Application of aloe-emodin or its combination with tylosin in inhibiting Mycoplasma gallisepticum and Mycoplasma synoviae

Abstract

The present invention belongs to the technical field of veterinary drugs, and specifically relates to the application of aloe-emodin or a composition thereof with tylosin in inhibiting Mycoplasma gallisepticum and Mycoplasma synoviae. The aloe-emodin composition provided by the present invention comprises: aloe-emodin and tylosin, and the optimal ratio concentration for Mycoplasma gallisepticum and Mycoplasma synoviae is: aloe-emodin: tylosin = 1.953 μg / mL: 0.244 μg / mL; the graded antibacterial concentration index is 0.375, and aloe-emodin and tylosin have a synergistic antibacterial effect on Mycoplasma gallisepticum and Mycoplasma synoviae. The composition reduces the antibacterial concentration of aloe-emodin and tylosin alone, saves costs, and has broad application prospects and development value in the fields of food processing, medicine, and aquaculture.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary drugs, and particularly relates to application of aloe-emodin or a composition thereof with tylosin in inhibiting Mycoplasma gallisepticum and Mycoplasma synoviae. Background Art

[0002] Mycoplasma gallisepticum and Mycoplasma synoviae are highly prevalent avian pathogens in poultry. Infection with Mycoplasma gallisepticum can cause respiratory diseases, decreased egg production, reduced hatchability, and abnormal eggshells in poultry. Infection with Mycoplasma synoviae can cause infectious synovitis, respiratory diseases, decreased egg production, and abnormal eggshells in poultry, causing huge economic losses to the poultry industry (Anneke Feberwee, Sjaak de Wit, Remco Dijkman. Clinical expression, epidemiology, and monitoring of Mycoplasma gallisepticum and Mycoplasma synoviae: an update. Avian Pathol. 2022 Feb; 51(1): 2-18.). In addition, mycoplasma infection can induce oxidative stress and apoptosis in the body, leading to immune damage and body damage, increasing the risk of mixed or secondary infection of bacteria and viruses in poultry, and greatly increasing the mortality rate of poultry (Wanying Hu, Wei Zhang, Syed Waqas Ali Shah, et al. Mycoplasma gallisepticum infection triggered histopathological changes, oxidative stress and apoptosis in chicken thymus and spleen. Dev Comp Immunol. 2021 Jan; 114: 103832.).

[0003] Mycoplasmas lack cell walls and are naturally resistant to antimicrobial drugs that target peptidoglycan, such as β-lactams, glycopeptides, and fosfomycins (Ken B Waites, Li Xiao, Yang Liu, et al. Mycoplasma pneumoniae from the Respiratory Tract and Beyond. Clin Microbiol Rev. 2017 Jul; 30(3): 747-809.). In addition, mycoplasmas lack the folate synthesis pathway and are resistant to sulfonamide antimicrobial drugs that target the folate synthesis pathway (Fabricio BM Arraes, Maria José A. de Carvalho, Andrea Q. etal.Differential metabolism of Mycoplasma species as revealed by theirgenomes.Genetics and Molecular Biology,30,1,182-189(2007);K KSchultz,E LStrait,BZ Erickson,et al.Optimization of an antibiotic sensitivity assay forMycoplasma hyosynoviae and susceptibility profiles of field isolates from1997to 2011.Vet Microbiol. 2012Jul 6;158(1-2):104-8.). In addition to the above drugs, polymyxin, nalidixic acid, rifampicin and some oxazolidinone drugs, such as linezolid, are also ineffective against mycoplasma (Ken B Waites, Li Xiao, Yang Liu, et al. Mycoplasma pneumoniae from the Respiratory Tract and Beyond. Clin Microbiol Rev. 2017 Jul; 30 (3): 747-809.). In summary, compared with bacteria, mycoplasma lacks some biosynthetic functions, and many drugs reported to be useful for bacteria or fungi are ineffective against mycoplasma, resulting in relatively few drugs that can be used to treat mycoplasma infections.

