Use of maslinic acid or a derivative thereof for the preparation of a product for the treatment of clostridium perfringens infection

Abstract

The application 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. The application verifies that the maslinic acid has a good inhibiting effect on Clostridium perfringens through in-vitro experiments and in-vivo infection models, and has a good treatment effect on intestinal infection of Clostridium perfringens in mammals and avian necrotic enteritis, thereby providing a new approach and thought for treatment of Clostridium perfringens infection, and having important clinical significance and application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to application of maslinic acid or a derivative thereof in preparation of a product for treating Clostridium perfringens infection. BACKGROUND

[0002] Clostridium perfringens is an important and most common zoonotic pathogenic bacterium, which mainly causes various severe systemic infections or intestinal diseases including gas gangrene, food poisoning, enteritis and necrotic enteritis in humans and animals, not only causing huge economic losses to the breeding industry, but also seriously endangering the public health and safety of humans and animals. In clinical treatment, the drug resistance of Clostridium perfringens to antibiotics is increasing, and the multiple drug resistance phenomenon is obvious, which seriously affects the clinical treatment effect of antibiotics. Moreover, in the breeding industry, the overuse of antibiotics not only promotes the occurrence and development of pathogenic bacteria drug resistance, but also leads to the residue of antibiotics in breeding products, which seriously threatens human health, so the implementation of antibiotic-free breeding is the development direction of healthy breeding. Therefore, it is of great significance to develop a new economic and effective anti-infection strategy to partially reduce the use of antibiotics for preventing and controlling Clostridium perfringens infection.

[0003] Maslinic acid is a pentacyclic triterpenoid compound mainly extracted from Olea europaea, which has various pharmacological activities such as anti-inflammatory, antioxidant, antitumor and neuroprotective effects, and has high safety and low toxicity. In terms of cost, a mature synthesis route of maslinic acid has been formed, and commercial supply can be realized. However, so far, there is no application of maslinic acid in treating Clostridium perfringens infection. SUMMARY

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

[0005] To achieve the above-mentioned purpose of the application, the technical solution adopted by the present application is as follows:

[0006] In a first aspect, the present application provides application of maslinic acid or a derivative thereof in preparation of a product for treating and / or improving Clostridium perfringens infection.

[0007] In an alternative embodiment, the application includes application in preparation of a product for treating and / or improving diseases caused by Clostridium perfringens infection and complications thereof.

[0008] In an alternative embodiment, the symptoms of the disease caused by the Clostridium perfringens infection include at least one of abdominal distension, abdominal pain, diarrhea, bloody stool, 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 alternative embodiment, the treating and / or ameliorating the Clostridium perfringens infection comprises:

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

[0012] And / or, ameliorating the colon inflammation disease activity index caused by the Clostridium perfringens infection;

[0013] And / or, ameliorating the colon tissue damage caused by the Clostridium perfringens infection;

[0014] And / or, ameliorating the decrease of body weight gain caused by the Clostridium perfringens infection.

[0015] In an alternative embodiment, the use comprises use in the manufacture of a product for treating and / or ameliorating Clostridium perfringens intestinal infection in a mammal and / or necrotic enteritis caused by Clostridium perfringens infection in poultry.

[0016] In an alternative embodiment, the product comprises at least one of a pharmaceutical product and a feed additive.

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

[0018] In an alternative embodiment, the dosage form of the pharmaceutical product comprises at least one of a pill, a capsule, a tablet, a powder, or a granule.

[0019] And / or, the pharmaceutical product has maslinic acid or a derivative thereof as the only active ingredient.

[0020] In a second aspect, the present application provides use of maslinic acid or a derivative thereof in the manufacture of a product for inhibiting and / or killing Clostridium perfringens.

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

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

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

[0024] wherein the chemical structural formula of the hawthorn acid involved in the present application is:

[0025] .

