Millettia sutchuenensis and composition containing Millettia sutchuenensis extract and application thereof

Abstract

The present invention relates to Herba Clematidis and an antibacterial composition containing Herba Clematidis extract and application thereof. The present invention provides an application of a composition comprising an antibiotic and Herba Clematidis extract for preventing and / or treating Staphylococcus aureus infection in the preparation of a preparation for preventing and treating Staphylococcus aureus infection. Through extensive research, the present invention unexpectedly discovered that Herba Clematidis has significant antibacterial activity against clinical multidrug-resistant MRSA strains, and can exert an anti-MRSA effect by inhibiting the proliferation of MRSA in the logarithmic growth phase; the antibacterial mechanism of Herba Clematidis extract may be to inhibit the synthesis of certain proteins of the bacteria, thereby inhibiting bacterial growth. Moreover, the test results show that Herba Clematidis extract can also produce a synergistic effect when used in combination with gentamicin. The present invention uses Herba Clematidis extract as a gentamicin enhancer and even in the preparation of antibacterial compositions, which has broad clinical applications and development prospects.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a scutellaria baicalensis extract and a composition containing a scutellaria baicalensis extract and applications thereof. Background Art

[0002] Drug-resistant bacterial infection has become a severe challenge faced by medical institutions and a serious threat to human health. Among them, methicillin-resistant Staphylococcus aureus (MRSA) is a common drug-resistant bacterium in clinical practice. With the abuse of antibiotics, the bacterial resistance rate has shown an upward trend year by year. The detection rate of MRSA in clinical practice is getting higher and higher, and it is showing a trend of multi-drug resistance. The monitoring data of the National Bacterial Resistance Monitoring Network and CHINET show that the infection rate of multi-drug resistant bacteria MRSA is more than 50%, and in ICU and burn wards, the infection has a tendency to increase. Because MRSA has multi-drug resistance, it faces difficulties such as high difficulty in treatment and high mortality in clinical practice, so it has become the focus of drug resistance detection worldwide. At present, the drugs that can be used to treat MRSA in clinical practice are very limited. The first-line drugs mainly include antibiotics such as vancomycin, teicoplanin and norvancomycin, and these drugs are expensive and have the disadvantages of large ototoxicity and nephrotoxicity. They need to be carefully selected during clinical use. Therefore, the development of more cost-effective anti-MRSA drugs is an urgent problem that needs to be solved at present. As the development of new antibiotics has also reached a bottleneck, it is an important measure to expand the source channels of antimicrobial drugs. Traditional Chinese medicine has the advantages of multi-function, multi-target action, not easy to produce drug resistance, low adverse reactions, and can reverse bacterial resistance when used in combination with antibiotics. In addition, thousands of years of practice have proved that traditional Chinese medicine has good efficacy in treating refractory infectious diseases. In recent years, it has been found that many traditional Chinese medicines and related active ingredients have significant anti-resistant bacteria effects. The development of antimicrobial drugs from traditional Chinese medicine will form a new research field.

[0003] As for the test methods for the in vitro, in vivo and in vitro and in vivo combined antibacterial activity of natural medicines, the applicable methods for different categories of natural medicines are different. Therefore, it is necessary to adopt appropriate methods to screen out suitable traditional Chinese medicines or natural medicines targeting specific bacterial groups or drug-resistant bacteria, so as to play an antibacterial or bactericidal role against bacteria or drug-resistant bacteria.

[0004] The herb is mild in nature, sweet and slightly bitter in taste, and has the effects of dispelling wind and dampness, relieving pain and detoxification, promoting blood circulation and reducing swelling, as well as analgesia and anti-inflammatory. It is clinically used to treat a variety of pain and gastrointestinal diseases. In addition, it can also be used to treat herpes zoster, tuberculosis, carbuncle, swelling, snake and insect bites, etc. It has high medicinal value. With the development and research of clinical practice, more functions of it are also being developed in the hope of obtaining more uses that are beneficial to humans. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide a new application of Cistanche deserticola or Cistanche deserticola extract in medicine. The Cistanche deserticola or Cistanche deserticola extract can be used alone or in combination with antibiotics to prevent and treat Staphylococcus aureus infection, especially to have an antibacterial or bactericidal effect on drug-resistant Staphylococcus aureus.