[0004] There are large differences in the drug resistance of mycoplasmas from different sources. For example, Mycoplasma synoviae from animals is seriously resistant to macrolides and fluoroquinolones (Zhang Huiru, Xiao Xia, Yang Tong, et al. Study on the resistance of Mycoplasma synoviae to commonly used antibiotics [J]. China Poultry. 2021, 43(04)); Genital Mycoplasmas from humans are resistant to quinolones and aminoglycosides to varying degrees, but are highly sensitive to macrolides and tetracyclines (Wei Jingwen, Liu Enling, Liu Zhihui, et al. Analysis of 1006 cases of female genital mycoplasma infection and drug resistance [J]. Journal of North China University of Technology (Medical Edition). 2020, 22(02)).

[0005] At present, there are some reports that aloe-emodin has antibacterial and antiviral effects. The patent application "Application of aloe-emodin in the preparation of drugs for the prevention and treatment of African swine fever" (patent number CN202210256584.3) discloses aloe-emodin (English name: Aloeemodin, molecular formula: C 15 H 10 O 5 , molecular weight: 270.24) can effectively protect cells infected with African swine fever virus, and has anti-African swine fever virus activity in a dose-dependent manner. The patent application "A fungicide composition containing rhubarb derivatives and benzisothiazolinone" (patent number CN201710112986.5) discloses a fungicide composition composed of rhubarb derivatives and benzisothiazolinone, which can prevent and control some bacterial or fungal diseases in crops. Tao Li et al. found that aloe-emodin has a good antibacterial effect on Gram-positive bacteria, with MIC of 4-32μg / mL and MBC of 32-128μg / mL; while the antibacterial effect on Gram-negative bacteria is worse than that on Gram-positive bacteria, with MIC of 128-256μg / mL and MBC higher than 1024μg / mL (Tao Li, Yan Lu, Hua Zhang, et al. Antibacterial Activity and Membrane-Targeting Mechanism of Aloe-Emodin Against Staphylococcus epidermidis. Front Microbiol. 2021Aug 16; 12: 621866.). The structural formula of the aloe-emodin is as follows:

[0006]

[0007] Tylosin is a 16-membered macrolide antibiotic that inhibits bacterial protein synthesis by binding to the ribosome 50s subunit (Mingfu Liu, Stephen Douthwaite,. Resistance to the macrolide antibiotic tylosin is conferred by single methylations at 23S rRNA nucleotides G748 and A2058 acting in synergy. PNAS. 2002 Nov 12; 99 (23): 14658-63.). Tylosin has a broad spectrum of antibacterial activity, has a good inhibitory effect on Gram-positive bacteria and mycoplasma, and has a certain antibacterial activity against most Gram-negative bacteria, so it is widely used to treat pneumonia, arthritis, respiratory tract infections, etc. (Sara T Elazab, Nahla S Elshater, Yousreya HHashem et al,. Pharmacokinetics, tissue residues, and ex vivo pharmacodynamics of tylosin against Mycoplasmaanatis in ducks. J Vet Pharmacol Ther. 2020Jan; 43(1):57-66.)

[0008] In summary, mycoplasma is different from bacteria, and antibacterial drugs are not necessarily effective against mycoplasma. At present, there is no research report on the effect of aloe-emodin and the combination of aloe-emodin and tylosin on mycoplasma. Summary of the invention

[0009] The technical problem to be solved by the present invention is to overcome the serious drug resistance of Mycoplasma gallisepticum and Mycoplasma synoviae, and provide application of aloe-emodin in preparing drugs against Mycoplasma galliscepticum and Mycoplasma synoviae.

[0010] Another key point of the present invention is to provide an aloe-emodin composition for use in a drug for treating and preventing Mycoplasma gallisepticum and Mycoplasma synoviae infection. The composition provided by the present invention has a good inhibitory and killing effect on Mycoplasma gallisepticum and Mycoplasma synoviae, and has a good application prospect in the prevention and treatment of avian mycoplasma disease.

[0011] To achieve the above object, the present invention adopts the following technical solutions:

[0012] The invention discloses an application of aloe-emodin in the preparation of a drug for treating or preventing Mycoplasma gallisepticum and Mycoplasma synoviae infection, comprising using aloe-emodin as an effective ingredient or one of the effective ingredients for the preparation of a drug for treating or preventing Mycoplasma gallisepticum and Mycoplasma synoviae infection, and for inhibiting or killing Mycoplasma gallisepticum and Mycoplasma synoviae in vitro.

