Application of madecassic acid in antibacterial treatment

Abstract

The present invention discloses an application of madecassic acid in antibacterial treatment, and belongs to the application field of effective ingredients of Chinese herbal medicine and natural compounds. The present invention systematically studies the antibacterial activity and related mechanisms of madecassic acid, and confirms that the antibacterial effect of madecassic acid on Gram-positive bacteria is better than that on Gram-negative bacteria, and the antibacterial effect on Staphylococcus aureus is the best. Through the experiment of combined use of madecassic acid and common β-lactam antibiotics, it is confirmed that madecassic acid and β-lactam antibiotics have a synergistic effect, and further evaluates the practical application of madecassic acid on the growth of MRSA bacteria in whole-fat high-temperature sterilized milk. The present invention systematically studies the antibacterial activity and related mechanisms of madecassic acid, which is helpful to develop its potential clinical medicinal value and edible value, and at the same time provides an important basis for the development of antibacterial drugs from Chinese herbal medicines and the formulation of clinical drug use specifications.

Description

Technical Field

[0001] The invention relates to the application field of effective components of Chinese herbal medicines and natural compounds, and in particular to the application of madecassic acid in antibacterial treatment. Background Art

[0002] Staphylococcus aureus is a common clinical pathogen that exists mostly on human skin, especially in the nasopharynx. It is one of the most common causes of infectious diseases in the world, and can cause a variety of infections ranging from mild skin infections to severe tissue infections and sepsis, posing a serious threat to human health. In addition, Staphylococcus aureus can also adhere to medical devices and equipment, causing a variety of hospital infections. The treatment of Staphylococcus aureus infection currently relies mainly on antibiotics. However, repeated and excessive use of antibiotics can lead to serious antibiotic residues and bacterial resistance, thereby reducing the effectiveness of treatment. Therefore, exploring low-toxicity and low-resistance drugs to replace antibiotics has become an important task in the current research and development of antibacterial drugs.

[0003] In order to slow down the evolution of bacterial resistance, the development of new antibacterial Chinese herbal medicines and their preparations has become an important hotspot in the field of antibacterial drug research and development. For example, Zhang et al. found that the water extract of Artemisia argyi leaves can destroy the integrity of the cell wall of Staphylococcus aureus at a certain concentration and increase the permeability of the cell membrane, but it cannot kill Staphylococcus aureus in a short time; Zhou et al. found that the n-butanol part and ethanol crude extract of Dendrobium fruit shell have strong antibacterial activity against Salmonella paratyphi A and Staphylococcus aureus, and its antibacterial mechanism may be to inhibit the synthesis of bacterial proteins. However, the chemical composition of Chinese herbal compound preparations and single Chinese medicines is complex, so the antibacterial mechanism is unclear and there is a lack of standard medication specifications. Therefore, it is very necessary to conduct in-depth research on the antibacterial activity and mechanism of key components in Chinese herbal medicines. The relevant data will provide an important basis for the development of antibacterial drugs from Chinese herbal medicines and the formulation of clinical medication specifications.

[0004] Centella asiatica (L.) Urb. is a dried herb of the Umbelliferae plant Centella asiatica (L.) Urb. It is cold in nature, bitter and pungent in taste, and has the effects of clearing away heat and dampness, detoxifying and reducing swelling. It is also often drunk as a herbal tea. Domestic and foreign literature reports that Centella asiatica has antibacterial, liver damage protection, neuroprotective, anti-tumor, and wound healing effects. The antibacterial activity of Centella asiatica is mainly attributed to asiatic acid. Related studies have shown that asiatic acid has significant broad-spectrum antibacterial activity (such as Escherichia coli, Salmonella typhimurium, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, etc.).

[0005] Madecassic acid (MA) is another pentacyclic triterpenoid in Centella asiatica extract. In the prior art, most studies on the activity of madecassic acid focus on anticancer, cardiovascular protection, antidiabetes and anti-inflammatory effects. There is currently no research on the antibacterial effect of madecassic acid. Therefore, a systematic study of the antibacterial activity and related mechanisms of madecassic acid will help develop its potential clinical medicinal value and provide an important basis for the development of antibacterial drugs derived from Chinese herbal medicines and the formulation of clinical drug use specifications. Summary of the invention

[0006] The purpose of the present invention is to provide an application of madecassic acid in antibacterial treatment to solve the problems existing in the above-mentioned prior art. The present invention systematically studies the antibacterial activity and related mechanisms of madecassic acid, which is helpful to develop its potential clinical medicinal value and edible value, and at the same time provides an important basis for the development of antibacterial drugs derived from Chinese herbal medicines and the formulation of clinical drug use specifications.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The invention provides use of madecassic acid or a composition comprising madecassic acid and beta-lactam antibiotics in preparing a product for inhibiting bacteria, wherein the bacteria include Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Gordonia, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Bacillus megaterium.

[0009] Furthermore, the bacteria include Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

[0010] Furthermore, the bacteria include Staphylococcus aureus.

[0011] Furthermore, the β-lactam antibiotics include cefepime and / or cephalexin.

[0012] Furthermore, in the composition of madecassic acid and β-lactam antibiotics, the concentration of madecassic acid is 7.8-62.5 μg / mL, and the concentration of β-lactam antibiotics is 125-3.9 μg / mL.

[0013] The present invention also provides an antibacterial product, comprising madecassic acid or a composition comprising madecassic acid and beta-lactam antibiotics.

[0014] The invention also provides an application of madecassic acid in prolonging the preservation of milk.

[0015] The invention also provides a method for prolonging the storage of milk, which comprises adding madecassic acid into the milk to inhibit the contamination of methicillin-resistant Staphylococcus aureus.

[0016] Furthermore, the concentration of madecassoic acid is 187.5-250 μg / mL.

[0017] Furthermore, after adding the madecassic acid, the mixture is stored at 28°C or 4°C.

[0018] The present invention discloses the following technical effects:

[0019] The present invention systematically studies the antibacterial activity and related mechanisms of madecassoic acid, and confirms that madecassoic acid has a better antibacterial effect on Gram-positive bacteria than on Gram-negative bacteria, has the best antibacterial effect on Staphylococcus aureus, can destroy the cell wall and cell membrane of Staphylococcus aureus, and reduce the activities of SDH and MDH in Staphylococcus aureus, and can also interact with DNA to cause relaxation and ring opening of supercoiled DNA, thereby inhibiting the activities of DNA topoisomerases I and II.