[0026] Based on the above technical scheme, the present application has at least the following beneficial effects: the hawthorn acid is a pentacyclic triterpenoid compound extracted from olea europaea, which has the characteristics of high safety and no drug resistance. The present application first discloses that the hawthorn acid has a good inhibitory effect on clostridium perfringens through in vitro experiments and in vivo infection models, and has a good therapeutic effect on intestinal infection of clostridium perfringens in mammals and necrotic enteritis in poultry. Therefore, the present application provides a new way and idea for the treatment of clostridium perfringens infection, which has important clinical significance and application value. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical scheme in the specific embodiments or prior art of the present application, the drawings needed to be used in the description of the specific embodiments or prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0028] Figure 1 is the growth curve graph of the hawthorn acid inhibiting clostridium perfringens in Example 1 of the present application;

[0029] Figure 2 is the result graph of the hawthorn acid inhibiting clostridium perfringens in the colony point plate experiment in Example 1 of the present application;

[0030] Figure 3 is the live bacteria / dead bacteria staining fluorescence graph of the hawthorn acid inhibiting the activity of clostridium perfringens in Example 1 of the present application;

[0031] Figure 4 is the result graph of the influence of the hawthorn acid on the abundance of clostridium perfringens in the cecal contents of clostridium perfringens intestinal infection mice in Example 2 of the present application;

[0032] Figure 5 is the result graph of the influence of the hawthorn acid on the colon inflammation disease activity index (DAI) score of clostridium perfringens intestinal infection mice in Example 2 of the present application;

[0033] Figure 6 is the result graph of the influence of the hawthorn acid on the colon histological injury of clostridium perfringens intestinal infection mice in Example 2 of the present application (hematoxylin-eosin staining graph);

[0034] Figure 7This is a graph showing the effect of hawthorn acid on body weight changes in broilers infected with Clostridium perfringens in Example 3 of this application;

[0035] Figure 8 This is a graph showing the effect of crataegolic acid on the abundance of Clostridium perfringens in the ileal contents of broiler chickens with necrotizing enteritis caused by Clostridium perfringens infection, as described in Example 3 of this application. Detailed Implementation

[0036] To further illustrate the technical means and results adopted by this application to achieve the intended inventive purpose, the following preferred embodiments are used to describe in detail the specific implementation methods, technical solutions, and features according to this application. Specific features, structures, or characteristics in the various embodiments described below can be combined in any suitable form.

[0037] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0038] The present application will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed in the present application.

[0039] Example 1: In vitro antibacterial experiment

[0040] 1. Experimental Materials

[0041] Crab 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 Solarbio Science & Technology Co., Ltd.; and the LIVE / DEAD BacLight Bacterial Viability Kit was purchased from Molecular Probes.

[0042] 2. Preparation of main reagents

[0043] (1) Preparation of hawthorn acid solution: Weigh hawthorn acid, prepare the corresponding concentration with dimethyl sulfoxide (DMSO), dilute in a gradient, and store at -80℃ in the dark;

[0044] (2) Preparation of brain heart infusion broth culture medium: Weigh 19.25 g of BHI powder, dissolve it in 500 mL of distilled water, autoclave at 121 °C for 15 min, and store at 4 °C;

[0045] (3) Preparation of soybean casein agar plates: Weigh 5g of TSA powder, dissolve it in 100mL of distilled water, autoclave at 121℃ for 15min, and add defibrinated sheep blood after the culture medium cools to 50℃. Store at 4℃ after coagulation.

[0046] 3. Resuscitation and culture of Clostridium perfringens

[0047] *Clostridium perfringens* (ATCC13124) was purchased from the American Type Culture Collection. *Clostridium perfringens* was inoculated onto soybean casein agar plates containing 10% defibrinated sheep blood and incubated anaerobically at 37°C for 24 h. The revived plates were then subjected to biological characteristics and biochemical tests to identify *Clostridium perfringens*. Identified and uncontaminated *Clostridium perfringens* were continuously passaged on brain heart broth and soybean casein agar plates under the same conditions to ensure bacterial viability.

[0048] 4. Experimental Methods

[0049] (1) The growth curve of Clostridium perfringens ATCC13124 inhibited by crataegolic acid.

[0050] Single colonies were picked from TSA agar plates and transferred to BHI medium for overnight incubation. The next day, the bacterial culture was diluted with BHI medium to a concentration of 1×10⁻⁶. 6 CFU / mL, and then hawthorn acid solution of gradient concentrations was added to make the final concentrations of hawthorn acid 0, 3, 4 and 5 μM respectively. Then, the mixture was incubated anaerobically at 37℃. The absorbance of the bacterial solution at OD600nm was measured every 1 hour until the bacterial growth reached the plateau phase. The absorbance of each treatment group at the corresponding time point was recorded and the growth curve was plotted.