[0006] The first aspect of the present invention provides the use of Cistanche deserticola or Cistanche deserticola extract in combination with antibiotics in the preparation of a preparation for preventing and treating Staphylococcus aureus infection, wherein the antibiotic is selected from at least one of polymyxin B sulfate, levofloxacin, gentamicin, vancomycin, and ampicillin.

[0007] In the present invention, the Cistanche deserticola extract can be a corresponding extract obtained by any extraction method conventionally known to those skilled in the art.

[0008] In some embodiments, the Cistanche deserticola extract is a water extract or an alcohol extract of Cistanche deserticola.

[0009] In some of the embodiments, the Cistanche deserticola extract is an extract of Cistanche deserticola obtained by ethanol combined with ultrasound.

[0010] The second aspect of the present invention is to provide a composition for preventing and / or treating Staphylococcus aureus infection, the active ingredients of which include antibiotics and Cistanche deserticola extract, wherein the antibiotics and the Cistanche deserticola extract respectively form independent dosing units, or the antibiotics and the Cistanche deserticola extract together form a combined dosing unit.

[0011] In some embodiments, the antibiotics include but are not limited to polymyxin B sulfate, levofloxacin, gentamicin sulfate, vancomycin, and ampicillin; preferably, the antibiotic is gentamicin.

[0012] In some of the embodiments, the Cistanche deserticola extract is prepared by alcohol extraction combined with ultrasonic extraction.

[0013] In some embodiments, the extract of Cnidium monnieri comprises: taking the whole dried Cnidium monnieri herb, crushing it, adding 8-12 times the amount of 50%-70% ethanol aqueous solution, ultrasonic extraction at 40℃-50℃ for 1-2 hours, stirring several times during the process; adding 8-12 times the amount of 50%-70% ethanol aqueous solution to the residue obtained after filtration, heating it for 1-2 hours, combining the filtrate, and concentrating it to obtain the extract.

[0014] The third aspect of the present invention provides use of any of the above compositions in the preparation of an anti-Staphylococcus aureus preparation.

[0015] In some of the embodiments, the preparation is a dosage unit in which the antibiotic and the Cistanche deserticola extract are combined, and may further contain a pharmaceutically acceptable carrier, solvent, diluent, excipient or other medium.

[0016] In some embodiments, the Staphylococcus aureus is a drug-resistant strain, and further, the drug-resistant strain is a multi-drug resistant strain; more preferably, the multi-drug resistant strain is MRSA.

[0017] The present invention unexpectedly found that the Chinese medicine Cistanche deserticola extract had different degrees of antibacterial effect (MIC was 62.5-250 mg / mL, MBC was 250-500 mg / mL) on 30 clinical multi-drug resistant strains of MRSA when conducting research on Chinese medicine anti-pathogenic microorganisms. Combined with dynamic antibacterial test and SDS-PAGE to measure the change of soluble total protein, the test showed that the MRSA bacterial growth curve after different concentrations (1 / 4×MIC-2×MIC) of Cistanche deserticola extract was significantly changed, and the Cistanche deserticola extract could play an anti-MRSA effect by inhibiting the proliferation of MRSA in the logarithmic growth phase. The strength of the MRSA inhibitory effect was positively correlated with the concentration of Cistanche deserticola extract, and the results showed that the antibacterial mechanism of Cistanche deserticola extract may hinder the synthesis of certain proteins of the bacterial body, thereby inhibiting the growth of bacteria.