[0020] The present invention verifies the synergistic effect of madecassic acid and β-lactam antibiotics through a combined drug administration experiment of madecassic acid and common β-lactam antibiotics, thus providing a new idea for supplementing the deficiencies of conventional antibacterial agent therapy and improving the efficacy of antibiotics.

[0021] The present invention further evaluated the practical application of madecassic acid on the growth of MRSA bacteria in whole-fat high-temperature sterilized milk by using whole-fat high-temperature sterilized milk as a matrix, indicating that 250 μg / mL MA can effectively inhibit the growth of MRSA and extend the shelf life of milk beverages. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 The effect of MA on the growth curve of Staphylococcus aureus, where Control is the control, MIC is the 31.25μg / mL MA action group, and 2MIC is the 62.5μg / mL MA action group;

[0024] Figure 2 The inhibitory effect of MA on the biofilm of Staphylococcus aureus, where ** indicates a significant difference compared with the control group (P<0.01);

[0025] Figure 3 The effect of MA on the change of conductivity of Staphylococcus aureus culture medium and the leakage of intracellular nucleic acid and protein of Staphylococcus aureus, where (a) is the change of conductivity of culture medium after MA acts on Staphylococcus aureus, (b) is the effect of MA on the leakage of intracellular nucleic acid of Staphylococcus aureus, (c) is the effect of MA on the leakage of intracellular protein of Staphylococcus aureus, Control is the control, MIC is the group acted with 31.25μg / mL MA, 2MIC is the group acted with 62.5μg / mL MA;

[0026] Figure 4 The effect of MA on the alkaline phosphatase leakage of Staphylococcus aureus, where * indicates a significant difference compared with the control group (P<0.05A), and ** indicates an extremely significant difference compared with the control group (P<0.01);

[0027] Figure 5 The effect of MA on the β-galactosidase content of Staphylococcus aureus, where Control is the control, MIC is the 31.25μg / mL MA action group, and 2MIC is the 62.5μg / mL MA action group;

[0028] Figure 6 It is the SDS-PAGE electrophoresis diagram of soluble protein of Staphylococcus aureus, where M is Mark, C is untreated sample, MIC is 31.25 μg / mL of NA-treated sample, 2MIC is 62.5 μg / mL of MA-treated sample;

[0029] Figure 7 It is the gel electrophoresis diagram after the direct action of MA and pET-28a DNA, wherein Form II represents gap or linear DNA, Form I represents supercoiled DNA, a represents the anhydrous ethanol control group, bh represent the treatment groups with final MA concentrations of 0.003906, 0.007812, 0.15625l, 0.3125, 0.625, 1.25 and 2.5 mg / mL, respectively;

[0030] Figure 8 The changes in the UV absorption spectrum after the interaction between MA and DNA, where the black line represents the control group without MA, the red line represents the group with a final MA concentration of 62.5 μg / mL, and the blue line represents the group with a final MA concentration of 31.25 μg / mL;

[0031] Fig. 9 TEM images of Staphylococcus aureus treated with MA, where Control is the control, MIC is the 31.25 μg / mL MA action group, and 2MIC is the 62.5 μg / mL MA action group;

[0032] Fig.10 The effect of MA on the activities of malate dehydrogenase (a) and succinate dehydrogenase (b) of Staphylococcus aureus, where ** represents a significant difference compared with the control group (P<0.01);

[0033] Fig.11 is the agarose gel electrophoresis image of pET28a;

[0034] Fig.12 The unwinding effect of topo enzyme with different enzyme amounts on pET-28a, where Form II represents gap or linear DNA, Form I represents supercoiled DNA, a represents the pET-28a control group, and bg represent the addition amount of crude enzyme extract of 2 μL, 2.8 μL, 3.6 μL, 4 μL, 6 μL and 8 μL respectively;

[0035] Fig.13 is the effect of MA on topoⅠ activity of Staphylococcus aureus, where Form II represents nicked or linear DNA, Form I represents supercoiled DNA, a represents the pET-28a control group, b represents the DNA topoisomerase group, c represents the DMSO control group, and dj represents MA concentrations of 0.156 mg / mL, 0.3125 mg / mL, 0.625 mg / mL, 1.25 mg / mL, 2.5 mg / mL, 5 mg / mL, and 10 mg / mL, respectively;

[0036] Fig.14 is the effect of MA on topo II activity of Staphylococcus aureus, where Form II represents nicked or linear DNA, Form I represents supercoiled DNA, a represents the pET-28a control group, b represents the DNA topoisomerase group, c represents the DMSO control group, and dj represents the MA concentrations of 0.156 mg / mL, 0.3125 mg / mL, 0.625 mg / mL, 1.25 mg / mL, 2.5 mg / mL, 5 mg / mL, and 10 mg / mL, respectively;

[0037] Fig.15 The inhibition mode of MA against Staphylococcus aureus;

[0038] Fig.16 The effect of MA combined with cephalexin (CEX) on the growth curve of MRSA;

[0039] Fig.17 The inhibition effect of different concentrations of MA on MRSA bacteria in whole-fat sterilized milk at 28°C, where (a) is the 24h inhibition and (b) is the 48h inhibition. ** indicates that the difference is extremely significant compared with the control group (P<0.01);

[0040] Fig.18 Figure 2 shows the inhibitory effect of different concentrations of MA on MRSA bacteria in whole-fat sterilized milk at 4°C. (a) shows the 24-hour inhibition and (b) shows the 48-hour inhibition. ** indicates a significant difference compared with the control group (P<0.01). DETAILED DESCRIPTION

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0043] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0044] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0045] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0046] The chemical structural formula of madecassoic acid used in the embodiments of the present invention is as follows:

[0047]

[0048] Example 1

[0049] 1. Experimental Materials

[0050] Staphylococcus aureus (S.aureus) CMCC (B) 26003 was purchased from Shanghai Luwei Technology Co., Ltd.; Escherichia coli (E.coli) ATCC 25922 was purchased from Shanghai Collection Biotechnology Center; Pseudomonas aeruginosa (P.aeruginosa) ATCC 27853 was purchased from Shanghai Collection Biotechnology Center; Gordinia sp. JD-4 was isolated and preserved by the Bioengineering Laboratory of Hunan University of Chinese Medicine; Candida albicans (C.albicans) CMCC (F) 98001 was purchased from Shanghai Collection Biotechnology Center; Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 was purchased from Beijing Collection Biotechnology Co., Ltd.; Bacillus megaterium (Bacillus Bacillus subtilis (B. subtilis) WB600 was purchased from Shanghai Collection Biotechnology Center.