[0013] The invention discloses an application of an aloe-emodin composition in preparing a medicine for treating or preventing infection with Mycoplasma gallisepticum or Mycoplasma synoviae. The invention comprises using the composition of the invention as the only effective ingredient for preparing a medicine for treating or preventing infection with Mycoplasma gallisepticum or Mycoplasma synoviae, and for inhibiting or killing Mycoplasma gallisepticum or Mycoplasma synoviae in vitro. The only effective ingredients of the aloe-emodin composition are aloe-emodin and tylosin.

[0014] The above-mentioned composition, preferably, when used to prepare a drug for treating or preventing Mycoplasma gallisepticum infection, the mass ratio of aloe-emodin to tylosin is 8:1;

[0015] The above-mentioned composition, preferably, when used to prepare a drug for treating or preventing chicken synoviae infection, the mass ratio of aloe-emodin to tylosin is 8-16:1;

[0016] The above-mentioned composition, preferably, when used for inhibiting Mycoplasma gallisepticum in vitro, the mass ratio of aloe-emodin to tylosin is 8:1;

[0017] Preferably, when the above-mentioned composition is used to inhibit Mycoplasma synoviae in vitro, the mass ratio of aloe-emodin to tylosin is 8-16:1.

[0018] Compared with the prior art, the present invention has the following advantages and effects:

[0019] The present invention provides for the first time that aloe-emodin or aloe-emodin and tylosin have the effect of synergistically inhibiting mycoplasma, and the composition can be used to resist Mycoplasma gallisepticum and Mycoplasma synoviae. The present invention studies and verifies through multiple methods that aloe-emodin or aloe-emodin and tylosin composition have good inhibitory and killing effects on Mycoplasma gallisepticum and Mycoplasma synoviae, lays a theoretical foundation for the development of new drugs against poultry mycoplasma disease, and even provides candidate substances for the exploration of antibiotic substitutes, and has good application prospects in the prevention and treatment of poultry mycoplasma disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a dynamic analysis of the growth curve of aloe-emodin against Mycoplasma gallisepticum.

[0021] Figure 2 This is a dynamic analysis of the growth curve of aloe-emodin against the growth of chicken synoviae Mycoplasma. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0023] Unless otherwise specified, the reagents and materials used in the following examples are commercially available. The present invention is described by taking Mycoplasma gallisepticum and Mycoplasma synoviae in avian mycoplasmas as examples. Mycoplasma gallisepticum and Mycoplasma synoviae used in the present invention are screened by the applicant. The applicant also screened multiple isolates of these two mycoplasmas. These isolates were tested using the same method as in the examples, and the results were the same as those disclosed in the examples.

[0024] The substance described in the present invention may also have an inhibitory and killing effect on other avian mycoplasmas. The structural formula of aloe-emodin is as follows:

[0025]

[0026] Statistical analysis of the following examples of the present invention: All experiments were repeated at least 3 times independently, and the results were expressed as mean values ​​and standard errors, and analyzed using one-way analysis of variance and T test. All statistical analyses used P<0.05 as the test standard for significant statistical differences, and the analysis software was GraphPad Prism 8.

[0027] Embodiment 1:

[0028] Determination of minimum inhibitory concentration and minimum bactericidal concentration of aloe-emodin against Mycoplasma gallisepticum and Mycoplasma synoviae:

[0029] 1. Materials

[0030] Modified Frey's medium is used for drug sensitivity experiments to detect the antibacterial efficacy of drugs.

[0031] 2. Test methods

[0032] The revived Mycoplasma gallisepticum and Mycoplasma synoviae of the third generation were inoculated into fresh modified Frey's medium at a ratio of 1:10 and incubated at 37°C with 5% CO 2 The cells were cultured in an incubator, and CCU was determined at 0, 12, 24, 36, and 48 hours to obtain the growth curves of Mycoplasma gallisepticum and Mycoplasma synoviae.