[0018] At the same time, it is worth noting that the combination of C. salsa extract and gentamicin can synergistically resist MRSA, significantly reduce the MIC of gentamicin, and increase the sensitivity of MRSA to gentamicin. The C. salsa extract cooperates with gentamicin to inhibit MRSA, which can effectively reduce the dosage of gentamicin and reduce its toxic and side effects. Although its specific synergistic mechanism needs further study, so far, there has been no report on the C. salsa extract improving the antibacterial effect of aminoglycoside antibiotics on Staphylococcus aureus. It fully shows that C. salsa extract has broad clinical application and development prospects as a gentamicin enhancer and even in the preparation of antibacterial compositions. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a partial structural representative diagram of the drug sensitivity test in Example 1, wherein AD is the test results of each drug sensitivity paper on methicillin-resistant Staphylococcus aureus M64; EH is the test results of each drug sensitivity paper on methicillin-resistant Staphylococcus aureus M66.

[0020] Figure 2 The figure is a statistical chart of the multi-drug resistance rates of 97 MRSA clinical isolates in Example 1.

[0021] Figure 3The graph is the result of the antibacterial test of the Cistanche deserticola extract using the Oxford cup method in Example 2, wherein AH are the Oxford cup antibacterial results of Cistanche deserticola against ATCC25923, ATCC43300, M29, M30, M37, M39, M43, and M46, respectively; wherein numbers 1, 4, 7, 10, 13, 16, 19, and 22 are the original solutions of Cistanche deserticola extract; 2, 5, 8, 11, 14, 17, 20, and 23 are Cistanche deserticola extracts with a concentration of 1 / 2; 3, 6, 9, 12, 15, 18, 21, and 24 are Cistanche deserticola extracts with a concentration of 1 / 4; C is a positive control: levofloxacin hydrochloride and sodium chloride injection (Hailijian, batch number 80120051202); NC is a negative control: sterile water.

[0022] Figure 4 This is the MIC result of the extract of Cnidium monnieri against MRSA determined by the microdilution method in Example 3.

[0023] Figure 5 The dynamic inhibition curve of the extract of C. salsa against MRSA in Example 4, wherein the test strain is the MRSA clinical strain M3. MIC: C. salsa extract 62.5 mg / mL.

[0024] Figure 6 This is a graph showing the detection results of the change in the total protein content of MRSA by the extract of Cnidium monnieri in Example 5, wherein M is a protein marker.

[0025] Figure 7 This is a diagram showing the antibacterial effect of the extract of Croton tiglium and gentamicin against ATCC43300 using the chessboard method in Example 6. DETAILED DESCRIPTION

[0026] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0027] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0028] Throughout the specification and claims, the following terms have the meanings clearly associated with this document unless the context clearly dictates otherwise. The phrase "in one embodiment" used in the present invention does not necessarily refer to the same embodiment, although it may be. In addition, the phrase "in another embodiment" used in the present invention does not necessarily refer to a different embodiment, although it may be. Therefore, as described below, the various embodiments of the present invention can be easily combined without departing from the scope or spirit of the present invention.

[0029] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0030] The present invention is further described in detail below with reference to specific embodiments.

[0031] Example 1 Determination of drug resistance spectrum of clinical strains

[0032] The test strains were 97 methicillin-resistant Staphylococcus aureus clinical isolates (MRSA) donated by Lingnan Hospital, the Third Affiliated Hospital of Sun Yat-sen University, and were isolated from the hospital from January to December 2020. The sources included sputum, secretions, pus, blood, urine and cerebrospinal fluid of patients, and were identified by VITEK 2Compact of BioMérieux. The quality control strain was the standard strain of Staphylococcus aureus ATCC25923 purchased from Huankai Biotech.