[0051] Madecassic acid was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Luria-Bertani (LB) agar medium, LB liquid medium, plate count agar medium (PCA medium), yeast extract, tryptone, and glucose were purchased from Guangdong Huankai Microbiological Technology Co., Ltd.; microplate reader (MB-530) was purchased from Shenzhen Huisong Technology Development Co., Ltd.; transmission electron microscope (HITACHI H-7650), purchased from Electronics Co., Ltd.; high-speed refrigerated centrifuge (D1524R), purchased from Thermo Fisher Scientific Inc.; electrophoresis instrument (DYY-6C), purchased from Beijing Liuyi Biotechnology Co., Ltd.; gel imager (WD-9413A), purchased from Junyi Oriental Biotechnology Co., Ltd.; cell disruptor (Biosafer900-92), purchased from Saifei Co., Ltd.; constant temperature incubator (SPX-150BⅢ), purchased from Tianjin Test Instrument Co., Ltd.; constant temperature culture oscillator (ZWY-1102C), purchased from Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.; clean bench (SW-CJ-ZFD), purchased from Sujing Antai; ultraviolet spectrophotometer (V-5100), purchased from Shanghai Yuanxi Instrument Co., Ltd.; high temperature sterilizer (GI54TW), purchased from Zhiwei Instrument Co., Ltd.; alkaline phosphatase (Alkaline The phosphatase (AKP) kit was purchased from Nanjing Jiancheng Bioengineering Institute; the succinate dehydrogenase (SDH) kit and the malate dehydrogenase (MDH) kit were purchased from Solebow Biotechnology Co., Ltd.; and pET-28a DNA (LM-1207) was purchased from Shanghai Lianmai Bioengineering Co., Ltd.

[0052] 2. Experimental Methods

[0053] 2.1 Preparation of test solution

[0054] The madecassoic acid monomer compound was prepared into a stock solution with a final concentration of 10 mg / mL using DMSO and stored at 4°C for later use.

[0055] 2.2 Activation culture of bacteria

[0056] The frozen bacterial suspensions of bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Gordonia, Bacillus subtilis, Bacillus megaterium and methicillin-resistant Staphylococcus aureus) stored in ultra-low temperature refrigerators (-80°C) for a long time were activated, and the frozen bacterial suspensions of the bacteria were inoculated into conical flasks containing the corresponding culture medium, and placed in a constant temperature culture shaker at 37°C for overnight culture. The bacterial suspension in the test tube was streaked and inoculated on the corresponding agar plate and cultured overnight in a constant temperature incubator. Finally, a single colony with good growth on the agar plate was picked and inoculated into a fresh corresponding liquid culture medium, and cultured in a constant temperature culture shaker at 37°C until the logarithmic growth phase.

[0057] 2.3 Preparation of bacterial suspension

[0058] A small amount of bacteria was picked from the purified Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Gordonia, Bacillus subtilis, Bacillus megaterium and methicillin-resistant Staphylococcus aureus and inoculated into LB and PCA liquid culture media. A small amount of single colony of Candida albicans was picked and inoculated into YPD medium (1% yeast extract, 2% tryptone, 2% glucose), and cultured in a constant temperature culture shaker at 37°C and 120rpm for 24h. The fungus Candida albicans was cultured in a constant temperature culture shaker at 28°C and 120rpm for 48h. The bacterial solution concentration was adjusted to 10 with LB liquid culture medium. 8 cfu / mL, and the concentration of Candida albicans was adjusted to 10 in YPD liquid medium. 8 cfu / mL.

[0059] 2.4 Antibacterial activity of madecassic acid

[0060] 2.4.1 Determination of the inhibition zone diameter of madecassic acid

[0061] The antibacterial activity of MA was evaluated by paper diffusion method. 10 8 cfu / mL concentration of bacterial solution 100 μL, and evenly spread it on the corresponding culture medium plate with a sterile coating rod, placed 6 mm filter paper soaked with MA solution (1 mg / mL) on the plate, and took the group with oxacillin (OXA) as the positive control, and selected a group with only solvent as the blank control group. The bacteria were cultured in a 37 ℃ constant temperature incubator for 12-18 h, and the fungus Candida albicans was cultured in a 28 ℃ constant temperature incubator for 24-48 h. The antibacterial effect was observed, and the diameter of the inhibition zone was measured with a vernier caliper.

[0062] 2.4.2 Minimum Inhibitory Concentration (MIC) Determination

[0063] The MIC values ​​of MA against five bacteria (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Bacillus megaterium) were determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining. The bacterial suspension cultured to the logarithmic phase was diluted to a concentration of 10 6 -10 7 cfu / mL, 180 μL of bacterial suspension and 20 μL of different concentrations of MA solution were added to a 96-well plate to make the final concentrations reach 500, 250, 125, 62.5, 31.25, 15.625, 7.8 and 3.9 μg / mL. Dimethyl sulfoxide (DMSO) was used as the control group and the plates were cultured in a 37°C constant temperature incubator for 16 h. After culture, TTC solution was added to each experimental well and cultured at 37°C in the dark for 4 h. The color changes were observed. The experiment was repeated 3 times and the average value was taken.

[0064] The antibacterial curve of 2.5-hydroxyasiatic acid against Staphylococcus aureus

[0065] Different volumes of MA solution were pipetted at 10 7 -10 8 cfu / mL of Staphylococcus aureus suspension, the final concentration of MA in the bacterial solution was 31.25μg / mL and 62.5μg / mL, and the control group without MA solution was placed in a constant temperature culture shaker at 37℃ and 120rpm. Samples were taken every 4 hours, and the optical density (OD) value at 600nm was measured to draw the growth curve of Staphylococcus aureus after MA action. Repeat the experiment 3 times and take the average value.

[0066] Inhibitory effect of 2.6-hydroxy-madecasic acid on biofilm formation of Staphylococcus aureus

[0067] Staphylococcus aureus was inoculated into LB medium and cultured at 37°C with constant temperature shaking at 120 rpm. 600nm When it reached about 0.3, MA solutions with different concentrations were mixed with the bacterial solution to make the final concentration of MA 31.25μg / mL and 62.5μg / mL, respectively. The control group was placed in a 37°C constant temperature incubator for 24 hours, the floating cells were removed, washed three times with phosphate buffered saline (PBS) solution, stained with 1% crystal violet for 10 minutes at room temperature, and then washed three times. 200μL of anhydrous ethanol was taken to dissolve the stained biofilm, and the OD value was measured at 570nm.