[0051] (2) Hawthorn acid inhibits the colony-forming Clostridium perfringens ATCC13124 colony-forming plate test

[0052] Take the overnight cultured Clostridium perfringens and dilute the bacterial culture with BHI medium to a concentration of 1×10⁻⁶. 6 CFU / mL, and then hawthorn acid solution of gradient concentrations was added to make the final concentrations of hawthorn acid 0, 3, 4 and 5 μM respectively. After anaerobic culture at 37℃ for 6 h, each group of bacterial solution was serially diluted, and 5 μL was spotted on TSA agar plates containing 10% defibrinated sheep blood. After anaerobic culture at 37℃ for 20 h, the colonies of Clostridium perfringens on the plates were photographed and recorded.

[0053] (3) Live / dead bacteria staining experiment to inhibit the activity of Clostridium perfringens ATCC13124 by crataegolic acid

[0054] Take the overnight cultured Clostridium perfringens and dilute the bacterial culture with BHI medium to a concentration of 1×10⁻⁶. 6 CFU / mL, and then gradient concentrations of hawthorn acid solution were added to make the final concentrations of hawthorn acid 0, 3, 4 and 5 μM respectively. After static anaerobic culture at 37℃ for 6 h, the bacteria were stained with a bacterial viability assay kit (LIVE / DEAD BacLight Bacterial Viability kit) and photographed with a fluorescence microscope to record the results.

[0055] 5. Experimental Results

[0056] (1) The growth curve of Clostridium perfringens ATCC13124 inhibited by crataegolic acid.

[0057] The results are as follows Figure 1 As shown, rutin can inhibit the growth of *Clostridium perfringens*. At a working concentration of 4 μM, rutin significantly prolongs the logarithmic growth phase of *Clostridium perfringens* and increases the time it takes to enter the stationary phase; at a working concentration of 5 μM, rutin completely inhibits the growth of *Clostridium perfringens*.

[0058] (2) Results of the colony spot test of Clostridium perfringens ATCC13124 inhibited by hawthorn acid

[0059] The results are as follows Figure 2 As shown, the formation of Clostridium perfringens colonies decreased in the hawthorn acid treatment group, and this was correlated with the concentration of hawthorn acid.

[0060] (3) Fluorescence staining of live / dead bacteria to inhibit the activity of Clostridium perfringens ATCC13124 by crataegolic acid

[0061] The results are as follows Figure 3 As shown, rutin can inhibit the activity of Clostridium perfringens. With increasing rutin concentration, the number of dead bacteria in Clostridium perfringens increases (red), while the number of live bacteria decreases (green).

[0062] Example 2: Treatment of Clostridium perfringens intestinal infection in mice with hawthorn acid

[0063] 1. Experimental Materials and Methods

[0064] (1) Experimental animals: SPF grade C57BL / 6 mice (female, weight 18-22g), purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0065] (2) Preparation of hawthorn acid drug suspension: hawthorn acid was dissolved in dimethyl sulfoxide (DMSO), and the resulting solution was stored at -80℃. Before each use, corn oil was added to make the final concentration of hawthorn acid 20mg / kg, of which the concentration of dimethyl sulfoxide (DMSO) was 3%, and the solution was thoroughly shaken and mixed.

[0066] (3) Animal grouping, modeling, and drug administration: After colonic injury was induced in mice with dextran sulfate sodium (DSS), they were infected with Clostridium perfringens. Female C57BL / 6J mice were randomly divided into control group, DSS group, DSS + Clostridium perfringens group, and DSS + Clostridium perfringens + hawthorn acid group, with 6 mice in each group.

[0067] Control group mice drank distilled water, while the other groups received 3% DSS on days 0-4, followed by distilled water on days 4-7. On day 4, overnight Clostridium perfringens cultures were collected, centrifuged (4000g, 15min), and resuspended in PBS to a concentration of 1×10⁻⁶. 7 CFU / mL was used to obtain bacterial suspensions. The DSS+Clostridium perfringens group and the DSS+Clostridium perfringens+crabapple group were administered 100 μL / animal of the above bacterial suspension by gavage, while the control group and the DSS group were administered an equal volume of PBS by gavage. Simultaneously, from days 4 to 7, the DSS+Clostridium perfringens+crabapple group was administered the prepared crataegapple drug suspension (20 mg / kg) by gavage, while the control group, the DSS group, and the DSS+Clostridium perfringens group were administered an equal volume of dimethyl sulfoxide (DMSO) + corn oil by gavage according to body weight.