[0033] The drug sensitivity test was conducted in strict accordance with the National Clinical Laboratory Operation Procedure (3rd Edition). The drug sensitivity test used the paper agar diffusion method (KB method) to detect the sensitivity of MRSA to different antimicrobial drugs. The diluted bacterial solution was dipped with a sterile cotton swab and evenly spread on the MH agar plate. After drying, drug sensitivity paper strips (all purchased from Oxoid, P (penicillin), E (erythromycin), TE (tetracycline), VA (vancomycin), DA (clindamycin), KZ (cefazolin), SXT (cotrimoxazole), TGC (tigecycline), CIP (ciprofloxacin), LZD (linezolid), CN (gentamicin), FOX (cefoxitin), OX (oxacillin)) were pasted on the plate, placed on the front for 30 minutes, and then inverted in a 35°C incubator for 18 hours. The size of the inhibition ring was measured, and the drug resistance was judged according to the CLSI 2013 M100-S23 drug resistance standard.

[0034] The statistical table and some result graphs of drug sensitivity test results are as follows: Table 1 and Figure 1As shown, the drug resistance rates of the above 97 clinical MRSA strains to penicillin (94.85%) and erythromycin (90.72%) were higher than 90.00%, and the drug resistance rates to oxacillin (77.32%), cefoxitin (68.04%), clindamycin (65.98%), ciprofloxacin (55.67%), tetracycline (51.55%) and cefazolin (50.52%) were also high, all exceeding 50%, and they were almost not resistant to trimethoprim-sulfamethoxazole, linezolid and vancomycin (RR≤5.00%).

[0035] Table 1. Resistance rate of clinically isolated MRSA to common antibiotics determined by KB method

[0036]

[0037] Furthermore, according to the calculation formula of multidrug resistance index MARI = a / bc (a is the total antimicrobial resistance value of bacteria, b is the number of antimicrobial drug types, and c represents the number of bacterial strains; 0 < MARI < 1, the larger the MARI value, the higher the multidrug resistance level), SPSS software was used for statistical analysis, and GraphPad Prism was used for graphing. The statistical graph of clinical strain resistance rate is shown in the figure. Figure 2 As shown, among the 97 clinical isolates of MRSA, 78 strains showed multidrug-resistant bacteria characteristics (showing significant resistance to three or more types of antibiotics), accounting for 80.41%, and the multidrug resistance index MARI was 0.5069.

[0038] Example 2 Detection of antibacterial effect of Cnidium monnieri extract by Oxford cup method

[0039] 1. Materials

[0040] Preparation of Cistanche deserticola extract: Accurately weigh 100g of Cistanche deserticola crushed material, add 10 times the amount of 60% ethanol, and use 45℃ ultrasonic extraction for 90min, stirring several times during the process; discard the filter residue after filtration, concentrate the filtrate by rotary evaporator, and add water to make up to 100ml; filter and sterilize with 0.22μm microporous filter membrane as the drug stock solution (1g crude drug / mL), and use it in divided packages.

[0041] Positive control drugs: levofloxacin hydrochloride and sodium chloride injection (Hailijian, batch number 80120051202), polymyxin B sulfate (Aladdin, batch number J2126657), levofloxacin (Aladdin, batch number J2122423), gentamicin sulfate (Aladdin, batch number H2106059), vancomycin (Aladdin, batch number H2125431), and ampicillin (Aladdin, batch number E2011092).

[0042] Pretreatment method of positive control drug: take an appropriate amount of polymyxin B, dilute and mix with normal saline to a working solution with a final concentration of 10 mg / mL; take an appropriate amount of levofloxacin and ampicillin, dilute and mix with normal saline to a working solution with a final concentration of 2 mg / mL; take an appropriate amount of gentamicin, dilute and mix with normal saline to a working solution with a final concentration of 1 mg / mL; take an appropriate amount of vancomycin, dilute and mix with normal saline to a working solution with a final concentration of 0.2 mg / mL.

[0043] Test strains: 56 random MRSA clinical isolates (MRSA) showing multi-drug resistance from 78 strains in Example 1 above.

[0044] Quality control strains: Staphylococcus aureus standard strain ATCC25923 and MRSA standard strain ATCC43300, both purchased from Huankai Biotechnology.