[0068] 2.7 Transmission electron microscopy (TEM) analysis of the effect of madecassoic acid on the cell morphology of Staphylococcus aureus

[0069] Staphylococcus aureus in the logarithmic growth phase was treated with MA at final concentrations of 31.25 μg / mL and 62.5 μg / mL, respectively. The control group was cultured for 6 hours without MA. After washing with sterile PBS, the cells were fixed with 2.5% glutaraldehyde and then fixed with 1% osmium tetroxide for 24 hours. After gradient dehydration with ethanol, the cells were embedded in resin for ultrathin sectioning, and the changes in the intracellular structure of Staphylococcus aureus were observed under a transmission electron microscope.

[0070] Effects of 2.8-hydroxyasiatic acid on the permeability of Staphylococcus aureus cell membrane and cell wall

[0071] 2.8.1 Determination of relative conductivity of bacterial solution, extravasation of macromolecules such as DNA and RNA, and alkaline phosphatase

[0072] Centrifuge the suspension of Staphylococcus aureus in the logarithmic growth phase for 10 minutes and wash with 5% glucose to isotonic concentration. Add an equal amount of sterile distilled water to 5% glucose, and measure the conductivity of the solution, which is recorded as L1. Add MA to the isotonic concentration bacterial solution so that the final concentration of MA is 31.25μg / mL and 62.5μg / mL, respectively. The control group is free of drugs. After mixing, place it in a 37°C constant temperature incubator and culture it. Measure the conductivity of the solution every 2 hours, which is recorded as L2. Put the 5% glucose isotonic concentration bacterial solution into boiling water for 5 minutes, measure the conductivity of the solution, which is recorded as L0, and calculate the relative conductivity = (L2-L1) / L0×100%. Take the suspension of Staphylococcus aureus in the logarithmic growth phase and centrifuge it for 10 minutes. Wash the bacteria twice with PBS and adjust the concentration to 10 7 cfu / mL, and then MA was added to make the final concentration of 31.25μg / mL and 62.5μg / mL. The control group did not contain drugs and was cultured in a 37℃ incubator. The bacterial solution was taken every 2h and the OD value in the culture supernatant at 260nm and 280nm was detected. Staphylococcus aureus in the logarithmic phase was treated with MA to a final concentration of 31.25μg / mL and 62.5μg / mL. The control group did not contain MA. The mixture was incubated at 37℃, taken out every 2h, centrifuged for 10min, and the AKP content in the supernatant was determined using an alkaline phosphatase (AKP) kit.

[0073] 2.8.2 Determination of β-galactosidase content after exposure to madecassic acid

[0074] β-galactosidase cannot pass through the cell membrane of bacteria. If the cell membrane is damaged, β-galactosidase leaks through the damaged membrane, and cytoplasmic β-galactosidase can be detected. Centrifuge the bacterial solution in the logarithmic growth period, resuspend it with M9 lactose induction medium, and culture it in a constant temperature culture shaker at 37°C and 120rpm for 8 hours. After centrifugation, resuspend the bacteria with β-galactosidase reaction buffer, add o-nitrophenyl-β-D-galactoside (ONPG) and mix well, and add MA to a final concentration of 31.25μg / mL and 62.5μg / mL. The control group without MA is cultured in a constant temperature culture shaker at 37°C and 120rpm. Take out samples every 2 hours and measure the OD value at 420nm.

[0075] 2.9 Effect of Madecassic Acid on Soluble Protein Content of Staphylococcus aureus by SDS-PAGE

[0076] The concentration of Staphylococcus aureus suspension in the logarithmic growth phase was adjusted with LB liquid medium, and MA solution was added to make the final concentration 31.25μg / mL and 62.5μg / mL. The control group was cultured at 37℃, 120rpm in a constant temperature culture shaker for 16h. Centrifuge, take the precipitate, add lysis buffer, and break it with a cell disruptor. Centrifuge, take the supernatant, add 5× loading buffer (1:4), and mix well. Boil in 100℃ boiling water for 10min, centrifuge, and take the supernatant. Perform SDS-PAGE electrophoresis.

[0077] 2.10 Determination of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) activities

[0078] Take the suspension of Staphylococcus aureus in the logarithmic growth phase, add MA to make the final concentration of 31.25μg / mL and 62.5μg / mL, and use the control group without MA. After culturing in a constant temperature culture shaker at 37°C and 120rpm for 20h, centrifuge for 10min, add PBS buffer to resuspend the precipitate, disrupt with a cell disruptor for 10 minutes, take the supernatant, and detect the activity of SDH and MDH according to the SDH and MDH activity detection kit.

[0079] 2.11 Study on the interaction between hydroxy-madecassoic acid and DNA

[0080] 2.11.1 Determination of the interaction between madecassic acid and DNA by agarose gel electrophoresis

[0081] MA was added to pET-28aDNA to make the final concentrations 0.003906, 0.007812, 0.15625l, 0.3125, 0.625, 1.25 and 2.5 mg / mL, respectively, and the buffer was added to 1.5 mL. After incubation at 37°C for 30 min, 1 μL 10 mg / mL proteinase K and 1 μL 10% sodium dodecyl sulfate (SDS) were added to terminate the reaction. After incubation at 37°C for 30 min, the reaction was electrophoresed in 0.8% agarose gel electrophoresis for 20 min at a voltage of 150 V. The gel was observed and photographed using a gel imaging system, and anhydrous ethanol was used as the control group.

[0082] 2.11.2 Determination of the interaction between madecassic acid and DNA by UV absorption spectroscopy

[0083] In a quartz cuvette, MA was added to pET-28a DNA to a final concentration of 31.25 μg / mL and 62.5 μg / mL. Anhydrous ethanol was used as the control group. The buffer was added to 1.5 mL and incubated at 37°C for 30 min. The UV absorption spectrum at 240-300 nm was detected using a UV spectrophotometer.

[0084] 2.12 Effects of Madecasic Acid on DNA Topoisomerase

[0085] (1) Reagent preparation

[0086] Unwinding buffer I: 200mmol / L Tris.HCl pH7.5, 40mmol / L magnesium chloride, 340mmol / L potassium chloride, 20mmol / L DTT, 5mmol / L EDTA, 0.12mg / mL BSA, without ATP.