[0068] Mice were weighed at the same time every day, and their fecal characteristics and fecal occult blood were observed. Disease Activity Index (DAI) scores were calculated (specific scoring criteria are shown in Table 1). DAI = weight score + fecal characteristics score + fecal occult blood score.

[0069] Table 1 Disease Activity Index (DAI) Scores for Mice

[0070]

[0071] On day 7, mice were dissected, and cecal contents were collected. Genomic DNA was extracted from the cecal contents using the SPINeasy DNA Pro Kit for Feces (MP Biomedicals). Using the obtained DNA as a template, bacterial 16S rDNA as an internal reference gene, and *Clostridium perfringens* as the target gene, the relative abundance of *Clostridium perfringens* in the mouse cecal contents was detected by qPCR. Simultaneously, the entire colon (from the terminal cecum to the anus) was harvested, and paraffin sections of the colon tissue were prepared. After hematoxylin-eosin staining, histological damage to the colon was assessed.

[0072] 2. Experimental Results

[0073] (1) Hawthorn acid reduces the abundance of Clostridium perfringens in the cecal contents of mice infected with Clostridium perfringens.

[0074] The results are as follows Figure 4 As shown, there are four bar charts, corresponding from left to right to the control group, DSS group, DSS + Clostridium perfringens group, and DSS + Clostridium perfringens + hawthorn acid group. Figure 4 It can be seen that, compared with the DSS group alone, 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 hawthorn acid administration group was significantly lower than that in the DSS + Clostridium perfringens group.

[0075] (2) The effect of crataegolic acid on the improvement of the Disease Activity Index (DAI) score of colitis in mice infected with Clostridium perfringens.

[0076] The results are as follows Figure 5 As shown, after DSS induction was stopped, compared with the DSS group alone, the fecal characteristics and bloody stools of mice in the DSS + Clostridium perfringens group continued to deteriorate, and the DAI score continued to rise. However, the administration of hawthorn acid could significantly improve the fecal characteristics and occult blood in mice caused by Clostridium perfringens infection, and the DAI score was significantly improved.

[0077] (3) The effect of crataegolic acid on the improvement of colonic histological damage in mice infected with Clostridium perfringens.

[0078] 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 + hawthorn acid group. Figure 6It can be seen that, compared with the DSS group alone, mice in the DSS + Clostridium perfringens group had more severe colitis pathological features, manifested as severe destruction of crypt structure, reduction of goblet cells and significant neutrophil infiltration. In contrast, mice in the hawthorn acid group had relatively intact intestinal mucosa, with a smaller degree and extent of neutrophil infiltration and milder crypt destruction. This indicates that hawthorn acid can improve the histological damage of mouse colon caused by Clostridium perfringens infection.

[0079] Example 3: Treatment of necrotic enteritis in broilers caused by Clostridium perfringens infection with hawthorn acid.

[0080] 1. Experimental Materials and Methods

[0081] (1) Experimental animals: 15-day-old AA+ broiler chickens were purchased from a large-scale chicken farm near Beijing.

[0082] (2) Preparation of hawthorn acid drug suspension: hawthorn acid was dissolved in dimethyl sulfoxide (DMSO), and the resulting solution was stored at -80℃. Before each use, corn oil was added to make the final concentration of hawthorn acid 20mg / kg, of which the concentration of dimethyl sulfoxide (DMSO) was 3%, and the solution was thoroughly shaken and mixed.