[0033] According to the growth curves of Mycoplasma gallisepticum and Mycoplasma synoviae determined above, the mycoplasma suspension cultured to the logarithmic growth phase was diluted to an appropriate concentration and added to sterile 96-well plates. 100 μL of the diluted mycoplasma suspension was added to the 1st to 11th wells to make the final concentration of the mycoplasma suspension in each well 10 5 CCU / mL;

[0034] The aloe-emodin solution was diluted in multiples with modified Frey's medium, and different concentrations of aloe-emodin solution were added to the above 96-well plate. The diluted aloe-emodin solution was added to wells 1 to 10, 100 μL per well, and 100 μL of modified Frey's medium was added to well 11 to form a 200 μL mycoplasma suspension as a growth control. Only 200 μL of modified Frey's medium was added to well 12 as a negative control. 37℃5% CO 2 Incubate in the incubator for 48 hours, observe the color change of the culture medium in the 96-well plate, and determine the MIC value. Take the bacterial solution from each well and spread it on the modified Frey solid medium. If the number of colonies is less than 5, it is the MBC value.

[0035] 3. Results

[0036] As shown in Table 1, when the concentration of aloe-emodin is 15.625 μg / mL, it has an inhibitory effect on the growth of Mycoplasma gallisepticum and Mycoplasma synoviae. When the concentration of aloe-emodin is 31.25 μg / mL, it has a bactericidal effect on Mycoplasma gallisepticum and Mycoplasma synoviae.

[0037] Table 1 Minimum inhibitory concentration and minimum bactericidal concentration of aloe-emodin against Mycoplasma gallisepticum and Mycoplasma synoviae

[0038]

[0039] Embodiment 2:

[0040] The inhibitory effect of aloe-emodin on the growth of Mycoplasma gallisepticum and Mycoplasma synoviae increases with the increase of concentration:

[0041] With reference to the minimum inhibitory concentration of aloe-emodin determined in Example 1, aloe-emodin solution was added to the suspensions of Mycoplasma gallisepticum and Mycoplasma synoviae grown to the logarithmic phase, respectively, to make the final concentrations 1 / 2MIC, 1MIC, 2MIC, 4MIC, 8MIC, respectively, the mycoplasma suspension without aloe-emodin was used as a positive control, and the modified Frey's medium alone was used as a negative control;

[0042] 5% CO at 37°C 2The cells were cultured in an incubator, and the CCU of mycoplasma was determined at different drug concentrations at 0, 12, 24, 36, and 48 h, respectively, to obtain the growth curves of Mycoplasma gallisepticum and Mycoplasma synoviae at different aloe-emodin concentrations.

[0043] Depend on Figure 1 , Figure 2 It can be seen that when the final concentration of aloe-emodin was 7.813 μg / mL and 15.625 μg / mL (i.e., 1 / 2MIC and 1MIC), compared with the control group without aloe-emodin, the growth of the two mycoplasmas was inhibited at each time point, and was dose-dependent with the concentration of the aloe-emodin solution. When the final concentration of aloe-emodin was greater than or equal to 31.25 μg / mL (i.e., 2MIC), both mycoplasmas were completely killed.

[0044] The above results show that aloe-emodin can not only inhibit the growth of Mycoplasma gallisepticum and Mycoplasma synoviae, but also kill Mycoplasma gallisepticum and Mycoplasma synoviae.

[0045] Embodiment 3:

[0046] Determination of the minimum inhibitory concentration and minimum bactericidal concentration of tylosin against Mycoplasma gallisepticum and Mycoplasma synoviae:

[0047] 1. Materials

[0048] Modified Frey's medium is used for drug sensitivity experiments to detect the antibacterial efficacy of drugs.

[0049] 2. Test methods

[0050] The mycoplasma suspension cultured to the logarithmic growth phase was diluted to an appropriate concentration and added to sterile 96-well plates. 100 μL of the diluted mycoplasma suspension was added to wells 1 to 11 to make the final concentration of the mycoplasma suspension in each well 10 5 CCU / mL;

[0051] Use modified Frey's medium to dilute the tylosin solution in multiples, add different concentrations of tylosin solution to the above 96-well plate, add the diluted tylosin solution to wells 1 to 10, 100 μL per well, add 100 μL of modified Frey's medium to well 11 to form a 200 μL mycoplasma suspension as a growth control, and add only 200 μL of modified Frey's medium to well 12 as a negative control. 37℃5% CO 2 Incubate in the incubator for 48 hours, observe the color change of the culture medium in the 96-well plate, and determine the MIC value. Take the bacterial solution from each well and spread it on the modified Frey solid medium. If the number of colonies is less than 5, it is the MBC value.