[0087] Unwinding buffer II: 4mmol / L ATP, 200mmol / LTris.HCl pH7.5, 40mmol / L magnesium chloride, 340mmol / L potassium chloride, 20mmol / L DTT, 5mmol / L EDTA, 0.12mg / mL BSA.

[0088] The effect of MA on the activity of DNA topoisomerase was detected by gel electrophoresis. Supercoiled pET-28aDNA, crude enzyme (DNA topoisomerase) extract and different concentrations of MA solution were added to DNA unwinding buffer I / Ⅱ, DMSO was used as the control group, distilled water was added to 20μL, 10% SDS and 10mg / mL proteinase K were added to terminate the reaction after incubation at 37℃ for 30min, and then 2μL of loading buffer was added. The reaction was electrophoresed in 0.8% agarose gel for 20min at a voltage of 150V, and the gel imaging system was used to observe and photograph.

[0089] 2.13 Statistical analysis

[0090] SPSS 22.0 statistical software was used to analyze the experimental data, and analysis of variance was used for pairwise comparisons between groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered extremely statistically significant.

[0091] 3. Results and Analysis

[0092] 3.1 Antibacterial activity of madecassic acid

[0093] The results of the disk diffusion method showed that the diameter of the inhibition zone of oxacillin against Staphylococcus aureus was the largest, and the DMSO group did not form an inhibition zone. MA had a better antibacterial effect on Staphylococcus aureus.

[0094] The antibacterial activities of MA against Staphylococcus aureus, MRSA, Escherichia coli, Gordonia, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans, and Bacillus megaterium are shown in Table 1 .

[0095] Table 1

[0096]

[0097] As shown in Table 1, the diameters of the inhibition zones of MA against Staphylococcus aureus, MRSA, Escherichia coli, Gordonia, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Bacillus megaterium are 13 mm, 14.5 mm, 10 mm, 13.5 mm, 11.5 mm, 10.5 mm, 13.5 mm and 10.5 mm, respectively; the MIC values ​​of MA against Staphylococcus aureus, Bacillus subtilis, MRSA, Escherichia coli, Bacillus megaterium and Pseudomonas aeruginosa are 31.25 μg / mL, 62.5 μg / mL, 62.5 μg / mL, 250 μg / mL, 62.5 μg / mL and 125 μg / mL, respectively. The MICs of oxacillin (positive control) against Staphylococcus aureus, MRSA, Bacillus subtilis and Bacillus megaterium were 0.048μg / mL, 7.8μg / mL, 3.9μg / mL and 1.9μg / mLμg / mL, respectively. The experimental results showed that MA had a better antibacterial effect against Gram-positive bacteria (Staphylococcus aureus, MRSA) than Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli).

[0098] 3.2 Antibacterial effect of hydroxy-madecassic acid on Staphylococcus aureus

[0099] The growth of Staphylococcus aureus in the control group was good, while the growth of Staphylococcus aureus after the action of 31.25μg / mL and 62.5μg / mL MA was significantly inhibited, and the OD value of the MA experimental group was significantly lower than that of the control group. This further confirmed that MA has an inhibitory effect on Staphylococcus aureus. 31.25μg / mL MA can inhibit the growth of Staphylococcus aureus within 28h; 62.5μg / mL MA can inhibit the growth of Staphylococcus aureus within 32h. Figure 1 .

[0100] 3.3 Effects of Madecasic Acid on Biofilm

[0101] When the concentration of MA was 31.25 μg / mL and 62.5 μg / mL, it had an inhibitory effect on the production of Staphylococcus aureus biofilm, and the inhibition rate increased with the increase of MA concentration, and the biofilm inhibition rate was 68.35% and 72.73%, respectively. Therefore, MA has an inhibitory effect on the production of Staphylococcus aureus biofilm, see Figure 2 .

[0102] 3.4 Effects of Madecasic Acid on the Cell Membrane and Cell Wall of Staphylococcus aureus

[0103] 3.4.1 Changes in conductivity in culture medium after exposure to madecassolic acid to Staphylococcus aureus

[0104] The experimental group with MA showed a faster growth trend, with the conductivity percentage reaching 67.17% and 76.79%, respectively, while the control group was 36.32%. The results showed that MA affected the function of the cell membrane of Staphylococcus aureus, causing the cell contents to leak. Figure 3 .

[0105] 3.4.2 Effect of Madecasic Acid on Macromolecules in Staphylococcus aureus Culture Medium

[0106] After treatment with 31.25 μg / mL and 62.5 μg / mL MA, the amount of nucleic acid leakage (OD 260nm ) and protein leakage (OD 280nm The results showed that the permeability of the cell membrane increased after the action of MA, and the intracellular macromolecules were released into the cell suspension. Figure 3 .

[0107] 3.4.3 Effects of Madecasic Acid on Staphylococcus aureus Cell Wall

[0108] The amount of alkaline phosphatase leakage in the MA-containing experimental group was significantly greater than that in the control group, suggesting that MA can increase the amount of alkaline phosphatase leakage in Staphylococcus aureus and that the integrity of the bacteria may be damaged, indicating that MA can destroy the cell wall of Staphylococcus aureus and cause the leakage of cell contents. Figure 4 .

[0109] 3.4.4 Effect of Madecasic Acid on β-Galactosidase Content

[0110] β-galactosidase exists on the bacterial inner membrane. When the inner membrane is damaged, the intracellular β-galactosidase will leak out through the cytoplasmic membrane. The change in the extracellular β-galactosidase content can infer the degree of damage to the bacterial inner membrane. The extracellular β-galactosidase content in the control group did not change significantly, but the extracellular β-galactosidase content in the MA 31.25μg / mL and 62.5μg / mL experimental groups was significantly higher than that in the control group, indicating that MA destroyed the integrity of the inner membrane of Staphylococcus aureus, thereby causing β-galactosidase to leak out of the cell, resulting in an increase in its extracellular content. Figure 5 .

[0111] Effects of 3.5-hydroxyasiatic acid on soluble protein of Staphylococcus aureus

[0112] The protein bands of Staphylococcus aureus that were not treated with MA were numerous and clear, while the protein bands of Staphylococcus aureus that were treated with MA (31.25 μg / mL and 62.5 μg / mL) became significantly lighter in color, indicating that MA can exert its antibacterial effect by affecting the protein expression in Staphylococcus aureus. The reason may be that MA affects the synthesis of Staphylococcus aureus nucleic acid and the expression of related genes, thereby hindering the synthesis of certain proteins and enzymes, resulting in a decrease in the protein expression in the bacteria, thereby exerting its antibacterial effect. Figure 6 .