[0083] (3) Animal grouping, modeling, and drug administration: Thirty-six healthy broiler chickens aged 15 days were selected and randomly divided into a control group, a Clostridium perfringens infection group, and a Clostridium perfringens infection + hawthorn acid group, with 12 chickens in each group. The Clostridium perfringens cultured overnight was centrifuged (4000g, 15min) and resuspended in PBS to a concentration of 1×10⁻⁶. 9 A bacterial suspension of CFU / mL was administered. From day 1 to 7, the *Clostridium perfringens* infection group and the *Clostridium perfringens* infection + hawthorn acid group were given 1 ml / animal of the above bacterial suspension by gavage, while the control group was given an equal volume of PBS by gavage. Simultaneously, from day 1 to 7, the *Clostridium perfringens* infection + hawthorn acid group was also given a hawthorn acid drug suspension (20 mg / kg) prepared above by gavage, while the control and *Clostridium perfringens* infection groups were given an equal volume of dimethyl sulfoxide (DMSO) + corn oil by gavage according to body weight. Weight was recorded on day 1 and day 8 as initial and final body weight, and weight gain was calculated. On day 8, all broilers were euthanized, their small intestines were dissected, and the ileum contents were collected. Genomic DNA was extracted from the ileum contents using the SPINeasy DNA Pro Kit for Feces (MP Biomedicals). Using the obtained DNA as a template, bacterial 16S rDNA as an internal reference gene, and Clostridium perfringens as the target gene, the relative abundance of Clostridium perfringens in the ileum contents of broilers was detected by qPCR.

[0084] 2. Experimental Results

[0085] (1) Hawthorn acid reduces the abundance of Clostridium perfringens in the ileal contents of broilers with necrotizing enteritis caused by Clostridium perfringens infection.

[0086] The results are as follows Figure 7 As shown, from left to right, they correspond to the control group, the *Clostridium perfringens* infection group, and the *Clostridium perfringens* infection + hawthorn acid group. Figure 7 It can be seen that, compared with the control group, the abundance of Clostridium perfringens in the ileal contents of broilers was significantly increased in the Clostridium perfringens infection group, while the administration of hawthorn acid significantly reduced the relative abundance of Clostridium perfringens in the ileal contents of broilers.

[0087] (2) Hawthorn acid improves weight changes in broilers with necrotizing enteritis caused by Clostridium perfringens infection.

[0088] The results are as follows Figure 8 As shown, from left to right, they correspond to the control group, the *Clostridium perfringens* infection group, and the *Clostridium perfringens* infection + hawthorn acid group. Figure 8 It can be seen that, compared with the control group, Clostridium perfringens infection led to a decrease in the weight gain of broilers, while the administration of hawthorn acid significantly improved the decrease in weight gain of broilers caused by Clostridium perfringens infection.

[0089] The above description is merely a preferred embodiment of this application; however, the scope of protection of this application is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in this application, based on the technical solution and its improved concept, should be covered within the scope of protection of this application.

Claims

1. The use of hawthorn acid as the sole active ingredient in the preparation of products for the treatment and / or improvement of Clostridium perfringens infection.

2. The application according to claim 1, characterized in that, The application includes its use in the preparation of products for treating and / or improving diseases caused by Clostridium perfringens infection.

3. The application according to claim 2, characterized in that, Symptoms of the disease caused by Clostridium perfringens infection include at least one of the following: abdominal distension, abdominal pain, diarrhea, bloody stools, fever, nausea and vomiting, tissue pain, and tissue necrosis.

4. The application according to claim 1, characterized in that, The treatment and / or improvement of Clostridium perfringens infection include: Reduce the abundance of Clostridium perfringens in the gut; And / or, improve the disease activity index of colitis caused by Clostridium perfringens infection; And / or, improve colonic tissue damage caused by Clostridium perfringens infection; And / or, improve the loss of weight gain caused by Clostridium perfringens infection.

5. The application according to claim 1, characterized in that, The application includes its use in the preparation of products for the treatment and / or improvement of Clostridium perfringens intestinal infection in mammals and / or necrotizing enteritis in birds caused by Clostridium perfringens infection.

6. The application according to any one of claims 1 to 5, characterized in that, The product includes at least one of pharmaceuticals and feed additives.

7. The application according to claim 6, characterized in that, The drug comprises an effective amount of hawthorn acid and a pharmaceutically acceptable carrier.

8. The application according to claim 7, characterized in that, The dosage form of the medicine includes at least one of pills, capsules, tablets, powders, or granules.

9. The application of hawthorn acid as the sole active ingredient in the preparation of products that inhibit and / or kill Clostridium perfringens.

10. The application according to claim 9, characterized in that, The inhibition and / or killing of Clostridium perfringens includes: inhibiting the growth and activity of Clostridium perfringens; and / or, the product includes at least one of disinfectant or bactericide.

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