[0052] 3. Results

[0053] It can be seen from Table 2 that when the concentration of tylosin is 0.977 μg / mL, it has an inhibitory effect on the growth of Mycoplasma gallisepticum and Mycoplasma synoviae, and when the concentration of tylosin is 1.953 μg / mL, it has a bactericidal effect on Mycoplasma gallisepticum and Mycoplasma synoviae.

[0054] Table 2 Minimum inhibitory concentration and minimum bactericidal concentration of tylosin against Mycoplasma gallisepticum and Mycoplasma synoviae

[0055]

[0056] Embodiment 4:

[0057] The synergistic antibacterial effect of aloe-emodin and tylosin against Mycoplasma gallisepticum and Mycoplasma synoviae was determined by checkerboard microdilution test:

[0058] 1. Materials

[0059] Modified Frey's medium is used for drug sensitivity experiments to detect the antibacterial efficacy of drugs.

[0060] 2. Test methods

[0061] Dilute the mycoplasma suspension cultured to the logarithmic growth phase to an appropriate concentration and add them to a sterile 96-well plate. Add 100 μL of the diluted mycoplasma suspension to rows 1 to 10 to make the final concentration of the mycoplasma suspension in each well 10. 5 CCU / mL, 200 μL of mycoplasma suspension was added to row 11 as a growth control, and only 200 μL of modified Frey's medium was added to row 12 as a negative control.

[0062] The two compounds used for the test were diluted in a 96-well plate in series, and 50 μL of diluted aloe-emodin was added to the 96-well plate from left to right (1-10), and 50 μL of diluted tylosin was added from top to bottom (AH) to make the final concentrations of aloe-emodin: 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.625 μg / mL, 7.813 μg / mL, respectively. / mL, 3.906μg / mL, 1.953μg / mL, 0.977μg / mL, and 0.488μg / mL, so that the final drug concentrations of tylosin were 15.625μg / mL, 7.813μg / mL, 3.906μg / mL, 1.953μg / mL, 0.977μg / mL, 0.488μg / mL, 0.244μg / mL, and 0.122μg / mL, respectively.

[0063] Place the 96-well plate in 37°C 5% CO 2Incubate in an incubator for 48 h, observe the color change of the culture medium in the 96-well plate, and calculate the synergy inhibition index according to the minimum combined inhibitory concentration of the antibiotic and the compound.

[0064] The determination method is as follows: for synergistic antibacterial effect, FICI ≤ 0.5; for additive effect, 0.5 < FICI ≤ 1; for no relevant effect, 1 < FICI ≤ 2; for antagonistic effect, FICI > 2.

[0065] 3. Results

[0066] As can be seen from Table 3, when emodin and tylosin are used in combination, the optimal concentrations of emodin and tylosin are 1.953 μg / mL and 0.244 μg / mL, respectively, and the fractional inhibitory concentration indices against Mycoplasma gallisepticum are both 0.375. When the concentration of emodin is 1.953 - 3.906 μg / mL and the concentration of tylosin is 0.244 μg / mL, they both have a synergistic antibacterial effect on the growth of Mycoplasma synoviae.

[0067] FICI calculation formula: FICI = MIC of drug A in combination / MIC of drug A alone + MIC of drug B in combination / MIC of drug B alone.

[0068] Table 3 Minimum inhibitory concentrations and fractional inhibitory concentration indices of emodin and tylosin against Mycoplasma gallisepticum and Mycoplasma synoviae

[0069]

[0070]

[0071] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of an aloe-emodin composition in preparing a drug for treating or preventing Mycoplasma gallisepticum or Mycoplasma synoviae infection, wherein the only active ingredients of the aloe-emodin composition are aloe-emodin and tylosin, and the mass ratio of aloe-emodin to tylosin is 8:1.

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