[0113] 3.6 Study on the interaction between hydroxy-madecassoic acid and DNA

[0114] 3.6.1 Changes in agarose gel electrophoresis after the action of madecassic acid on DNA

[0115] Agarose gel electrophoresis results Figure 7 As shown, when the MA concentration is greater than 0.007812 mg / mL, Form I (supercoiled DNA) gradually decreases and Form II (linear DNA) gradually increases, indicating that MA can cause relaxation and ring opening of supercoiled DNA.

[0116] 3.6.2 UV spectroscopy study of the binding of madecassic acid to DNA

[0117] Ultraviolet absorption spectroscopy is often used to study the interaction between compounds and DNA. Generally, after the compound interacts with DNA, a red shift and a hypochromic effect appear, indicating that the compound and DNA are intercalated. As the concentration of MA increases, the maximum absorbance of pET-28a DNA gradually decreases, a hypochromic effect occurs, and its wavelength appears a red shift, which further proves that MA can bind to pET-28a DNA. The insertion of MA causes the separation of DNA base pairs, resulting in changes in DNA conformation, as shown in Figure 2. Figure 8 .

[0118] 3.7 Effects of Madecasic Acid on the Morphology of Staphylococcus aureus Cells

[0119] Transmission electron microscopy results showed that MA had an effect on the morphology of S. aureus. The untreated S. aureus strain had a normal morphology with intact septa. However, compared with the untreated S. aureus, the cytoplasmic membrane of the S. aureus strain exposed to 31.25 μg / mL MA was damaged and the surface was rougher. The cells of S. aureus exposed to 62.5 μg / mL MA lysed and released the cytoplasmic contents, as shown in Figure 2. Fig. 9 .

[0120] 3.8 Effect of Madecasic Acid on the Activity of Succinate Dehydrogenase and Malate Dehydrogenase

[0121] Malate dehydrogenase (MDH) and succinate dehydrogenase (SDH) are key regulatory enzymes in the tricarboxylic acid cycle. Detecting the activity of MDH and SDH can indirectly reflect the energy metabolism in bacteria. After Staphylococcus aureus was treated with 31.25μg / mL and 62.5μg / mL MA, the activities of SDH and MDH in Staphylococcus aureus were significantly decreased. This suggests that MA can inhibit the activity of MDH and SDH in Staphylococcus aureus. Fig.10 .

[0122] 3.9 Effect of Madecasic Acid on the Activity of DNA Topoisomerase in Staphylococcus aureus

[0123] 3.9.1pET-28a purity test results

[0124] Agarose gel electrophoresis pattern Fig.11 , pET-28a is a commonly used fusion protein type prokaryotic efficient expression vector, with a size of 5369bp and containing a kanamycin resistance gene. The electrophoresis pattern shows that the DNA is between 4000bp and 5000bp, and it can be determined that the extracted DNA is the target gene pET-28a.

[0125] 3.9.2 DNA topoisomerase activity assay of crude enzyme extract from Staphylococcus aureus

[0126] The crude enzyme extract can unwind the supercoil of pET-28a into open-circular or linear DNA (Form Ⅱ). With the increase of the amount of crude enzyme extract, the Form Ⅱ DNA gradually increases and the supercoiled DNA (Form I) gradually decreases, indicating that the DNA topoisomerase in the crude enzyme extract is active and has a strong unwinding effect. Fig.12 .

[0127] 3.9.3 Results of the assay of DNA topoisomerase I activity of madecola acid against Staphylococcus aureus

[0128] DNA topoisomerase I is one of the key enzymes in nucleic acid metabolism in organisms. It is an enzyme that catalyzes the instantaneous disconnection and reconnection of DNA single strands, thereby mediating DNA unwinding. It participates in all key reactions such as DNA replication, transcription, recombination and repair, and this process does not require the participation of the energy auxiliary factor ATP. Under the catalysis of DNA topoisomerase I, pET-28a DNA unwinds into Form II. When the concentration of MA is greater than 1.25 mg / mL, as the concentration of MA increases, Form I gradually increases and Form II gradually decreases, indicating that MA can inhibit the activity of Staphylococcus aureus topoisomerase I, see Fig.13 .

[0129] 3.9.4 Results of the assay of madecassoic acid on the activity of DNA topoisomerase II of Staphylococcus aureus

[0130] DNA topoisomerase II is an important target enzyme that catalyzes the two strands of a DNA double helix by breaking them. The role of topoisomerase II is to mediate the unwinding, breaking and reconnection of DNA double helices to affect the structure of DNA. When the concentration of MA is greater than 2.5 mg / mL, as the concentration of MA increases, Form I gradually increases and Form II gradually decreases, indicating that MA can inhibit the activity of Staphylococcus aureus topoisomerase II, see Fig.14 .

