and / or improving diseases caused by Clostridium perfringens infection and complications thereof.

3. The use according to claim 2, characterized in that The symptoms of the disease caused by Clostridium perfringens infection include at least one of abdominal distension, abdominal pain, diarrhea, bloody stools, fever, nausea and vomiting, tissue pain and tissue necrosis; And / or, the complications of the disease caused by the Clostridium perfringens infection include at least one of severe dehydration, hypoproteinemia, shock, toxic megacolon, intestinal obstruction, intestinal perforation, systemic inflammatory response syndrome and sepsis.

4. The use according to claim 1, characterized in that The treatment and / or improvement of Clostridium perfringens infection includes: reducing the abundance of Clostridium perfringens in the intestine; and / or, improving the disease activity index of colitis caused by Clostridium perfringens infection; and / or, improving colon tissue damage caused by Clostridium perfringens infection; and / or, improving decreased weight gain caused by Clostridium perfringens infection.

5. The use according to claim 1, characterized in that The application includes application in preparing products for treating and / or improving intestinal infection of mammals caused by Clostridium perfringens and / or necrotic enteritis in poultry caused by infection with Clostridium perfringens.

6. The use according to any one of claims 1 to 5, characterized in that The product includes at least one of medicines, health products and feed additives.

7. The use according to claim 6, characterized in that The medicine comprises an effective amount of maslinic acid or a derivative thereof, and a pharmaceutically acceptable carrier.

8. The use according to claim 7, characterized in that The dosage form of the drug includes at least one of pills, capsules, tablets, powders or granules; And / or, the medicine uses maslinic acid or its derivatives as the only active ingredient.

9. Use of maslinic acid or its derivatives in preparing products for inhibiting and / or killing Clostridium perfringens.

10. The use according to claim 9, characterized in that The inhibition and / or killing of Clostridium perfringens comprises: Inhibits the growth and viability of Clostridium perfringens; And / or, the product includes at least one of an inhibitor, a disinfectant, or a bactericide.

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