[0045] 2. Methods

[0046] The standard strain of Staphylococcus aureus ATCC25923, the standard strain of MRSA ATCC43300 and 56 clinical strains of MRSA were inoculated into MH broth medium respectively, and cultured in a constant temperature air bath shaking incubator at 37°C and 200r / min for 12h. The above bacterial liquid was streaked on MH agar medium, and a single clone colony was picked in MH broth medium, and cultured at 37°C and 200r / min to complete the activation of the strain. The activated bacterial liquid was centrifuged at 8000r / min for 5min, the supernatant was discarded, and the bacterial precipitate was diluted to 1×10 with sterilized MH broth medium. 6 CFU / mL, as the experimental bacterial solution.

[0047] First, pour the sterilized MH agar medium into the culture dish and wait for it to solidify. In addition, wait for the MH agar medium to cool to about 45°C, mix in 5μl of the corresponding experimental bacterial solution, mix evenly, accurately take 5.0mL of the mixed bacterial culture medium and completely cover the solidified culture medium and ensure its surface is smooth, and wait for solidification. Place an Oxford cup that has been sterilized by high pressure on the surface of the culture medium, add different concentrations of Millettia reticulata extract to the cup, use sterile double distilled water as a negative control, and levofloxacin working solution as a positive control, place the plate in a constant temperature incubator at 37°C and culture overnight, then take out the plate. Use the "cross method" to determine the diameter of the inhibition zone, take pictures and record the relevant results, and judge that the inhibition zone diameter is >7mm as having an antibacterial effect. The test results are shown in Table 2, Figure 3 shown.

[0048] Table 2 Statistics on the antibacterial effect of the extracts of Cistanche deserticola on MRSA clinical strains

[0049]

[0050] The antibacterial effect of the extract of C. salsa was evaluated on Staphylococcus aureus ATCC25923, MRSA standard strain ATCC43300 and 56 MRSA clinical strains. The results of the Oxford cup antibacterial test showed that the extract of C. salsa had different degrees of antibacterial activity against ATCC25923, ATCC43300 and 30 MRSA clinical strains (inhibition ring diameter > 7mm), and the antibacterial rate against MRSA clinical strains was 53.57% (30 / 56). Figure 3 And Table 2. Figure 3 It can be seen that the extract of C. salsa showed significant antibacterial activity at concentrations of 500mg / ml and 250mg / ml, and the sensitivity of MRSA to different concentrations of C. salsa extract showed a certain concentration dependence. The comprehensive results showed that the extract of C. salsa had a certain antibacterial effect on MRSA.

[0051] Example 3 Determination of MIC and MBC of the extract of Cnidium monnieri against MRSA

[0052] The extract of C. salsa and five antibiotics (polymyxin B, levofloxacin, gentamicin, ampicillin, and vancomycin) were diluted from 1:2 to 1:512 by the doubling dilution method. The corresponding concentrations of C. salsa extract after doubling dilution were 500, 250, 125, 62.5, 31.25, 15.625, 7.813, and 3.906 mg / mL (1 / 2-1 / 256), respectively. 100 μL of different concentrations of diluted drug solution and antibiotics were added to a 96-well plate, and then 1×10 6 CFU / mL MRSA bacterial suspension 100μL, while making a blank control, culture at 37℃ for 18h, observe the growth of the test bacteria (observe turbidity with naked eye), add 20μL 0.2% TTC to each well, protect from light, culture for 4h, repeat the experiment 3 times, take the average value, and take the group without drug addition as the blank control. The lowest drug concentration without the production of red reduced formazan is the minimum inhibitory concentration (MIC). After measuring the MIC, take 100μL of culture from each well without bacterial growth and inoculate it on the nutrient agar plate, place it in a constant temperature incubator at 37℃ for 18-24h, and the corresponding drug concentration when the number of colonies growing on the plate is less than 5 is the minimum bactericidal concentration (MBC).