[0131] In this example, the filter paper method and TTC staining method were used to determine the antibacterial activity of MA and its MIC value, and the antibacterial activity of MA against 8 common pathogens (Staphylococcus aureus, MRSA, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Gordonia, Bacillus subtilis, and Bacillus megaterium) was determined. The antibacterial effect of MA on Gram-positive bacteria was better than that on Gram-negative bacteria, and its MIC for Staphylococcus aureus was 31.25 μg / mL. At the same time, the growth curves of 31.25 μg / mL and 62.5 μg / mL MA on Staphylococcus aureus were determined, which further confirmed that MA had a significant inhibitory effect on the growth of Staphylococcus aureus and could completely inhibit the growth of Staphylococcus aureus within 28 hours. At the same time, 31.25 μg / mL and 62.5 μg / mL MA could significantly inhibit the formation of Staphylococcus aureus biofilm. Bacteria are composed of cell membrane, cell wall, cytoplasm and nucleoplasm, among which the cell membrane is an important medium for bacteria to exchange substances and energy with the outside world. When the cell membrane permeability of pathogenic bacteria is destroyed, the electrolytes in the bacteria will seep into the culture medium, and the conductivity of the supernatant of the Staphylococcus aureus culture medium can indirectly reflect the permeability of the bacterial cell membrane. In this embodiment, the relative conductivity change of the bacteria after the action of MA was detected. The study found that the relative conductivity of the experimental group was significantly higher than that of the control group after MA was used for 4 hours, which means that the permeability of the cell membrane of Staphylococcus aureus was changed after the action of MA, causing the K + 、Na + The leakage of small molecules such as OD 260nm ) and protein leakage (OD 280nm) and found that the OD values ​​of nucleic acid leakage and protein leakage of the bacterial suspension after MA action increased significantly, which confirmed that the cell membrane of Staphylococcus aureus was damaged after MA action, and macromolecular substances such as DNA and RNA in the bacteria were released into the culture medium, which led to the death of Staphylococcus aureus. At the same time, by measuring the effect of MA on the content of β-galactosidase, it was found that the extracellular β-galactosidase content in the MA-treated group was significantly higher than that in the control group, which fully confirmed that MA destroyed the cell membrane of Staphylococcus aureus. AKP is an enzyme existing between cell walls. Under normal circumstances, the activity of AKP cannot be detected outside the cell, but when the cell wall is damaged, AKP goes outside the cell and the activity of AKP can be detected. The experimental results show that the AKP content in the experimental group is significantly higher than that in the blank group, indicating that MA destroys the bacterial cell wall and can increase the permeability of the bacterial cell wall. At the same time, MA can inhibit the synthesis of soluble proteins of Staphylococcus aureus, and the cell morphology of Staphylococcus aureus treated with 31.25μg / mL and 62.5μg / mL MA was observed by transmission electron microscopy. The results showed that MA can damage the cell wall and cell membrane of Staphylococcus aureus, thereby causing leakage of bacterial cell contents. This result is consistent with the previous data on the effect of MA on cell walls and cell membranes. SDH and MDH are key metabolic enzymes in the tricarboxylic acid cycle of pathogenic bacteria. Detection of SDH and MDH activity in Staphylococcus aureus can reflect the energy metabolism of Staphylococcus aureus. In this embodiment, the activity of SDH and MDH in Staphylococcus aureus before and after the action of MA was detected, and it was found that MA can reduce the activity of SDH and MDH in Staphylococcus aureus, which means that MA can exert an antibacterial effect by acting on metabolic enzymes in Staphylococcus aureus. In addition, the interaction mode between MA and DNA was also detected to determine whether the drug can interact with DNA. The results of agarose gel electrophoresis showed that MA interacted with DNA to cause relaxation and ring opening of supercoiled DNA, and ultraviolet absorption spectroscopy further proved that MA could bind to pET-28aDNA. The effect of MA on the activity of DNA topoisomerase of Staphylococcus aureus was studied by the unwinding of DNA. The experimental results showed that MA could inhibit the activity of DNA topoisomerase I and II. The antibacterial mode of MA on Staphylococcus aureus is as follows Fig.15 shown.

[0132] Example 2

[0133] 1. Experimental Materials

[0134] MRSA ATCC 43300, purchased from Beijing Baozang Biotechnology Co., Ltd.; madecassic acid, purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Luria-Bertani (LB) agar medium and LB liquid medium, purchased from Guangdong Huankai Microbiological Technology Co., Ltd.; oxacillin, purchased from China Food and Drug Administration; cephalexin and cefepime, purchased from Shanghai Yuanye Biotechnology Co., Ltd.; Baird-Parker medium, purchased from Beijing Luqiao Technology Co., Ltd.; yolk potassium diazinon, purchased from Qingdao Haibo Biotechnology Co., Ltd.; constant temperature incubator (SPX-150BⅢ), purchased from Tianjin Test Instrument Co., Ltd.; constant temperature culture oscillator (ZWY-1102C), purchased from Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.; clean bench (SW-CJ-ZFD), purchased from Sujing Antai; UV spectrophotometer (V-5100), purchased from Shanghai Yuanxi Instrument Co., Ltd.; high temperature sterilizer (GI54TW), purchased from Zhiwei Instrument Co., Ltd.

[0135] 2. Test methods

[0136] 2.1 Chessboard test method

[0137] The chessboard test was used to evaluate the MIC value of the combination of MA and β-lactam antibiotics (cefepime, cephalexin, and oxacillin). The MRSA bacterial suspension cultured to the logarithmic phase was diluted to a concentration of 10 6 -10 7 cfu / mL, 180 μL of bacterial suspension and 20 μL of the serial dilution combination of MA and antibiotics were added to the 96-well plate, with DMSO as the control group, and cultured in a 37°C constant temperature incubator for 16 hours. After culture, TTC solution was added to each experimental well, and cultured at 37°C in the dark for 4 hours. The color change was observed, and the experiment was repeated 3 times to take the average value. The partial inhibitory concentration index (FICI) is used to determine the mode of action between MA and β-lactam antibiotics, as shown below:

[0138] FICI=MIC A Combined / MIC A Single use + MIC B Combined / MIC B Single use

[0139] In the formula, MICA combination: the MIC value of MA when used in combination; MICA alone: ​​the MIC value of MA when used alone; MICB combination: the MIC value of β-lactam antibiotics when used in combination; MICB alone: ​​the MIC value of β-lactam antibiotics when used alone.

[0140] The judgment criteria are as follows: Synergistic effect: FICI ≤ 0.5; Antagonistic effect: FICI ≥ 4; Additive effect: 0.5 < FICI ≤ 1; No relationship: 1 < FICI < 4. In addition, the MIC values of three β-lactam antibiotics against MRSA and the reduction multiples of MIC after combination with MA were measured, and all experiments were carried out three times.

[0141] 2.2 Determination of combined antibacterial growth curve

[0142] Respectively pipette different volumes of MA solution, the mixture of MA and cephalexin, and cephalexin into the Staphylococcus aureus suspension at 10 7 -10 8 cfu / mL, so that the final concentration of MA in the bacterial suspension is 1 / 4MA, the final concentration of the mixture of MA and cephalexin is 1 / 4MA and 1 / 32 cephalexin, and the final concentration of cephalexin is 1 / 32 cephalexin. Take the non-drug added group as the control group, put it in a constant temperature culture oscillator at 37 °C and 120 rpm for culture, sample every 4 h, measure its optical density (OD) value at 600 nm, and draw the growth curve of Staphylococcus aureus after the action. Repeat the experiment 3 times and take the average value.

[0143] 2.3 Effect of asiatic acid on the growth of MRSA under the condition of whole milk

[0144] Preparation of Baird-Parker agar plate: Weigh 58 g of Baird-Parker medium, stir until completely dissolved, make up the volume to 950 mL, dispense into Erlenmeyer flasks, sterilize the prepared medium by high temperature and high pressure, wait until it cools to about 50 °C, take 1 tube of potassium tellurite egg yolk enrichment agent and add it to 95 mL of the medium, pour it into disposable bacteriological culture dishes, and after the plate cools and solidifies, seal the plate and store it in a 4 °C refrigerator for later use.

[0145] Dilute the MRSA bacterial suspension with fresh sterilized whole milk to about 1×10 8 CFU / mL, mix the bacterial suspension with MA solution containing different concentrations so that the final concentration of MA is MIC and 3MIC respectively, and store them at 28 °C or 4 °C respectively. Take DMSO as the control group, after culturing for 24 h and 48 h, take 200 μL each and coat them on the Baird-Parker agar plate, place them in a 37 °C constant temperature incubator for incubation for 24 h, and use the colony counting method to measure the microbial concentration.

[0146] 3 Experimental results

[0147] 3.1 Synergistic effect of asiatic acid and β-lactam antibiotics

[0148] The chessboard test method was used to evaluate the dual combination of MA and three β-lactam antibiotics. The experimental results are shown in Table 2. Among the three combinations studied, two showed synergistic effects, one showed irrelevant effects, and no antagonism was detected. The MIC of MA against MRSA strains was 62.5μg / mL; when MA was combined with cephalexin, the MIC of cephalexin decreased from 125μg / mL to 3.9μg / mL, and the MIC of MA decreased from 62.5μg / mL to 15.625μg / mL. After MA was combined with oxacillin, the MIC of MA decreased from 62.5μg / mL to 31.25μg / mL, and the MIC of oxacillin did not change. When MA was combined with cefepime, the MIC of cefepime decreased from 62.5μg / mL to 3.9μg / mL, and the MIC of MA decreased from 62.5μg / mL to 7.8μg / mL. The FICI values ​​of the dual combination of MA and three β-lactam antibiotics ranged from 0.187 to 1.52, and the MIC values ​​of two β-lactam antibiotics were reduced by 16- to 32-fold.

[0149] Table 2

[0150]

[0151] 3.2 Growth curve

[0152] like Fig.16 As shown, the combination group showed an inhibitory effect within 32 hours. After 20 hours of culture, the use of 1 / 32 cephalexin and 1 / 4MA alone could not inhibit the growth of MRSA bacteria. Among them, 1 / 32 cephalexin could inhibit the growth of MRSA bacteria within 16 hours, and the combination of the two drugs could inhibit the growth of MRSA bacteria within 32 hours.

[0153] 3.3 Application of Madecassic Acid in Food

[0154] like Fig.17 As shown in the results, when whole-fat sterilized milk was stored at 28°C, the use of the 3MIC group at different exposure times had an inhibitory effect on the reproduction of MRSA bacteria in whole-fat sterilized milk, but there was no statistical difference between the MIC group at 24h and 48h and the control group (P>0.05). Compared with the control group, the 3MIC group inhibited the growth of MRSA within 24h and 48h (P<0.01). The concentration of microorganisms decreased significantly by 0.51log 10 CFU / mL, 1.23log 10 The results showed that under the infection mode of whole-fat sterilized milk at 28°C, the 3MIC MA group could inhibit the growth of MRSA in whole-fat sterilized milk at 24h and 48h.

[0155] like Fig.18As shown. When incubated at 4°C, the antibacterial activity of 3MIC MA showed significant differences after 24h and 48h of storage compared with the control group (P<0.01), and it can inhibit the growth of MRSA in whole-fat sterilized milk. However, there was no significant difference between the MIC group and the control group after 24h and 48h of storage (P>0.05). The results showed that the 3MIC MA experimental group achieved the expected effect, among which the MRSA bacterial concentration in the 48h group was significantly inhibited, which can prolong the storage of milk.

[0156] As a new source of antibacterial agents, Chinese herbal medicine can be used in combination with antibiotics to provide new ideas for supplementing the deficiencies of conventional therapies and improving the efficacy of antibiotics. MA is an active ingredient found in Centella asiatica and is a compound with multiple pharmacological effects. Previous studies have shown that MA has good antibacterial effects on Staphylococcus aureus, MRSA, Bacillus megaterium, etc. Therefore, this embodiment selects MA as a research object, and common β-lactam antibiotics are used in combination with it. The MIC of MA and β-lactam antibiotics to MRSA is determined by microbroth dilution method, and a combined drug sensitivity test is performed by chessboard test method to analyze the antibacterial effect on MRSA after combined use. It was found that MA and cefepime and cefalexin acted in combination, respectively, and had an additive effect, and the growth curve was determined by ultraviolet spectrophotometry, which further confirmed that MA and cefalexin had a synergistic effect. Experiments have confirmed that MA can reduce the MIC of cefepime and cefalexin alone to MRSA.

[0157] In this example, whole-fat high-temperature sterilized milk was used as a matrix to further evaluate the practical application of MA on the growth of MRSA bacteria in whole-fat high-temperature sterilized milk. The antibacterial activity of MA in whole-fat sterilized milk was tested at 28°C and ℃, respectively, simulating ideal refrigeration conditions (4°C) and room temperature conditions (28°C), and the antibacterial activity of MA in whole-fat sterilized milk was measured using the colony counting method. Among them, the total number of bacteria in the control group and the experimental group under ideal refrigeration conditions (4°C) was significantly lower than that under room temperature, which may be caused by low temperature stress. Compared with the control group, the microbial concentration of the group adding 3MIC was significantly reduced under ideal refrigeration conditions and room temperature conditions, indicating that 250μg / mLMA can effectively inhibit MRSA growth and extend the shelf life of milk beverages.

[0158] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a composition consisting of madecassic acid and β-lactam antibiotics in the preparation of a drug for inhibiting bacteria, characterized in that: The bacteria include Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Gordonia, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Bacillus megaterium; the β-lactam antibiotic is cefepime or cephalexin; When the β-lactam antibiotic is cefepime, the concentration of cefepime is 3.9 μg / mL, and the concentration of madecassic acid is 7.8 μg / mL; When the β-lactam antibiotic is cephalexin, the concentration of cephalexin is 3.9 μg / mL, and the concentration of madecassic acid is 15.625 μg / mL.

2. The use according to claim 1, characterized in that: The bacteria include Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

3. The use according to claim 1, characterized in that: The bacteria include Staphylococcus aureus.

4. An antibacterial drug, characterized in that: The invention comprises the composition consisting of madecassic acid and β-lactam antibiotics according to any one of claims 1 to 3.

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