[0053] Table 3 MIC and MBC test results of the extract of Cnidium monnieri against MRSA

[0054]

[0055] Note: “+” means the culture medium is turbid; “-” means the culture medium is clear

[0056] The results of microdilution method showed that the MIC of the extract of Cistanche deserticola against Staphylococcus aureus ATCC25923 and MRSA standard bacteria ATCC43300 were both 125 mg / mL, and the MBC was 500 mg / mL. The MIC against MRSA clinical isolates was 62.5-250 mg / mL, and the MBC was 250-500 mg / mL (e.g. Figure 4 , as shown in Table 3).

[0057] Example 4 Effect of the extract of Cnidium monnieri on the growth curve of MRSA

[0058] After MRSA was activated, it was prepared with physiological saline to 0.5 McFarland turbidity for use. The experiment was divided into a positive control group (only bacterial solution was added without drug solution) and a group of Millettia serrata extract (containing 1 / 4×MIC, 1 / 2×MIC, 1×MIC and 2×MIC). The negative control group was a group without bacterial solution but with drug solution of the same concentration, which was used to reduce drug color interference. 1% bacterial solution and corresponding drug solution were added to the MH liquid culture medium, respectively, and cultured at 37°C and 180r / min for 24h. Samples were taken aseptically every 2 hours, and the absorbance (A) at a wavelength of 600nm was measured with an enzyme marker. The OD600 of the experimental group was the difference between the absorbance of the experimental treatment group and the negative group of the same concentration and the same period, and the average value of 3 repeated experiments was taken. With t as the horizontal axis (X) and A600 as the vertical axis (Y), the MRSA growth curve was drawn to analyze the effect of different concentrations of Millettia serrata extract on MRSA growth. SPSS software was used for statistical analysis, and GraphPadPrism was used for drawing.

[0059] The inhibitory curve of the extract of Cistanche deserticola on the growth of MRSA is shown in Figure 5 As shown. Compared with the control group, the 1 / 4×MIC, 1 / 2×MIC, 1×MIC and 2×MIC groups treated with the extract of C. salsa all showed different degrees of inhibition of growth, and the optical density of the bacterial solution was significantly reduced. The higher the concentration of C. salsa extract, the greater the effect on bacterial growth. From the changes in the growth curve, the extract of C. salsa shortened the logarithmic growth phase of MRSA, prolonged the growth stagnation phase, and led to a significant decrease in the number of bacteria. The results showed that the extract of C. salsa had an inhibitory effect on the normal growth and reproduction of MRSA, and the strength of the inhibitory effect was positively correlated with the concentration of C. salsa extract.

[0060] Example 5 Effect of the Extract of Croton tiglium on the Change of Total Protein Content of MRSA

[0061] Sample preparation: Add 1% (v / v) MRSA cultured to the logarithmic phase to 30 mL MH medium. After the bacterial enrichment culture, add different concentrations of the extract of the scutellaria baicalensis (1 / 16×MIC~2×MIC), and add the same volume of sterile water without the extract of the scutellaria baicalensis as a blank group. After culturing at 37°C for 4 hours, collect 100 mg of bacteria by low-temperature centrifugation, suspend in 100 μL sterile water, and add 25 μL 4×SDS-PAGE Loading Buffer. Boil the sample in a water bath for 8 minutes, centrifuge at 8500 rpm for 5 minutes, and take the supernatant for SDS-PAGE electrophoresis.

[0062] Protein has the functions of maintaining collective metabolism and transporting various substances in the body, and is the main bearer of life activities. If the drug destroys the specific spatial structure of bacterial protein, its biological function will be lost, and the drug will play an antibacterial role. Figure 6 It can be seen that compared with the control group, the MRSA soluble protein content was significantly reduced after the action of Cistanche deserticola extract, but the protein type did not change significantly. As the concentration of Cistanche deserticola extract increased, the MRSA soluble protein band of the corresponding experimental group became lighter, indicating that the protein content was reduced. That is, the strength of the inhibitory effect on MRSA was positively correlated with the concentration of Cistanche deserticola extract. The results showed that the antibacterial mechanism of Cistanche deserticola extract may hinder the synthesis of certain proteins in the bacteria, thereby inhibiting bacterial growth.

[0063] Example 6 Effect of combined use of Cistanche deserticola extract and antibiotics

[0064] According to the MIC results of the Cistanche deserticola extract and four commonly used antibiotics (reference Example 2 detection method, the detection results are shown in Table 5), the multiple dilution method was used to dilute them respectively so that their final concentrations were 1 / 32MIC to 4MIC. The micro-chessboard dilution method was used to determine the MIC of the Cistanche deserticola extract in combination with antibiotics (polymyxin B, levofloxacin, gentamicin, ampicillin and vancomycin), and the FIC was analyzed. Among them, the FIC index = MIC of drug A when used in combination / MIC when drug A is used alone + MIC when drug B is used in combination / MIC when drug B is used alone. Judgment criteria: FIC index ≤ 0.5 is a synergistic effect, 0.5 < FIC index ≤ 1 is an additive effect, 1 < FIC index ≤ 2 is an irrelevant effect, and FIC index > 2 is an antagonistic effect. Take the experiment of the combined inhibition of M3 by Cistanche deserticola extract and gentamicin as an example:

[0065] The working solution of the extract of C. salsa and gentamicin was diluted to 4 times the MIC (i.e., 1 / 4 of C. salsa and 1 / 8 of gentamicin). Each row of the 96-well plate was numbered 1-12 from left to right and AH from top to bottom.

[0066] 1) Add 100 μL of Psoralea corylifolia to 9A-9G and 100 μL of antibiotics to 12A-12H.

[0067] 2) Add 50 μL of culture medium to 2A-8G.

[0068] 3) Pipette 50 μL of the Millettia repens in the 9th column to the 8th column, pipette and shake repeatedly for 3 times, then pipette 50 μL to the 7th column, and so on to the 2nd column, and discard the last 50 μL.

[0069] 4) Pipette 50 μL of the antibiotics in the 12th column into row G to fill it up to 100 μL, then pipette 50 μL of the culture medium into column 10, dilute the antibiotics in multiple proportions, and pipette 50 μL into row F to fill it up to 100 μL, and so on to row A.

[0070] 5) Add 200 μL of Croton tiglium to 10A, add 200 μL of antibiotics to 11A, add 100 μL of culture medium to 10B and below 11B, and dilute 10A and 11A in turn.

[0071] 6) Add 110 μL of M3 bacterial suspension and culture in a 37°C incubator overnight for 16-20 hours. Observe bacterial growth and record the results. Add 20 μL of 0.2% TTC to each well and culture for 4 hours in dark. Repeat the experiment 3 times and take the average value. The group without drug addition was used as blank control.

[0072] Table 4. Chessboard method combined drug sensitivity diagram

[0073]

[0074]

[0075] Table 5. MIC of antibiotics and extracts of Cnidium monnieri against test strains

[0076]

[0077] Table 6. FIC of the combination of Cnidium monnieri extract and antibiotics against the test strains

[0078]

[0079]

[0080] Table 7. Changes in MIC of test strains before and after the combination of C.

[0081]

[0082] The antibacterial effect of the combined use of C. salsa extract and different antibiotics was determined by the chessboard method. The results are shown in Tables 6 and 7. Among them, C. salsa extract can significantly improve the antibacterial effect of gentamicin (FIC=0.375) on ATCC43300, and the combined use shows a synergistic effect (such as Figure 7 As shown, well D5 in the 96-well plate is clear and does not turn red after adding TTC for culture, indicating that no bacteria grow in this well, that is, when the extract of Croton tiglium is at a concentration of 1 / 8 MIC, the MIC of gentamicin can be reduced from 1 / 16 to 1 / 4×1 / 16=1 / 64).

[0083] In addition, the antibacterial effect of levofloxacin (FIC=0.625), ampicillin (FIC=0.531) and vancomycin (FIC=0.531) on ATCC43300 was improved to a certain extent, and the combination showed an additive effect, while the combination with polymyxin B had no effect on ATCC43300. In addition, the extract of the genus Centella asiatica combined with polymyxin B (FIC=0.625), ampicillin (FIC=1.0), levofloxacin (FIC=0.531) and gentamicin (FIC=0.75) had an additive effect on the MRSA clinical strain M3, while the combination with vancomycin had no effect on the MRSA clinical strain, and the combination with levofloxacin (FIC=1.0), gentamicin (FIC=0.516) and vancomycin (FIC=0.625) had an additive antibacterial effect on Staphylococcus aureus ATCC25923.

[0084] The results of this experiment showed that the combined use of C. salsa extract and gentamicin can synergistically resist the MRSA standard strain ATCC43300, significantly reduce the MIC of gentamicin, increase the sensitivity of ATCC43300 to gentamicin, and improve the antibacterial effect on Staphylococcus aureus ATCC25923 and MRSA clinical strains to a certain extent. The combined use showed an additive effect, but the specific synergistic and additive mechanisms need further study. Gentamicin is an aminoglycoside antibiotic that can bind to the 30S subunit of the bacterial ribosome and block bacterial protein synthesis to exert antibacterial effects. It has strong ear and kidney toxicity. C. salsa extract synergizes with gentamicin to inhibit ATCC43300, which can effectively reduce the dosage of gentamicin and reduce its toxic and side effects. So far, there has been no report on the use of C. salsa extract to enhance the antibacterial effect of aminoglycoside antibiotics on Staphylococcus aureus. Therefore, C. salsa extract has potential development value as a gentamicin enhancer.

[0085] In addition, the combined use of the extract of C. salsa with polymyxin B, ampicillin, levofloxacin and vancomycin did not significantly improve the effect on Staphylococcus aureus ATCC25923 and MRSA. The combination of polymyxin B, ampicillin and levofloxacin on MRSA clinical strains, the combination of ampicillin, levofloxacin and vancomycin on MRSA standard strain ATCC43300, and the combination of levofloxacin and vancomycin on Staphylococcus aureus ATCC25923 all showed an additive effect, showing a certain antibacterial effect and the effectiveness of combined medication. In the results, no antagonistic effect was found in the combined medication, indicating that the combination of C. salsa extract and the above antibiotics is feasible. Although the drugs used in the current study are crude extracts of C. salsa, the specific active ingredients and their antibacterial mechanisms need further in-depth research. The above results all suggest that the natural antibacterial components of C. salsa extract may open up new areas for solving the problem of the sharp increase in antibiotic-resistant bacteria.

[0086] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of a Cistanche deserticola extract combined with an antibiotic in the preparation of a preparation for preventing and treating methicillin-resistant Staphylococcus aureus infection, wherein the antibiotic is gentamicin; wherein in the preparation, the Cistanche deserticola extract is 15.63 mg / mL and the gentamicin is 0.0156 mg / mL.

2. The use according to claim 1, It is characterized in that The extract of Cistanche deserticola is a water extract or an alcohol extract of Cistanche deserticola.

3. A preparation for preventing and / or treating Staphylococcus aureus infection, It is characterized in that The active ingredients of the preparation are antibiotics and the Cistanche deserticola extract, and the antibiotics and the Cistanche deserticola extract respectively form independent administration units, or the antibiotics and the Cistanche deserticola extract together form a combined administration unit; The antibiotic is gentamicin; In the preparation, the extract of Croton tiglium is 15.63 mg / mL, and the gentamicin is 0.0156 mg / mL.

4. The preparation according to claim 3, It is characterized in that The extract of Cistanche deserticola is a water extract or an alcohol extract of Cistanche deserticola.

5. The preparation according to claim 4, It is characterized in that The extract of Cnidium sutchuenensis is an alcohol extract, and its preparation method comprises: taking the whole herb of Cnidium sutchuenensis, crushing it, adding 8-12 times the amount of 50%-70% ethanol aqueous solution, ultrasonically extracting at 40℃-50℃ for 1-2 hours, stirring several times during the process; filtering to obtain a filtrate.

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